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Sample records for embryonic endothelial progenitor

  1. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.

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    Moretti, Alessandra; Caron, Leslie; Nakano, Atsushi; Lam, Jason T; Bernshausen, Alexandra; Chen, Yinhong; Qyang, Yibing; Bu, Lei; Sasaki, Mika; Martin-Puig, Silvia; Sun, Yunfu; Evans, Sylvia M; Laugwitz, Karl-Ludwig; Chien, Kenneth R

    2006-12-15

    Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

  2. Endothelial progenitor cells physiology and metabolic plasticity in brain angiogenesis and blood-brain barrier modeling

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    Natalia Malinovskaya

    2016-12-01

    Full Text Available Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons. Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies.

  3. Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

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    Reichman, David E; Park, Laura; Man, Limor; Redmond, David; Chao, Kenny; Harvey, Richard P; Taketo, Makoto M; Rosenwaks, Zev; James, Daylon

    2018-01-08

    Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs. © 2018. Published by The Company of Biologists Ltd.

  4. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

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    Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

    2017-01-01

    Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

  5. Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

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    Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

    2012-01-01

    Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

  6. Strategies to Reverse Endothelial Progenitor Cell Dysfunction in Diabetes

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    Alessandra Petrelli

    2012-01-01

    Full Text Available Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial, their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs. Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

  7. Obstructive sleep apnea and endothelial progenitor cells

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    Wang Q

    2013-10-01

    Full Text Available Qing Wang,1,* Qi Wu,2,* Jing Feng,3,4 Xin Sun5 1The Second Respiratory Department of the First People's Hospital of Kunming, Yunnan, People's Republic of China; 2Tianjin Haihe Hospital, Tianjin, People's Republic of China; 3Respiratory Department of Tianjin Medical University General Hospital, Tianjin, People's Republic of China; 4Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, NC, USA; 5Respiratory Department of Tianjin Haihe Hospital, Tianjin, People's Republic of China *These authors contributed equally to this work Background: Obstructive sleep apnea (OSA occurs in 4% of middle-aged men and 2% of middle-aged women in the general population, and the prevalence is even higher in specific patient groups. OSA is an independent risk factor for a variety of cardiovascular diseases. Endothelial injury could be the pivotal determinant in the development of cardiovascular pathology in OSA. Endothelial damage ultimately represents a dynamic balance between the magnitude of injury and the capacity for repair. Bone marrow–derived endothelial progenitor cells (EPCs within adult peripheral blood present a possible means of vascular maintenance that could home to sites of injury and restore endothelial integrity and normal function. Methods: We summarized pathogenetic mechanisms of OSA and searched for available studies on numbers and functions of EPCs in patients with OSA to explore the potential links between the numbers and functions of EPCs and OSA. In particular, we tried to elucidate the molecular mechanisms of the effects of OSA on EPCs. Conclusion: Intermittent hypoxia cycles and sleep fragmentation are major pathophysiologic characters of OSA. Intermittent hypoxia acts as a trigger of oxidative stress, systemic inflammation, and sympathetic activation. Sleep fragmentation is associated with a burst of sympathetic activation and systemic inflammation. In most studies, a reduction in circulating EPCs has

  8. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors

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    Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Evan Manuel

    2014-01-01

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages...... expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how...... cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess...

  9. Impairment of circulating endothelial progenitors in Down syndrome

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    Costa Valerio

    2010-09-01

    Full Text Available Abstract Background Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. Methods Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. Results We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. Conclusions Our data provide evidences for a reduced number and altered

  10. Endothelial progenitor cell-based neovascularization : implications for therapy

    NARCIS (Netherlands)

    Krenning, Guido; van Luyn, Marja J. A.; Harmsen, Martin C.

    Ischemic cardiovascular events are a major cause of death globally. Endothelial progenitor cell (EPC)-based approaches can result in improvement of vascular perfusion and might offer clinical benefit. However, although functional improvement is observed, the lack of long-term engraftment of EPCs

  11. Characterization of vascular endothelial progenitor cells from chicken bone marrow

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    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  12. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

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    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  13. Endothelial Progenitor Cells as Shuttle of Anticancer Agents.

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    Laurenzana, Anna; Margheri, Francesca; Chillà, Anastasia; Biagioni, Alessio; Margheri, Giancarlo; Calorini, Lido; Fibbi, Gabriella; Del Rosso, Mario

    2016-10-01

    Cell therapies are treatments in which stem or progenitor cells are stimulated to differentiate into specialized cells able to home to and repair damaged tissues. After their discovery, endothelial progenitor cells (EPCs) stimulated worldwide interest as possible vehicles to perform autologous cell therapy of tumors. Taking into account the tumor-homing properties of EPCs, two different approaches to control cancer progression have been pursued by combining cell-based therapy with gene therapy or with nanomedicine. The first approach is based on the possibility of engineering EPCs to express different transgenes, and the second is based on the capacity of EPCs to take up nanomaterials. Here we review the most important progress covering the following issues: the characterization of bona fide endothelial progenitor cells, their role in tumor vascularization and metastasis, and preclinical data about their use in cell-based tumor therapy, considering antiangiogenic, suicide, immune-stimulating, and oncolytic virus gene therapy. The mixed approach of EPC cell therapy and nanomedicine is discussed in terms of plasmonic-dependent thermoablation and molecular imaging.

  14. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

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    Caterina Oriana Aragona

    2016-01-01

    Full Text Available Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”. Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included. Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers. Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: Prospero CRD42015023717.

  15. Ewing's sarcoma precursors are highly enriched in embryonic osteochondrogenic progenitors.

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    Tanaka, Miwa; Yamazaki, Yukari; Kanno, Yohei; Igarashi, Katsuhide; Aisaki, Ken-ichi; Kanno, Jun; Nakamura, Takuro

    2014-07-01

    Ewing's sarcoma is a highly malignant bone tumor found in children and adolescents, and the origin of this malignancy is not well understood. Here, we introduced a Ewing's sarcoma-associated genetic fusion of the genes encoding the RNA-binding protein EWS and the transcription factor ETS (EWS-ETS) into a fraction of cells enriched for osteochondrogenic progenitors derived from the embryonic superficial zone (eSZ) of long bones collected from late gestational murine embryos. EWS-ETS fusions efficiently induced Ewing's sarcoma-like small round cell sarcoma formation by these cells. Analysis of the eSZ revealed a fraction of a precursor cells that express growth/differentiation factor 5 (Gdf5), the transcription factor Erg, and parathyroid hormone-like hormone (Pthlh), and selection of the Pthlh-positive fraction alone further enhanced EWS-ETS-dependent tumor induction. Genes downstream of the EWS-ETS fusion protein were quite transcriptionally active in eSZ cells, especially in regions in which the chromatin structure of the ETS-responsive locus was open. Inhibition of β-catenin, poly (ADP-ribose) polymerase 1 (PARP1), or enhancer of zeste homolog 2 (EZH2) suppressed cell growth in a murine model of Ewing's sarcoma, suggesting the utility of the current system as a preclinical model. These results indicate that eSZ cells are highly enriched in precursors to Ewing's sarcoma and provide clues to the histogenesis of Ewing's sarcoma in bone.

  16. Endothelial progenitor cell subsets and preeclampsia: Findings and controversies

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    Armin Attar

    2017-10-01

    Full Text Available Vascular remodeling is an essential component of gestation. Endothelial progenitor cells (EPCs play an important role in the regulation of vascular homeostasis. The results of studies measuring the number of EPCs in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of some variations in technical issues and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC, colony forming unit endothelial cells (CFU-ECs, and endothelial colony-forming cells (ECFCs. In general, most studies have shown an increase in the number of CACs in the maternal circulation with a progression in the gestational age in normal pregnancies, while functional capacities measured by CFU-ECs and ECFCs remain intact. In the case of preeclampsia, mobilization of CACs and ECFCs occurs in the peripheral blood of pregnant women, but the functional capacities shown by culture of the derived colony-forming assays (CFU-EC and ECFC assays are altered. Furthermore, the number of all EPC subsets will be reduced in umbilical cord blood in the case of preeclampsia. As EPCs play an important role in the homeostasis of vascular networks, the difference in their frequency and functionality in normal pregnancies and those with preeclampsia can be expected. In this review, there was an attempt to provide a justification for these controversies.

  17. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

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    Yu, Cai-Guo; Zhang, Ning; Yuan, Sha-Sha; Ma, Yan; Yang, Long-Yan; Feng, Ying-Mei; Zhao, Dong

    2016-01-01

    Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs) in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF) treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on "VEGF uncoupling with nitric oxide" and "competitive angiopoietin 1/angiopoietin 2" mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics.

  18. Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

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    Young Yu

    Full Text Available The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

  19. [Circulating endothelial progenitor cell levels in treated hypertensive patients].

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    Maroun-Eid, C; Ortega-Hernández, A; Abad, M; García-Donaire, J A; Barbero, A; Reinares, L; Martell-Claros, N; Gómez-Garre, D

    2015-01-01

    Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP<140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

  20. Vascular Endothelial Growth Factor from Embryonic Status to Cardiovascular Pathology

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    Mohsen Azimi-Nezhad

    2014-05-01

    Full Text Available Vascular endothelial growth factor (VEGF is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF121, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases.

  1. Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs.

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    Wechsler, S J; Luedke, A J

    1991-01-01

    Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

  2. Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

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    Im, Wooseok; Chung, Jin-Young; Bhan, Jaejun; Lim, Jiyeon; Lee, Soon-Tae; Chu, Kon; Kim, Manho

    2012-01-01

    Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verified that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms. PMID:23717107

  3. Endothelial progenitor cells regenerate infracted myocardium with neovascularisation development.

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    Abd El Aziz, M T; Abd El Nabi, E A; Abd El Hamid, M; Sabry, D; Atta, H M; Rahed, L A; Shamaa, A; Mahfouz, S; Taha, F M; Elrefaay, S; Gharib, D M; Elsetohy, Khaled A

    2015-03-01

    We achieved possibility of isolation, characterization human umbilical cord blood endothelial progenitor cells (EPCs), examination potency of EPCs to form new blood vessels and differentiation into cardiomyoctes in canines with acute myocardial infarction (AMI). EPCs were separated and cultured from umbilical cord blood. Their phenotypes were confirmed by uptake of double stains dioctadecyl tetramethylindocarbocyanine-labeled acetylated LDL and FITC-labeled Ulex europaeus agglutinin 1 (DILDL-UEA-1). EPCs of cord blood were counted. Human VEGFR-2 and eNOS from the cultured EPCs were assessed by qPCR. Human EPCs was transplanted intramyocardially in canines with AMI. ECG and cardiac enzymes (CK-MB and Troponin I) were measured to assess severity of cellular damage. Histopathology was done to assess neovascularisation. Immunostaining was done to detect EPCs transdifferentiation into cardiomyocytes in peri-infarct cardiac tissue. qPCR for human genes (hVEGFR-2, and eNOS) was done to assess homing and angiogenic function of transplanted EPCs. Cultured human cord blood exhibited an increased number of EPCs and significant high expression of hVEGFR-2 and eNOS genes in the culture cells. Histopathology showed increased neovascularization and immunostaining showed presence of EPCs newly differentiated into cardiomyocyte-like cells. Our findings suggested that hEPCs can mediate angiogenesis and differentiate into cardiomyoctes in canines with AMI.

  4. Endothelial progenitor cells regenerate infracted myocardium with neovascularisation development

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    M.T. Abd El Aziz

    2015-03-01

    Full Text Available We achieved possibility of isolation, characterization human umbilical cord blood endothelial progenitor cells (EPCs, examination potency of EPCs to form new blood vessels and differentiation into cardiomyoctes in canines with acute myocardial infarction (AMI. EPCs were separated and cultured from umbilical cord blood. Their phenotypes were confirmed by uptake of double stains dioctadecyl tetramethylindocarbocyanine-labeled acetylated LDL and FITC-labeled Ulex europaeus agglutinin 1 (DILDL-UEA-1. EPCs of cord blood were counted. Human VEGFR-2 and eNOS from the cultured EPCs were assessed by qPCR. Human EPCs was transplanted intramyocardially in canines with AMI. ECG and cardiac enzymes (CK-MB and Troponin I were measured to assess severity of cellular damage. Histopathology was done to assess neovascularisation. Immunostaining was done to detect EPCs transdifferentiation into cardiomyocytes in peri-infarct cardiac tissue. qPCR for human genes (hVEGFR-2, and eNOS was done to assess homing and angiogenic function of transplanted EPCs. Cultured human cord blood exhibited an increased number of EPCs and significant high expression of hVEGFR-2 and eNOS genes in the culture cells. Histopathology showed increased neovascularization and immunostaining showed presence of EPCs newly differentiated into cardiomyocyte-like cells. Our findings suggested that hEPCs can mediate angiogenesis and differentiate into cardiomyoctes in canines with AMI.

  5. Bone marrow endothelial progenitors in atherosclerotic plaque resolution

    Science.gov (United States)

    Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Barlic-Dicen, Jana

    2013-01-01

    Atherosclerosis is a major cause of morbidity and mortality in the United States. Persistently elevated circulating low-density lipoprotein, or hypercholesterolemia, and deposition of low-density lipoprotein in the vascular wall are the main inducers of atherosclerosis, which manifests itself as arterial lesions or plaques. Some plaques become thrombosis-prone and rupture, causing acute myocardial infarction or stroke. Lowering plasma cholesterol through the use of statins is the primary intervention against atherosclerosis. Treatment with statins slows progression of atherosclerosis but can only support limited plaque regression. Partially regressed plaques continue to pose a serious threat due to their remaining potential to rupture. Thus, new interventions inducing complete reversal of atherosclerosis are being sought. Implementation of new therapies will require clear understanding of the mechanisms driving plaque resolution. In this Commentary, we highlight the role of bone marrow endothelial progenitors in atherosclerotic plaque regression and discuss how regenerative cell-based interventions could be used in combination with plasma lipid-lowering to induce plaque reversal in order to prevent and/or reduce adverse cardiovascular events. PMID:23538778

  6. Effect of vitamin D on endothelial progenitor cells function.

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    Yoav Hammer

    Full Text Available Endothelial progenitor cells (EPCs are a population of bone marrow-derived cells, which have an important role in the process of endothelialization and vascular repair following injury. Impairment of EPCs, which occurs in patients with diabetes, was shown to be related to endothelial dysfunction, coronary artery disease (CAD and adverse clinical outcomes. Recent evidence has shown that calcitriol, the active hormone of vitamin D, has a favorable impact on the endothelium and cardiovascular system. There is limited data on the effect of vitamin D on EPCs function.To examine the in vitro effects of Calcitriol on EPCs from healthy subjects and patients with diabetes.Fifty-one patients with type 2 diabetes (60±11 years, 40% women, HbA1C: 9.1±0.8% and 23 healthy volunteers were recruited. EPCs were isolated and cultured with and without calcitriol. The capacity of the cells to form colony-forming units (CFUs, their viability (measured by MTT assay, KLF-10 levels and angiogenic markers were evaluated after 1 week of culture.In diabetic patients, EPC CFUs and cell viability were higher in EPCs exposed to calcitriol vs. EPCs not exposed to calcitriol [EPC CFUs: 1.25 (IQR 1.0-2.0 vs. 0.5 (IQR 0.5-1.9, p < 0.001; MTT:0.62 (IQR 0.44-0.93 vs. 0.52 (IQR 0.31-0.62, p = 0.001]. KLF-10 levels tended to be higher in EPCs exposed to vitamin D, with no differences in angiopoietic markers. In healthy subjects, calcitriol supplementation also resulted in higher cell viability [MTT: 0.23 (IQR 0.11-0.46 vs. 0.19 (0.09-0.39, p = 0.04], but without differences in CFU count or angiopoietic markers.In patients with diabetes mellitus, in vitro vitamin D supplementation improved EPCs capacity to form colonies and viability. Further studies regarding the mechanisms by which vitamin D exerts its effect are required.

  7. Interactions between endothelial progenitor cells (EPC) and titanium implant surfaces.

    Science.gov (United States)

    Ziebart, Thomas; Schnell, Anne; Walter, Christian; Kämmerer, Peer W; Pabst, Andreas; Lehmann, Karl M; Ziebart, Johanna; Klein, Marc O; Al-Nawas, Bilal

    2013-01-01

    Endothelial cells play an important role in peri-implant angiogenesis during early bone formation. Therefore, interactions between endothelial progenitor cells (EPCs) and titanium dental implant surfaces are of crucial interest. The aim of our in vitro study was to investigate the reactions of EPCs in contact with different commercially available implant surfaces. EPCs from buffy coats were isolated by Ficoll density gradient separation. After cell differentiation, EPC were cultured for a period of 7 days on different titanium surfaces. The test surfaces varied in roughness and hydrophilicity: acid-etched (A), sand-blasted-blasted and acid-etched (SLA), hydrophilic A (modA), and hydrophilic SLA (modSLA). Plastic and fibronectin-coated plastic surfaces served as controls. Cell numbers and morphology were analyzed by confocal laser scanning microscopy. Secretion of vascular endothelial growth factor (VEGF)-A was measured by enzyme-linked immunosorbent assay and expressions of iNOS and eNOS were investigated by real-time polymerase chain reaction. Cell numbers were higher in the control groups compared to the cells of titanium surfaces. Initially, hydrophilic titanium surfaces (modA and modSLA) showed lower cell numbers than hydrophobic surfaces (A and SLA). After 7 days smoother surfaces (A and modA) showed increased cell numbers compared to rougher surfaces (SLA and modSLA). Cell morphology of A, modA, and control surfaces was characterized by a multitude of pseudopodia and planar cell soma architecture. SLA and modSLA promoted small and plump cell soma with little quantity of pseudopodia. The lowest VEGF level was measured on A, the highest on modSLA. The highest eNOS and iNOS expressions were found on modA surfaces. The results of this study demonstrate that biological behaviors of EPCs can be influenced by different surfaces. The modSLA surface promotes an undifferentiated phenotype of EPCs that has the ability to secrete growth factors in great quantities. In

  8. Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

    Science.gov (United States)

    Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

    2015-07-01

    Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p cell level correlated positively with the number of patient colonies (r = 0.762, p Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research

  9. Endothelial progenitor cells display clonal restriction in multiple myeloma

    International Nuclear Information System (INIS)

    Braunstein, Marc; Özçelik, Tayfun; Bağişlar, Sevgi; Vakil, Varsha; Smith, Eric LP; Dai, Kezhi; Akyerli, Cemaliye B; Batuman, Olcay A

    2006-01-01

    In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V H ). In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V H primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI

  10. Norepinephrine stimulates mobilization of endothelial progenitor cells after limb ischemia.

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    Qijun Jiang

    Full Text Available OBJECTIVE: During several pathological processes such as cancer progression, thermal injury, wound healing and hindlimb ischemia, the mobilization of endothelial progenitor cells (EPCs mobilization was enhanced with an increase of sympathetic nerve activity and norepinephrine (NE secretion, yet the cellular and molecular mechanisms involved in the effects of NE on EPCs has less been investigated. METHODS AND RESULTS: EPCs from BMs, peripheral circulation and spleens, the VEGF concentration in BM, skeletal muscle, peripheral circulation and spleen and angiogenesis in ischemic gastrocnemius were quantified in mice with hindlimbs ischemia. Systemic treatment of NE significantly increased EPCs number in BM, peripheral circulation and spleen, VEGF concentration in BM and skeletal muscle and angiogenesis in ischemic gastrocnemius in mice with hind limb ischemia, but did not affair VEGF concentration in peripheral circulation and spleen. EPCs isolated from healthy adults were cultured with NE in vitro to evaluate proliferation potential, migration capacity and phosphorylations of Akt and eNOS signal moleculars. Treatment of NE induced a significant increase in number of EPCs in the S-phase in a dose-dependent manner, as well as migrative activity of EPCs in vitro (p<0.05. The co-treatment of Phentolamine, I127, LY294002 and L-NAME with NE blocked the effects of NE on EPCs proliferation and migration. Treatment with NE significantly increased phosphorylation of Akt and eNOS of EPCs. Addition of phentolamine and I127 attenuated the activation of Akt/eNOS pathway, but metoprolol could not. Pretreatment of mice with either Phentolamine or I127 significantly attenuated the effects of NE on EPCs in vivo, VEGF concentration in BM, skeletal muscle and angiogenesis in ischemic gastrocnemius, but Metoprolol did not. CONCLUSION: These results unravel that sympathetic nervous system regulate EPCs mobilization and their pro-angiogenic capacity via α adrenoceptor

  11. A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors

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    Huixuan Liang

    2012-09-01

    Nestin-cre transgenic mice have been widely used to direct recombination to neural stem cells (NSCs and intermediate neural progenitor cells (NPCs. Here we report that a readily utilized, and the only commercially available, Nestin-cre line is insufficient for directing recombination in early embryonic NSCs and NPCs. Analysis of recombination efficiency in multiple cre-dependent reporters and a genetic mosaic line revealed consistent temporal and spatial patterns of recombination in NSCs and NPCs. For comparison we utilized a knock-in Emx1cre line and found robust recombination in NSCs and NPCs in ventricular and subventricular zones of the cerebral cortices as early as embryonic day 12.5. In addition we found that the rate of Nestin-cre driven recombination only reaches sufficiently high levels in NSCs and NPCs during late embryonic and early postnatal periods. These findings are important when commercially available cre lines are considered for directing recombination to embryonic NSCs and NPCs.

  12. The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis

    NARCIS (Netherlands)

    Polus, A.; Kiec-wilk, B.; Hartwich, J.; Balwierz, A.; Stachura, J.; Dyduch, G.; Laidler, P.; Zagajewski, J.; Langman, T.; Schmitz, G.; Goralcsky, R.; Wertz, K.; Riss, G.; Keijer, J.; Dembinska-Kiec, A.

    2006-01-01

    Objectives: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect

  13. Human endothelial progenitor cells internalize high-density lipoprotein.

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    Kaemisa Srisen

    Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

  14. Mechanosensitivity of Embryonic Neurites Promotes Their Directional Extension and Schwann Cells Progenitors Migration

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    Gonzalo Rosso

    2017-11-01

    Full Text Available Background/Aims: Migration of Schwann cells (SCs progenitors and neurite outgrowth from embryonic dorsal root ganglions (DRGs are two central events during the development of the peripheral nervous system (PNS. How these two enthralling events preceding myelination are promoted is of great relevance from basic research and clinical aspects alike. Recent evidence demonstrates that biophysical cues (extracellular matrix stiffness and biochemical signaling act in concert to regulate PNS myelination. Microenvironment stiffness of SCs progenitors and embryonic neurites dynamically changes during development. Methods: DRG explants were isolated from day 12.5 to 13.5 mice embryos and plated on laminin-coated substrates with varied stiffness values. After 4 days in culture and immunostaining with specific markers, neurite outgrowth pattern, SCs progenitors migration, and growth cone shape and advance were analyzed with confocal fluorescence microscopy. Results: We found out that growing substrate stiffness promotes directional neurite outgrowth, SCs progenitors migration, growth cone advance and presumably axons fasciculation. Conclusions: DRG explants are in vitro models for the research of PNS development, myelination and regeneration. Consequently, we conclude the following: Our observations point out the importance of mechanosensitivity for the PNS. At the same time, they prompt the investigation of the important yet unclear links between PNS biomechanics and inherited neuropathies with myelination disorders such as Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies. Finally, they encourage the consideration of mechanosensitivity in bioengineering of scaffolds to aid nerve regeneration after injury.

  15. Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels

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    Barleon Bernhard

    2010-07-01

    Full Text Available Abstract Background Postnatal endothelial progenitor cells (EPCs have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. Results In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels. Mouse lung microvascular endothelial cells (MLMVECs were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony. These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. Conclusion The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

  16. Identification of human embryonic progenitor cell targeting peptides using phage display.

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    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  17. Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells.

    Science.gov (United States)

    Chen, Bai-Song; Xie, Hua; Zhang, Sheng-Li; Geng, Hong-Quan; Zhou, Jun-Mei; Pan, Jun; Chen, Fang

    2011-12-01

    This study assessed the use of vascular endothelial growth factor (VEGF) gene-modified endothelial progenitor cells (EPCs) seeded onto bladder acellular matrix grafts (BAMGs), to enhance the blood supply in tissue-engineered bladders in a porcine model. Autologous porcine peripheral EPCs were isolated, cultured, expanded, characterized, and modified with the VEGF gene using an adenovirus vector. The expression of VEGF was examined using reverse transcriptase polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). VEGF gene modified EPCs were seeded onto BAMG and cultured for 3 days before implantation into pigs for bladder tissue engineering. A partial bladder cystectomy was performed in 12 pigs. The experimental group (6 pigs) received VEGF gene-modified EPC-seeded BAMG. The control group (6 pigs) received BAMG without seeded EPCs. The resulting tissue-engineered bladders were subject to a general and histological analysis. Microvessel density (MVD) was assessed using immunohistochemistry. The ex vivo transfection efficiency of EPCs was greater than 60%-70% when concentrated adenovirus was used. The genetically modified cells expressed both VEGF and green fluorescent protein (GFP). Scanning electron microscopy (SEM) and Masson's trichrome staining of cross sections of the cultured cells seeded to BAMG showed cell attachment and proliferation on the surface of the BAMG. Histological examination revealed bladder regeneration in a time-dependent fashion. Significant increases in MVD were observed in the experimental group, in comparison with the control group. VEGF-modified EPCs significantly enhanced neovascularization, compared with BAMG alone. These results indicate that EPCs, combined with VEGF gene therapy, may be a suitable approach for increasing blood supply in the tissue engineering of bladders. Thus, a useful strategy to achieve a tissue-engineered bladder is indicated.

  18. Dynamics of circulating endothelial cells and endothelial progenitor cells in breast cancer patients receiving cytotoxic chemotherapy

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    Kuo Yu-Hsuan

    2012-12-01

    Full Text Available Abstract Background The abundance of circulating endothelial cells (CECs and circulating endothelial progenitor cells (CEPs, which serve as surrogate markers for angiogenesis, may be affected by chemotherapy. We studied their dynamic change during consecutive cycles of chemotherapy. Methods We collected blood samples from 15 breast cancer patients, who received a total of 56 courses of systemic chemotherapy, and measured the CECs, viable CECs (V-CECs, and CEPs by six-color flow cytometry within the seven days prior to chemotherapy, twice a week during the first and second cycles of chemotherapy, and then once a week during the subsequent cycles. Results The CEC, V-CEC, and CEP levels all significantly decreased from day 1 of treatment to the first week of chemotherapy. After one week of chemotherapy, the CEC and V-CEC levels returned to a level similar to day 1. The CEP level remained significantly reduced after the first week of chemotherapy, but gradually rebounded until the next course of chemotherapy. After six cycles of chemotherapy, the total number of CEC and V-CEC cells trended toward a decrease and the CEP cells toward an increase. Clinical factors, including the existence of a tumor, chemotherapy regimens, and the use of granulocyte colony stimulating factor, did not significantly affect these results. Conclusions The CEC and CEP counts change dynamically during each course of chemotherapy and after the chemotherapy cycles, providing background data for any future study planning to use CECs and CEPs as surrogate markers of angiogenesis in antiangiogenesis treatments combined with chemotherapy.

  19. Adhesion behavior of endothelial progenitor cells to endothelial cells in simple shear flow

    Science.gov (United States)

    Gong, Xiao-Bo; Li, Yu-Qing; Gao, Quan-Chao; Cheng, Bin-Bin; Shen, Bao-Rong; Yan, Zhi-Qiang; Jiang, Zong-Lai

    2011-12-01

    The adhesion of endothelial progenitor cells (EPCs) on endothelial cells (ECs) is one of the critical physiological processes for the regenesis of vascular vessels and the prevention of serious cardiovascular diseases. Here, the rolling and adhesion behavior of EPCs on ECs was studied numerically. A two-dimensional numerical model was developed based on the immersed boundary method for simulating the rolling and adhesion of cells in a channel flow. The binding force arising from the catch bond of a receptor and ligand pair was modeled with stochastic Monte Carlo method and Hookean spring model. The effect of tumor necrosis factor alpha (TNF- α) on the expression of the number of adhesion molecules in ECs was analyzed experimentally. A flow chamber system with CCD camera was set up to observe the top view of the rolling of EPCs on the substrate cultivated with ECs. Numerical results prove that the adhesion of EPC on ECs is closely related to membrane stiffness of the cell and shear rate of the flow. It also suggests that the adhesion force between EPC and EC by P-selectin glycoprotein ligand-1 only is not strong enough to bond the cell onto vessel walls unless contributions of other catch bond are considered. Experimental results demonstrate that TNF- α enhanced the expressions of VCAM, ICAM, P-selectin and E-selectin in ECs, which supports the numerical results that the rolling velocity of EPC on TNF- α treated EC substrate decreases obviously compared with its velocity on the untreated one. It is found that because the adhesion is affected by both the rolling velocity and the deformability of the cell, an optimal stiffness of EPC may exist at a given shear rate of flow for achieving maximum adhesion rates.

  20. Exercise training improves in vivo endothelial repair capacity of early endothelial progenitor cells in subjects with metabolic syndrome.

    Science.gov (United States)

    Sonnenschein, Kristina; Horváth, Tibor; Mueller, Maja; Markowski, Andrea; Siegmund, Tina; Jacob, Christian; Drexler, Helmut; Landmesser, Ulf

    2011-06-01

    Endothelial dysfunction and injury are considered to contribute considerably to the development and progression of atherosclerosis. It has been suggested that intense exercise training can increase the number and angiogenic properties of early endothelial progenitor cells (EPCs). However, whether exercise training stimulates the capacity of early EPCs to promote repair of endothelial damage and potential underlying mechanisms remain to be determined. The present study was designed to evaluate the effects of moderate exercise training on in vivo endothelial repair capacity of early EPCs, and their nitric oxide and superoxide production as characterized by electron spin resonance spectroscopy analysis in subjects with metabolic syndrome. Twenty-four subjects with metabolic syndrome were randomized to an 8 weeks exercise training or a control group. Superoxide production and nitric oxide (NO) availability of early EPCs were characterized by using electron spin resonance (ESR) spectroscopy analysis. In vivo endothelial repair capacity of EPCs was examined by transplantation into nude mice with defined carotid endothelial injury. Endothelium-dependent, flow-mediated vasodilation was analysed using high-resolution ultrasound. Importantly, exercise training resulted in a substantially improved in vivo endothelial repair capacity of early EPCs (24.0 vs 12.7%; p exercise training, but not in the control group. Moreover, exercise training reduced superoxide production of EPCs, which was not observed in the control group. The present study suggests for the first time that moderate exercise training increases nitric oxide production of early endothelial progenitor cells and reduces their superoxide production. Importantly, this is associated with a marked beneficial effect on the in vivo endothelial repair capacity of early EPCs in subjects with metabolic syndrome.

  1. The Use of Endothelial Progenitor Cells for the Regeneration of Musculoskeletal and Neural Tissues

    OpenAIRE

    Kamei, Naosuke; Atesok, Kivanc; Ochi, Mitsuo

    2017-01-01

    Endothelial progenitor cells (EPCs) derived from bone marrow and blood can differentiate into endothelial cells and promote neovascularization. In addition, EPCs are a promising cell source for the repair of various types of vascularized tissues and have been used in animal experiments and clinical trials for tissue repair. In this review, we focused on the kinetics of endogenous EPCs during tissue repair and the application of EPCs or stem cell populations containing EPCs for tissue regenera...

  2. Endothelial progenitor cell dysfunction in patients with progressive chronic kidney disease

    NARCIS (Netherlands)

    Krenning, Guido; Dankers, Patricia Y. W.; Drouven, Johannes W.; Waanders, Femke; Franssen, Casper F. M.; van Luyn, Marja J. A.; Harmsen, Martin C.; Popa, Eliane R.

    Krenning G, Dankers PY, Drouven JW, Waanders F, Franssen CF, van Luyn MJ, Harmsen MC, Popa ER. Endothelial progenitor cell dysfunction in patients with progressive chronic kidney disease. Am J Physiol Renal Physiol 296: F1314-F1322, 2009. First published April 1, 2009; doi:

  3. Endothelial progenitors encapsulated in bioartificial niches are insulated from systemic cytotoxicity and are angiogenesis competent.

    Science.gov (United States)

    Ratliff, B B; Ghaly, T; Brudnicki, P; Yasuda, K; Rajdev, M; Bank, M; Mares, J; Hatzopoulos, A K; Goligorsky, M S

    2010-07-01

    Intrinsic stem cells (SC) participate in tissue remodeling and regeneration in various diseases and following toxic insults. Failure of tissue regeneration is in part attributed to lack of SC protection from toxic stress of noxious stimuli, thus prompting intense research efforts to develop strategies for SC protection and functional preservation for in vivo delivery. One strategy is creation of artificial SC niches in an attempt to mimic the requirements of endogenous SC niches by generating scaffolds with properties of extracellular matrix. Here, we investigated the use of hyaluronic acid (HA) hydrogels as an artificial SC niche and examined regenerative capabilities of encapsulated embryonic endothelial progenitor cells (eEPC) in three different in vivo models. Hydrogel-encapsulated eEPC demonstrated improved resistance to toxic insult (adriamycin) in vitro, thus prompting in vivo studies. Implantation of HA hydrogels containing eEPC to mice with adriamycin nephropathy or renal ischemia resulted in eEPC mobilization to injured kidneys (and to a lesser extent to the spleen) and improvement of renal function, which was equal or superior to adoptively transferred EPC by intravenous infusion. In mice with hindlimb ischemia, EPC encapsulated in HA hydrogels dramatically accelerated the recovery of collateral circulation with the efficacy superior to intravenous infusion of EPC. In conclusion, HA hydrogels protect eEPC against adriamycin cytotoxicity and implantation of eEPC encapsulated in HA hydrogels supports renal regeneration in ischemic and cytotoxic (adriamycin) nephropathy and neovascularization of ischemic hindlimb, thus establishing their functional competence and superior capabilities to deliver stem cells stored in and released from this bioartificial niche.

  4. The level of circulating endothelial progenitor cells may be associated with the occurrence and recurrence of chronic subdural hematoma

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    Yan Song

    2013-01-01

    Full Text Available OBJECTIVES: The onset of chronic subdural hematoma may be associated with direct or indirect minor injuries to the head or a poorly repaired vascular injury. Endothelial progenitor cells happen to be one of the key factors involved in hemostasis and vascular repair. This study was designed to observe the levels of endothelial progenitor cells, white blood cells, platelets, and other indicators in the peripheral blood of patients diagnosed with chronic subdural hematoma to determine the possible relationship between the endothelial progenitor cells and the occurrence, development, and outcomes of chronic subdural hematoma. METHOD: We enrolled 30 patients with diagnosed chronic subdural hematoma by computer tomography scanning and operating procedure at Tianjin Medical University General Hospital from July 2009 to July 2011. Meanwhile, we collected 30 cases of peripheral blood samples from healthy volunteers over the age of 50. Approximately 2 ml of blood was taken from veins of the elbow to test the peripheral blood routine and coagulation function. The content of endothelial progenitor cells in peripheral blood mononuclear cells was determined by flow cytometry. RESULTS: The level of endothelial progenitor cells in peripheral blood was significantly lower in preoperational patients with chronic subdural hematomas than in controls. There were no significant differences between the two groups regarding the blood routine and coagulation function. However, the levels of circulating endothelial progenitor cells were significantly different between the recurrent group and the non-recurrent group. CONCLUSIONS: The level of circulating endothelial progenitor cells in chronic subdural hematoma patients was significantly lower than the level in healthy controls. Meanwhile, the level of endothelial progenitor cells in recurrent patients was significantly lower than the level in patients without recurrence. Endothelial progenitor cells may be related to the

  5. The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells.

    Science.gov (United States)

    Liberda, Eric N; Cuevas, Azita K; Qu, Qingshan; Chen, Lung Chi

    2014-08-01

    The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species. Following an acute whole body inhalation exposure to 500 µg/m(3) of nickel nanoparticles for 5 h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure. Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.

  6. Wnt signaling positively regulates endothelial cell fate specification in the Fli1a-positive progenitor population via Lef1.

    Science.gov (United States)

    Hübner, Kathleen; Grassme, Kathrin S; Rao, Jyoti; Wenke, Nina K; Zimmer, Cordula L; Korte, Laura; Mu Ller, Katja; Sumanas, Saulius; Greber, Boris; Herzog, Wiebke

    2017-10-01

    During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of β-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel β-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2 BAC :Venus-Pest) mu288 ; Tg(14TCF:loxP-STOP-loxP-dGFP) mu202 ). We therefore can detect β-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos. Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1. Our data indicate a novel role of β-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis. Copyright © 2017. Published by Elsevier Inc.

  7. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

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    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  8. Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.

    Science.gov (United States)

    Hamed, Saher; Ben-Nun, Ohad; Egozi, Dana; Keren, Aviad; Malyarova, Nastya; Kruchevsky, Danny; Gilhar, Amos; Ullmann, Yehuda

    2012-10-01

    Bone marrow-derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that was isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell-treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell-treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.

  9. Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Jacobsen, J.; Gunnarsson, A.

    2011-01-01

    Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis......Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis...

  10. Endothelial progenitor cells in mothers of low-birthweight infants: a link between defective placental vascularization and increased cardiovascular risk?

    LENUS (Irish Health Repository)

    King, Thomas F J

    2013-01-01

    Offspring birthweight is inversely associated with future maternal cardiovascular mortality, a relationship that has yet to be fully elucidated. Endothelial progenitor cells (EPCs) are thought to play a key role in vasculogenesis, and EPC numbers reflect cardiovascular risk.

  11. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  12. Androgen Modulates Functions of Endothelial Progenitor Cells through Activated Egr1 Signaling

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    Yizhou Ye

    2016-01-01

    Full Text Available Researches show that androgens have important effects on migration of endothelial cells and endothelial protection in coronary heart disease. Endothelial progenitor cells (EPCs as a progenitor cell type that can differentiate into endothelial cells, have a critical role in angiogenesis and endothelial protection. The relationship between androgen and the functions of EPCs has animated much interest and controversy. In this study, we investigated the angiogenic and migratory functions of EPCs after treatment by dihydrotestosterone (DHT and the molecular mechanisms as well. We found that DHT treatment enhanced the incorporation of EPCs into tubular structures formed by HUVECs and the migratory activity of EPCs in the transwell assay dose dependently. Moreover, microarray analysis was performed to explore how DHT changes the gene expression profiles of EPCs. We found 346 differentially expressed genes in androgen-treated EPCs. Angiogenesis-related genes like Egr-1, Vcan, Efnb2, and Cdk2ap1 were identified to be regulated upon DHT treatment. Furthermore, the enhanced angiogenic and migratory abilities of EPCs after DHT treatment were inhibited by Egr1-siRNA transfection. In conclusion, our findings suggest that DHT markedly enhances the vessel forming ability and migration capacity of EPCs. Egr1 signaling may be a possible pathway in this process.

  13. Capture of circulatory endothelial progenitor cells and accelerated re-endothelialization of a bio-engineered stent in human ex vivo shunt and rabbit denudation model

    NARCIS (Netherlands)

    K. Larsen (Katarína); C. Cheng (Caroline (Ka Lai)); D. Tempel (Dennie); S. Parker (Sherry); S. Yazdani (Saami); W.K. den Dekker (Wijnand); H.J. Houtgraaf (Jaco); R. de Jong (Renate); S. Swager-ten Hoor (Stijn); E. Ligtenberg (Erik); S.R. Hanson (Stephen); R. Rowland (Steve); F. Kolodgie (Frank); P.W.J.C. Serruys (Patrick); R. Virmani (Renu); H.J. Duckers (Henricus)

    2012-01-01

    textabstractThe Genous™ Bio-engineered R™ stent (GS) aims to promote vascular healing by capture of circulatory endothelial progenitor cells (EPCs) to the surface of the stent struts, resulting in accelerated re-endothelialization. Here, we assessed the function of the GS in comparison to bare-metal

  14. Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

    Science.gov (United States)

    Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

    2013-01-01

    Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

  15. Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

    Directory of Open Access Journals (Sweden)

    Peter E Westerweel

    Full Text Available Circulating Endothelial Progenitor Cell (EPC levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment.Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+Flk-1(+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+ hematopoietic progenitor cells (HPC and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed.In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro.EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

  16. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects.

    Science.gov (United States)

    Mikirova, Nina A; Jackson, James A; Hunninghake, Ron; Kenyon, Julian; Chan, Kyle W H; Swindlehurst, Cathy A; Minev, Boris; Patel, Amit N; Murphy, Michael P; Smith, Leonard; Ramos, Famela; Ichim, Thomas E; Riordan, Neil H

    2010-04-08

    The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  17. Nutraceutical augmentation of circulating endothelial progenitor cells and hematopoietic stem cells in human subjects

    Directory of Open Access Journals (Sweden)

    Minev Boris

    2010-04-01

    Full Text Available Abstract The medical significance of circulating endothelial or hematopoietic progenitors is becoming increasing recognized. While therapeutic augmentation of circulating progenitor cells using G-CSF has resulted in promising preclinical and early clinical data for several degenerative conditions, this approach is limited by cost and inability to perform chronic administration. Stem-Kine is a food supplement that was previously reported to augment circulating EPC in a pilot study. Here we report a trial in 18 healthy volunteers administered Stem-Kine twice daily for a 2 week period. Significant increases in circulating CD133 and CD34 cells were observed at days 1, 2, 7, and 14 subsequent to initiation of administration, which correlated with increased hematopoietic progenitors as detected by the HALO assay. Augmentation of EPC numbers in circulation was detected by KDR-1/CD34 staining and colony forming assays. These data suggest Stem-Kine supplementation may be useful as a stimulator of reparative processes associated with mobilization of hematopoietic and endothelial progenitors.

  18. Bone marrow endothelial progenitors augment atherosclerotic plaque regression in a mouse model of plasma lipid lowering

    Science.gov (United States)

    Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Iida, Ryuji; Wang, Qilong; Zou, Ming-Hui; Barlic-Dicen, Jana

    2012-01-01

    The major event initiating atherosclerosis is hypercholesterolemia-induced disruption of vascular endothelium integrity. In settings of endothelial damage, endothelial progenitor cells (EPCs) are mobilized from bone marrow into circulation and home to sites of vascular injury where they aid endothelial regeneration. Given the beneficial effects of EPCs in vascular repair, we hypothesized that these cells play a pivotal role in atherosclerosis regression. We tested our hypothesis in the atherosclerosis-prone mouse model in which hypercholesterolemia, one of the main factors affecting EPC homeostasis, is reversible (Reversa mice). In these mice normalization of plasma lipids decreased atherosclerotic burden; however, plaque regression was incomplete. To explore whether endothelial progenitors contribute to atherosclerosis regression, bone marrow EPCs from a transgenic strain expressing green fluorescent protein under the control of endothelial cell-specific Tie2 promoter (Tie2-GFP+) were isolated. These cells were then adoptively transferred into atheroregressing Reversa recipients where they augmented plaque regression induced by reversal of hypercholesterolemia. Advanced plaque regression correlated with engraftment of Tie2-GFP+ EPCs into endothelium and resulted in an increase in atheroprotective nitric oxide and improved vascular relaxation. Similarly augmented plaque regression was also detected in regressing Reversa mice treated with the stem cell mobilizer AMD3100 which also mobilizes EPCs to peripheral blood. We conclude that correction of hypercholesterolemia in Reversa mice leads to partial plaque regression that can be augmented by AMD3100 treatment or by adoptive transfer of EPCs. This suggests that direct cell therapy or indirect progenitor cell mobilization therapy may be used in combination with statins to treat atherosclerosis. PMID:23081735

  19. A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Yijie Geng

    2016-07-01

    Full Text Available The emerging models of human embryonic stem cell (hESC self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin+CD31+CD34+KDR+CD43− putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL+ multi-cellular modules and a VEGFR3+ sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

  20. A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

    Science.gov (United States)

    Geng, Yijie; Feng, Bradley

    2016-07-01

    The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

  1. Benfotiamine counteracts glucose toxicity effects on endothelial progenitor cell differentiation via Akt/FoxO signaling.

    Science.gov (United States)

    Marchetti, Valentina; Menghini, Rossella; Rizza, Stefano; Vivanti, Alessia; Feccia, Tiziana; Lauro, Davide; Fukamizu, Akiyoshi; Lauro, Renato; Federici, Massimo

    2006-08-01

    Dysfunction of mature endothelial cells is thought to play a major role in both micro- and macrovascular complications of diabetes. However, recent advances in biology of endothelial progenitor cells (EPCs) have highlighted their involvement in diabetes complications. To determine the effect of glucotoxicity on EPCs, human EPCs have been isolated from peripheral blood mononuclear cells of healthy donors and cultured in the presence or absence of high glucose (33 mmol/l) or high glucose plus benfotiamine to scavenge glucotoxicity. Morphological analysis revealed that high glucose significantly affected the number of endothelial cell colony forming units, uptake and binding of acLDL and Lectin-1, and the ability to differentiate into CD31- and vascular endothelial growth factor receptor 2-positive cells. Functional analysis outlined a reduced EPC involvement in de novo tube formation, when cocultured with mature endothelial cells (human umbilical vein endothelial cells) on matrigel. To explain the observed phenotypes, we have investigated the signal transduction pathways known to be involved in EPC growth and differentiation. Our results indicate that hyperglycemia impairs EPC differentiation and that the process can be restored by benfotiamine administration, via the modulation of Akt/FoxO1 activity.

  2. Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

    Institute of Scientific and Technical Information of China (English)

    Liu-mei Hu; Guo-xu Xu; Guo-tong XU; Wei-ye Li; Xia Lei; Bo Ma; Yu Zhang; Yan Yan; Ya-lan Wu; Ge-zhi Xu; Wen Ye; Ling Wang

    2011-01-01

    Objective To investigate the possible involvement of erythropoietin (EPO)/erythropoietin receptor(EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR).Methods EPOR positive circulating progenitor cells (CPCs: CD34+) and endothelial progenitor cells (EPCs: CD34+KDR+) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes (n=7), non-prolif-erative DR (NPDR, n=7), proliferative DR (PDR, n=8), and PDR complicated with diabetic nephropathy (PDR-DN, n=7). Results The numbers of EPOR+ CPCs and EPOR+ EPCs were reduced remarkably in NPDR compared with the control group (both P<0.01), whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR+ CPCs in CPCs (NPDR vs.control, P< 0.01) and that of EPOR+ EPCs in EPCs (NPDR vs. control, P< 0.05). Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the im-paired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR+ EPCs associated with ischemia.

  3. FGFR3 regulates brain size by controlling progenitor cell proliferation and apoptosis during embryonic development.

    Science.gov (United States)

    Inglis-Broadgate, Suzanne L; Thomson, Rachel E; Pellicano, Francesca; Tartaglia, Michael A; Pontikis, Charlie C; Cooper, Jonathan D; Iwata, Tomoko

    2005-03-01

    Mice with the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3(+/K644E)) exhibited a marked enlargement of the brain. The brain size was increased as early as E11.5, not secondary to the possible effect of Fgfr3 activity in the skeleton. Furthermore, the mutant brains showed a dramatic increase in cortical thickness, a phenotype opposite to that in FGF2 knockout mice. Despite this increased thickness, cortical layer formation was largely unaffected and no cortical folding was observed during embryonic days 11.5-18.5 (E11.5-E18.5). Measurement of cortical thickness revealed an increase of 38.1% in the EIIa;Fgfr3(+/K644E) mice at E14.5 and the advanced appearance of the cortical plate was frequently observed at this stage. Unbiased stereological analysis revealed that the volume of the ventricular zone (VZ) was increased by more than two fold in the EIIa;Fgfr3(+/K644E) mutants at E14.5. A relatively mild increase in progenitor cell proliferation and a profound decrease in developmental apoptosis during E11.5-E14.5 most likely accounts for the dramatic increase in total telecephalic cell number. Taken together, our data suggest a novel function of Fgfr3 in controlling the development of the cortex, by regulating proliferation and apoptosis of cortical progenitors.

  4. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  5. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thomas C Schulz

    Full Text Available Development of a human embryonic stem cell (hESC-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

  6. Late Release of Circulating Endothelial Cells and Endothelial Progenitor Cells after Chemotherapy Predicts Response and Survival in Cancer Patients

    Directory of Open Access Journals (Sweden)

    Jeanine M. Roodhart

    2010-01-01

    Full Text Available We and others have previously demonstrated that the acute release of progenitor cells in response to chemotherapy actually reduces the efficacy of the chemotherapy. Here, we take these data further and investigate the clinical relevance of circulating endothelial (progenitor cells (CE(PCs and modulatory cytokines in patients after chemotherapy with relation to progression-free and overall survival (PFS/OS. Patients treated with various chemotherapeutics were included. Blood sampling was performed at baseline, 4 hours, and 7 and 21 days after chemotherapy. The mononuclear cell fraction was analyzed for CE(PC by FACS analysis. Plasma was analyzed for cytokines by ELISA or Luminex technique. CE(PCs were correlated with response and PFS/OS using Cox proportional hazard regression analysis. We measured CE(PCs and cytokines in 71 patients. Only patients treated with paclitaxel showed an immediate increase in endothelial progenitor cell 4 hours after start of treatment. These immediate changes did not correlate with response or survival. After 7 and 21 days of chemotherapy, a large and consistent increase in CE(PC was found (P < .01, independent of the type of chemotherapy. Changes in CE(PC levels at day 7 correlated with an increase in tumor volume after three cycles of chemotherapy and predicted PFS/OS, regardless of the tumor type or chemotherapy. These findings indicate that the late release of CE(PC is a common phenomenon after chemotherapeutic treatment. The correlation with a clinical response and survival provides further support for the biologic relevance of these cells in patients' prognosis and stresses their possible use as a therapeutic target.

  7. Effect of onion peel extract on endothelial function and endothelial progenitor cells in overweight and obese individuals.

    Science.gov (United States)

    Choi, Eun-Yong; Lee, Hansongyi; Woo, Jong Shin; Jang, Hyun Hee; Hwang, Seung Joon; Kim, Hyun Soo; Kim, Woo-Sik; Kim, Young-Seol; Choue, Ryowon; Cha, Yong-Jun; Yim, Jung-Eun; Kim, Weon

    2015-09-01

    Acute or chronic intake of polyphenol-rich foods has been reported to improve endothelial function. Quercetin, found abundantly in onion, is a potent antioxidant flavonoid. The aim of this study was to investigate whether consumption of onion peel extract (OPE) improves endothelial function in healthy overweight and obese individuals. This was a randomized double-blind, placebo-controlled study. Seventy-two healthy overweight and obese participants were randomly assigned to receive a red, soft capsule of OPE (100 mg quercetin/d, 50 mg quercetin twice daily; n = 36 participants) or an identical placebo capsule (n = 36) for 12 wk. Endothelial function, defined by flow-mediated dilation (FMD), circulating endothelial progenitor cells (EPCs) by flow cytometry, and laboratory test were determined at baseline and after treatment. Baseline characteristics and laboratory findings did not significantly differ between the two groups. Compared with baseline values, the OPE group showed significantly improved FMD at 12 wk (from 12.5 ± 5.2 to 15.2 ± 6.1; P = 0.002), whereas the placebo group showed no difference. Nitroglycerin-mediated dilation did not change in either group. EPC counts (44.2 ± 25.6 versus 52.3 ± 18.6; P = 0.005) and the percentage of EPCs were significantly increased in the OPE group. When FMD was divided into quartiles, rate of patients with endothelial dysfunction defined as lowest quartile (cutoff value, 8.6%) of FMD improved from 26% to 9% by OPE. Medium-term administration of OPE an improvement in FMD and circulating EPCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Osteocalcin expression by circulating endothelial progenitor cells in patients with coronary atherosclerosis.

    Science.gov (United States)

    Gössl, Mario; Mödder, Ulrike I; Atkinson, Elizabeth J; Lerman, Amir; Khosla, Sundeep

    2008-10-14

    This study was designed to test whether patients with coronary atherosclerosis have increases in circulating endothelial progenitor cells (EPCs) expressing an osteogenic phenotype. Increasing evidence indicates a link between bone and the vasculature, and bone marrow and circulating osteogenic cells have been identified by staining for the osteoblastic marker, osteocalcin (OCN). Endothelial progenitor cells contribute to vascular repair, but repair of vascular injury may result in calcification. Using cell surface markers (CD34, CD133, kinase insert domain receptor [KDR]) to identify EPCs, we examined whether patients with coronary atherosclerosis had increases in the percentage of EPCs expressing OCN. We studied 72 patients undergoing invasive coronary assessment: control patients (normal coronary arteries and no endothelial dysfunction, n = 21) versus 2 groups with coronary atherosclerosis-early coronary atherosclerosis (normal coronary arteries but with endothelial dysfunction, n = 22) and late coronary atherosclerosis (severe, multivessel coronary artery disease, n = 29). Peripheral blood mononuclear cells were analyzed using flow cytometry. Compared with control patients, patients with early or late coronary atherosclerosis had significant increases (approximately 2-fold) in the percentage of CD34+/KDR+ and CD34+/CD133+/KDR+ cells costaining for OCN. Even larger increases were noted in the early and late coronary atherosclerosis patients in the percentage of CD34+/CD133-/KDR+ cells costaining for OCN (5- and 2-fold, p < 0.001 and 0.05, respectively). A higher percentage of EPCs express OCN in patients with coronary atherosclerosis compared with subjects with normal endothelial function and no structural coronary artery disease. These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary atherosclerosis.

  9. Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.

    Directory of Open Access Journals (Sweden)

    Karin Huizer

    Full Text Available The characterization of circulating endothelial progenitor cells (EPCs is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs, circulating endothelial cells (CECs, and culture-generated outgrowth endothelial cells (OECs from blood samples of healthy adults (AB and umbilical cord (UCB. Peripheral blood mononuclear cells (PBMCs were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.

  10. Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue

    Science.gov (United States)

    Kopp, Hans-Georg; Ramos, Carlos A.; Rafii, Shahin

    2010-01-01

    Purpose of review During the last several years, a substantial amount of evidence from animal as well as human studies has advanced our knowledge of how bone marrow derived cells contribute to neoangiogenesis. In the light of recent findings, we may have to redefine our thinking of endothelial cells as well as of perivascular mural cells. Recent findings Inflammatory hematopoietic cells, such as macrophages, have been shown to promote neoangiogenesis during tumor growth and wound healing. Dendritic cells, B lymphocytes, monocytes, and other immune cells have also been found to be recruited to neoangiogenic niches and to support neovessel formation. These findings have led to the concept that subsets of hematopoietic cells comprise proangiogenic cells that drive adult revascularization processes. While evidence of the importance of endothelial progenitor cells in adult vasculogenesis increased further, the role of these comobilized hematopoietic cells has been intensely studied in the last few years. Summary Angiogenic factors promote mobilization of vascular endothelial growth factor receptor 1-positive hematopoietic cells through matrix metalloproteinase-9 mediated release of soluble kit-ligand and recruit these proangiogenic cells to areas of hypoxia, where perivascular mural cells present stromal-derived factor 1 (CXCL-12) as an important retention signal. The same factors are possibly involved in mobilization of vascular endothelial growth factor receptor 2-positive endothelial precursors that may participate in neovessel formation. The complete characterization of mechanisms, mediators and signaling pathways involved in these processes will provide novel targets for both anti and proangiogenic therapeutic strategies. PMID:16567962

  11. Macrophage-Mediated Lymphangiogenesis: The Emerging Role of Macrophages as Lymphatic Endothelial Progenitors

    International Nuclear Information System (INIS)

    Ran, Sophia; Montgomery, Kyle E.

    2012-01-01

    It is widely accepted that macrophages and other inflammatory cells support tumor progression and metastasis. During early stages of neoplastic development, tumor-infiltrating macrophages (TAMs) mount an immune response against transformed cells. Frequently, however, cancer cells escape the immune surveillance, an event that is accompanied by macrophage transition from an anti-tumor to a pro-tumorigenic type. The latter is characterized by high expression of factors that activate endothelial cells, suppress immune response, degrade extracellular matrix, and promote tumor growth. Cumulatively, these products of TAMs promote tumor expansion and growth of both blood and lymphatic vessels that facilitate metastatic spread. Breast cancers and other epithelial malignancies induce the formation of new lymphatic vessels (i.e., lymphangiogenesis) that leads to lymphatic and subsequently, to distant metastasis. Both experimental and clinical studies have shown that TAMs significantly promote tumor lymphangiogenesis through paracrine and cell autonomous modes. The paracrine effect consists of the expression of a variety of pro-lymphangiogenic factors that activate the preexisting lymphatic vessels. The evidence for cell-autonomous contribution is based on the observed tumor mobilization of macrophage-derived lymphatic endothelial cell progenitors (M-LECP) that integrate into lymphatic vessels prior to sprouting. This review will summarize the current knowledge of macrophage-dependent growth of new lymphatic vessels with specific emphasis on an emerging role of macrophages as lymphatic endothelial cell progenitors (M-LECP)

  12. Enhanced adhesion of early endothelial progenitor cells to radiation-induced senescence-like vascular endothelial cells in vitro

    International Nuclear Information System (INIS)

    Sermsathanasawadi, N.; Inoue, Yoshinori; Iwai, Takehisa; Ishii, Hideto; Yoshida, Masayuki; Igarashi, Kaori; Miura, Masahiko

    2009-01-01

    The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation. (author)

  13. The Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

    Science.gov (United States)

    Liberda, Eric N.

    Introduction. Particulate air pollution, specifically nickel found on or in particulate matter, has been associated with an increased risk of mortality in human population studies and can cause increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiate atherosclerosis in murine exposures. With the discovery of endothelial progenitor cells (EPCs), a door has been opened which may explain these observed cardiovascular effects associated with inhaled air particles and nickel exposure. In order to further quantify the effects of inhaled nickel nanoparticles and attempt to elucidate how the observed findings from other studies may occur, several whole body inhalation exposure experiments to nickel nanoparticles were performed. Methods. Following whole body exposure to approximately 500mug/m3 of nickel nanoparticles for 5 hrs, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation, and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells, circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the inhalation exposure. Plasma proteins were assessed using the 2D DIGE proteomic approach and commercially available ELISAs. Results and Conclusions. Exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation. CECs were significantly upregulated suggesting that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. This decrease in EPC function

  14. Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

    Science.gov (United States)

    Powell, Tiffany M; Paul, Jonathan D; Hill, Jonathan M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; McCoy, J Philip; Read, Elizabeth J; Khuu, Hanh M; Leitman, Susan F; Finkel, Toren; Cannon, Richard O

    2005-02-01

    Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair

  15. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

    Directory of Open Access Journals (Sweden)

    Kathryn L McCabe

    Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

  16. Myocardial regeneration by transplantation of modified endothelial progenitor cells expressing SDF-1 in a rat model

    DEFF Research Database (Denmark)

    Schuh, A.; Kroh, A.; Konschalla, S.

    2012-01-01

    Cell based therapy has been shown to attenuate myocardial dysfunction after myocardial infarction (MI) in different acute and chronic animal models. It has been further shown that stromal-cell derived factor-1a (SDF-1a) facilitates proliferation and migration of endogenous progenitor cells...... into injured tissue. The aim of the present study was to investigate the role of exogenously applied and endogenously mobilized cells in a regenerative strategy for MI therapy. Lentivirally SDF-1a-infected endothelial progenitor cells (EPCs) were injected after 90 min. of ligation and reperfusion of the left...... compared to medium control. Intracoronary application of cells did not lead to significant differences compared to medium injected control hearts. Histology showed a significantly elevated rate of apoptotic cells and augmented proliferation after transplantation of EPCs and EPCs + SDF-1 alpha in infarcted...

  17. Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co-Transplantation.

    Science.gov (United States)

    Souidi, Naima; Stolk, Meaghan; Rudeck, Juliane; Strunk, Dirk; Schallmoser, Katharina; Volk, Hans-Dieter; Seifert, Martina

    2017-05-01

    Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8 + T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245. © 2017 AlphaMed Press.

  18. Effects of benazepril on functional activity of endothelial progenitor cells from hypertension patients.

    Science.gov (United States)

    Li, Yongdong; Alatan, Gaole; Ge, Zhiping; Liu, Dan

    2014-01-01

    The effect of angiotensin-converting enzyme inhibitors on hypertension patients regarding endothelial progenitor cell (EPC) functions is poorly understood. The aim of this study was to investigate the effects of benazepril on the proliferation, adhesion and migration capacity of EPCs and its possible mechanism. The functions of EPCs from hypertension patients were obviously reduced compared with control group, and this could be improved by benazepril in a dose-dependent manner, whereas this improvement were obviously blocked when AMD3100 were used together. Therefore, benazepril could obviously improve functions of EPCs from hypertension patients, and the potential mechanism may be related to SDF-1/CXCR4 axis.

  19. Endothelial Progenitor Cell Dysfunction in Polycystic Ovary Syndrome: Implications for The Genesis of Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Yu-Hsun Kao

    2013-01-01

    Full Text Available Polycystic ovary syndrome (PCOS, the most common endocrine disorder affecting women ofreproductive age, is characterized by hyperandrogenism and insulin resistance. Women withPCOS have a higher risk for cardiovascular diseases (CVDs and endothelial dysfunction. Themechanisms underlying these risks are unclear. Human peripheral blood contains circulatingendothelial progenitor cells (EPCs derived from bone marrow that have the ability to proliferate anddifferentiate into mature endothelial cells, which may contribute to vessel homeostasis and repair.PCOS is associated with insulin resistance, hyperinsulinemia, and dyslipidemia, which may resultin EPC dysfunction. In this review, we summarize the potential mechanisms of EPC dysfunction inPCOS, which possibly result in a higher genesis of CVDs in PCOS-affected subjects.

  20. Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

    Science.gov (United States)

    Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

    2013-08-20

    Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Endothelial progenitor cell mobilization and increased intravascular nitric oxide in patients undergoing cardiac rehabilitation.

    Science.gov (United States)

    Paul, Jonathan D; Powell, Tiffany M; Thompson, Michael; Benjamin, Moshe; Rodrigo, Maria; Carlow, Andrea; Annavajjhala, Vidhya; Shiva, Sruti; Dejam, Andre; Gladwin, Mark T; McCoy, J Philip; Zalos, Gloria; Press, Beverly; Murphy, Mandy; Hill, Jonathan M; Csako, Gyorgy; Waclawiw, Myron A; Cannon, Richard O

    2007-01-01

    We investigated whether cardiac rehabilitation participation increases circulating endothelial progenitor cells (EPCs) and benefits vasculature in patients already on stable therapy previously shown to augment EPCs and improve endothelial function. Forty-six of 50 patients with coronary artery disease completed a 36-session cardiac rehabilitation program: 45 were treated with HMG-CoA reductase inhibitor (statin) therapy > or = 1 month (average baseline low-density lipoprotein cholesterol = 81 mg/dL). Mononuclear cells isolated from blood were quantified for EPCs by flow cytometry (CD133/VEGFR-2 cells) and assayed in culture for EPC colony-forming units (CFUs). In 23 patients, EPCs were stained for annexin-V as a marker of apoptosis, and nitrite was measured in blood as an indicator of intravascular nitric oxide. Endothelial progenitor cells increased from 35 +/- 5 to 63 +/- 10 cells/mL, and EPC-CFUs increased from 0.9 +/- 0.2 to 3.1 +/- 0.6 per well (both P < .01), but 11 patients had no increase in either measure. Those patients whose EPCs increased from baseline showed significant increases in nitrite and reduction in annexin-V staining (both P < .01) versus no change in patients without increase in EPCs. Over the course of the program, EPCs increased prior to increase in nitrite in the blood. Cardiac rehabilitation in patients receiving stable statin therapy and with low-density lipoprotein cholesterol at goal increases EPC number, EPC survival, and endothelial differentiation potential, associated with increased nitric oxide in the blood. Although this response was observed in most patients, a significant minority showed neither EPC mobilization nor increased nitric oxide in the blood.

  2. Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells

    DEFF Research Database (Denmark)

    Jantzen, Kim; Møller, Peter Horn; Karottki, Dorina Gabriela

    2016-01-01

    . Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate......Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked...... to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related...

  3. Endothelial and circulating progenitor cells in hematological diseases and allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Ruggeri, Annalisa; Paviglianiti, Annalisa; Volt, Fernanda; Kenzey, Chantal; Rafii, Hanadi; Rocha, Vanderson; Gluckman, Eliane

    2017-10-12

    Circulating endothelial cells (CECs), originated form endothelial progenitors (EPCs) are mature cells which are not associated with vessel walls, and that are detached from the endothelium. Normally, they are present in insignificant amounts in the peripheral blood of healthy individuals. On the other hand, elevated CECs and EPCs levels have been reported in the peripheral blood of patients with different types of cancers and some other diseases. Consequently, CECs and EPCs represent a potential biomarker in several clinical conditions involving endothelial turnover and remodeling, such as hematological diseases. These cells may be involved in disease progression and the neoplastic angiogenesis process. Moreover, CESs and EPCs are probably involved in endothelial damage that is a marker of several complications following allogeneic hematopoietic stem cell transplantation. This review aims to provide an overview on the characterization of CECs and EPCs, describe isolation methods and to identify the potential role of these cells in hematological diseases and hematopoietic stem cell transplantation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

    Science.gov (United States)

    Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

    2006-07-01

    Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

  5. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  6. Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina

    DEFF Research Database (Denmark)

    Friis, Tina; Haack-Sørensen, Mandana; Mathiasen, Anders B

    2011-01-01

    Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods and resu......Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods...... and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6-8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra......-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p angina attacks (p Angina Questionnaire (SAQ) evaluations (p

  7. Extracellular histones reduce survival and angiogenic responses of late outgrowth progenitor and mature endothelial cells.

    Science.gov (United States)

    Mena, H A; Carestia, A; Scotti, L; Parborell, F; Schattner, M; Negrotto, S

    2016-02-01

    ESSENTIALS: Extracellular histones are highly augmented in sites of neovessel formation, such as regeneration tissues. We studied histone effect on survival and angiogenic activity of mature and progenitor endothelial cells. Extracellular histones trigger apoptosis and pyroptosis and reduce angiogenesis in vivo and in vitro. Histone blockade can be useful as a therapeutic strategy to improve angiogenesis and tissue regeneration. Extracellular histones are highly augmented in sites of neovessel formation, like regeneration tissues. Their cytotoxic effect has been studied in endothelial cells, although the mechanism involved and their action on endothelial colony-forming cells (ECFCs) remain unknown. To study the effect of histones on ECFC survival and angiogenic functions and compare it with mature endothelial cells. Nuclear morphology analysis showed that each human recombinant histone triggered both apoptotic-like and necrotic-like cell deaths in both mature and progenitor endothelial cells. While H1 and H2A exerted a weak toxicity, H2B, H3 and H4 were the most powerful. The percentage of apoptosis correlated with the percentage of ECFCs exhibiting caspase-3 activation and was zeroed by the pan-caspase inhibitor Z-VAD-FMK. Necrotic-like cell death was also suppressed by this compound and the caspase-1 inhibitor Ac-YVAD-CMK, indicating that histones triggered ECFC pyroptosis. All histones, at non-cytotoxic concentrations, reduced migration and H2B, H3 and H4 induced cell cycle arrest and impaired tubulogenesis via p38 activation. Neutrophil-derived histones exerted similar effects. In vivo blood vessel formation in the quail chorioallantoic membrane was also reduced by H2B, H3 and H4. Their cytotoxic and antiangiogenic effects were suppressed by unfractioned and low-molecular-weight heparins and the combination of TLR2 and TLR4 blocking antibodies. Histones trigger both apoptosis and pyroptosis of ECFCs and inhibit their angiogenic functions. Their cytotoxic and

  8. Human endothelial progenitor cells rescue cortical neurons from oxygen-glucose deprivation induced death.

    Science.gov (United States)

    Bacigaluppi, Susanna; Donzelli, Elisabetta; De Cristofaro, Valentina; Bragazzi, Nicola Luigi; D'Amico, Giovanna; Scuteri, Arianna; Tredici, Giovanni

    2016-09-19

    Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    International Nuclear Information System (INIS)

    Gong, Xi; Zhang, Kunshan; Wang, Yanlu; Wang, Junbang; Cui, Yaru; Li, Siguang; Luo, Yuping

    2013-01-01

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome

  10. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xi [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Zhang, Kunshan [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Yanlu; Wang, Junbang; Cui, Yaru [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Li, Siguang, E-mail: siguangli@163.com [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Luo, Yuping, E-mail: luoyuping@163.com [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China)

    2013-10-04

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.

  11. Red wine consumption improves in vitro migration of endothelial progenitor cells in young, healthy individuals.

    Science.gov (United States)

    Hamed, Saher; Alshiek, Jonia; Aharon, Anat; Brenner, Benjamin; Roguin, Ariel

    2010-07-01

    Endothelial progenitor cells (EPCs) contribute to the maintenance of vascular endothelial function. The moderate consumption of red wine provides cardiovascular protection. We investigated the underlying molecular mechanism of EPC migration in young, healthy individuals who drank red wine. Fourteen healthy volunteers consumed 250 mL red wine daily for 21 consecutive days. Vascular endothelial function, plasma stromal cell-derived factor 1alpha (SDF1alpha) concentrations, and the number, migration, and nitric oxide production of EPCs were determined before and after the daily consumption of red wine. EPCs were glucose stressed to study the effect of red wine on EPC migration, proliferation, and senescence and to study the expressions of CXC chemokine receptor 4 (CXCR4) and members of the Pi3K/Akt/eNOS (phosphatidylinositol 3-kinase/protein kinase B/endothelial nitric oxide synthase) signaling pathway by Western blotting. Daily red wine consumption for 21 consecutive days significantly enhanced vascular endothelial function. Although plasma SDF1alpha concentrations were unchanged, EPC count and migration were significantly increased after this 21-d consumption period. Red wine increased the migration, proliferation, CXCR4 expression, and activity of the Pi3K/Akt/eNOS signaling pathway and decreased the extent of apoptosis in glucose-stressed EPCs. The results of the present study indicate that red wine exerts its effect through the up-regulation of CXCR4 expression and activation of the SDF1alpha/CXCR4/Pi3K/Akt/eNOS signaling pathway, which results in increased EPC migration and proliferation and decreased extent of apoptosis. Our findings suggest that these effects could be linked to the mechanism of cardiovascular protection that is associated with the regular consumption of red wine.

  12. MRI of the transplanted endothelial progenitor cells for prevent atherosclerotic plaque formation

    International Nuclear Information System (INIS)

    Ma Zhanlong; Teng Gaojun; Mai Xiaoli; Chen Jun; Sun Junhui; Zhang Hongying; Yu Hui; Li Guozhao

    2007-01-01

    Objective: To evaluate the 1.5 T magnetic resonance imaging system to depict and track in vivo of magnetically labeled endothelial progenitor cells (EPCs), and to study the possibility for preventing the atherosclerotic plaque formation in New Zealand rabbit model of carotid arterial injury after transplantation. Methods: New Zealand rabbit EPCs were isolated, confirmed, expanded and then incubated with home synthesized Fe 2 O 3 -PLL, Prussian blue stain was performed for showing intracellular irons. The model of carotid arterial injury was performed by 2.5F balloons, the group A of 8 rabbits received magnetically labeled EPCs, group B of 3 rabbits received fluorescent-labeled EPCs and the group C of 5 rabbits were given same volume saline injection after endothelial injury of the carotid artery. MR imaging and histology were performed and compared 4 days later for randomly chosen three rabbit, each from one of the three group; all the other rabbits were fed with high lipid diet and examed using MR imaging and histology after 15 weeks. Results: Epcs labeling efficiency was more than 95% by Prussian blue stain, 4 days after transplantation of EPCs, only in group A, the injured endothelium of carotid artery had signal intensity loss in T 2 * WI, which were correlated well with the area where the most Prussian blue staining positive cells were found in histopathology analyses. The rabbits of group A and B which received EPCs transplantation exhibited fewer plaques formation than those of the group C (P 2 O 3 -PLL. The 1.5 T magnetic resonance imaging system could depict and monitor the magnetically labeled endothelial progenitor cells homing to the injured endothelium of the artery, and EPCs contribute to preventing atherosclerotic plaque formation in New Zealand rabbit model of atherosclerosis. (authors)

  13. The Use of Endothelial Progenitor Cells for the Regeneration of Musculoskeletal and Neural Tissues

    Directory of Open Access Journals (Sweden)

    Naosuke Kamei

    2017-01-01

    Full Text Available Endothelial progenitor cells (EPCs derived from bone marrow and blood can differentiate into endothelial cells and promote neovascularization. In addition, EPCs are a promising cell source for the repair of various types of vascularized tissues and have been used in animal experiments and clinical trials for tissue repair. In this review, we focused on the kinetics of endogenous EPCs during tissue repair and the application of EPCs or stem cell populations containing EPCs for tissue regeneration in musculoskeletal and neural tissues including the bone, skeletal muscle, ligaments, spinal cord, and peripheral nerves. EPCs can be mobilized from bone marrow and recruited to injured tissue to contribute to neovascularization and tissue repair. In addition, EPCs or stem cell populations containing EPCs promote neovascularization and tissue repair through their differentiation to endothelial cells or tissue-specific cells, the upregulation of growth factors, and the induction and activation of endogenous stem cells. Human peripheral blood CD34(+ cells containing EPCs have been used in clinical trials of bone repair. Thus, EPCs are a promising cell source for the treatment of musculoskeletal and neural tissue injury.

  14. A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Laura Giusti

    2017-01-01

    Full Text Available Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs, bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG, on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.

  15. Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

    Science.gov (United States)

    Guo, Peipei; Poulos, Michael G; Palikuqi, Brisa; Badwe, Chaitanya R; Lis, Raphael; Kunar, Balvir; Ding, Bi-Sen; Rabbany, Sina Y; Shido, Koji; Butler, Jason M; Rafii, Shahin

    2017-12-01

    Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

  16. Haploinsufficiency of the insulin-like growth factor-1 receptor enhances endothelial repair and favorably modifies angiogenic progenitor cell phenotype.

    Science.gov (United States)

    Yuldasheva, Nadira Y; Rashid, Sheikh Tawqeer; Haywood, Natalie J; Cordell, Paul; Mughal, Romana; Viswambharan, Hema; Imrie, Helen; Sukumar, Piruthivi; Cubbon, Richard M; Aziz, Amir; Gage, Matthew; Mbonye, Kamatamu Amanda; Smith, Jessica; Galloway, Stacey; Skromna, Anna; Scott, D Julian A; Kearney, Mark T; Wheatcroft, Stephen B

    2014-09-01

    Defective endothelial regeneration predisposes to adverse arterial remodeling and is thought to contribute to cardiovascular disease in type 2 diabetes mellitus. We recently demonstrated that the type 1 insulin-like growth factor receptor (IGF1R) is a negative regulator of insulin sensitivity and nitric oxide bioavailability. In this report, we examined partial deletion of the IGF1R as a potential strategy to enhance endothelial repair. We assessed endothelial regeneration after wire injury in mice and abundance and function of angiogenic progenitor cells in mice with haploinsufficiency of the IGF1R (IGF1R(+/-)). Endothelial regeneration after arterial injury was accelerated in IGF1R(+/-) mice. Although the yield of angiogenic progenitor cells was lower in IGF1R(+/-) mice, these angiogenic progenitor cells displayed enhanced adhesion, increased secretion of insulin-like growth factor-1, and enhanced angiogenic capacity. To examine the relevance of IGF1R manipulation to cell-based therapy, we transfused IGF1R(+/-) bone marrow-derived CD117(+) cells into wild-type mice. IGF1R(+/-) cells accelerated endothelial regeneration after arterial injury compared with wild-type cells and did not alter atherosclerotic lesion formation. Haploinsufficiency of the IGF1R is associated with accelerated endothelial regeneration in vivo and enhanced tube forming and adhesive potential of angiogenic progenitor cells in vitro. Partial deletion of IGF1R in transfused bone marrow-derived CD117(+) cells enhanced their capacity to promote endothelial regeneration without altering atherosclerosis. Our data suggest that manipulation of the IGF1R could be exploited as novel therapeutic approach to enhance repair of the arterial wall after injury. © 2014 American Heart Association, Inc.

  17. Estradiol-induced, endothelial progenitor cell-mediated neovascularization in male mice with hind-limb ischemia

    NARCIS (Netherlands)

    Ruifrok, Willem-Peter T.; de Boer, Rudolf A.; Iwakura, Atsushi; Silver, Marcy; Kusano, Kengo; Tio, Rene A.; Losordo, Douglas W.

    We investigated whether administration of estradiol to male mice augments mobilization of bone marrow-derived endothelial progenitor cells (EPC) and incorporation into foci of neovascularization after hind-limb ischemia, thereby contributing to blood flow restoration. Mice were randomized and

  18. Alternative splicing events identified in human embryonic stem cells and neural progenitors.

    Directory of Open Access Journals (Sweden)

    Gene W Yeo

    2007-10-01

    Full Text Available Human embryonic stem cells (hESCs and neural progenitor (NP cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hESC to NP, very little work has been performed to understand post-transcriptional changes during neuronal differentiation. Alternative RNA splicing (AS, a major form of post-transcriptional gene regulation, is important in mammalian development and neuronal function. Human ESC, hESC-derived NP, and human central nervous system stem cells were compared using Affymetrix exon arrays. We introduced an outlier detection approach, REAP (Regression-based Exon Array Protocol, to identify 1,737 internal exons that are predicted to undergo AS in NP compared to hESC. Experimental validation of REAP-predicted AS events indicated a threshold-dependent sensitivity ranging from 56% to 69%, at a specificity of 77% to 96%. REAP predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved AS events. Our results also reveal that focusing on differentially expressed genes between hESC and NP will overlook 14% of potential AS genes. In addition, we found that REAP predictions are enriched in genes encoding serine/threonine kinase and helicase activities. An example is a REAP-predicted alternative exon in the SLK (serine/threonine kinase 2 gene that is differentially included in hESC, but skipped in NP as well as in other differentiated tissues. Lastly, comparative sequence analysis revealed conserved intronic cis-regulatory elements such as the FOX1/2 binding site GCAUG as being proximal to candidate AS exons, suggesting that FOX1/2 may participate in the regulation of AS in NP and hESC. In summary, a new methodology for exon array analysis was introduced

  19. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2016-11-15

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  20. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    International Nuclear Information System (INIS)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-01-01

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  1. Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells

    International Nuclear Information System (INIS)

    Jantzen, Kim; Møller, Peter; Karottki, Dorina Gabriela; Olsen, Yulia; Bekö, Gabriel; Clausen, Geo; Hersoug, Lars-Georg; Loft, Steffen

    2016-01-01

    Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34 + KDR + cells) or early (CD34 + CD133 + KDR + cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2′,7′-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health.

  2. Mobilization of endothelial progenitor cells after endovascular interventions in patients with type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Marina Sergeevna Michurova

    2014-12-01

    Full Text Available Aim. To investigate the mobilisation of endothelial progenitor cells (EPC in patients with type 2 diabetes mellitus (T2DM after endovascular interventions for coronary and peripheral arteries. Materials and Methods. The levels of EPC in peripheral blood were determined by flow cytometry in 42 patients prior to endovascular intervention and 2?4 days after surgery. EPC were defined as CD34+ VEGFR2+ CD45- and CD34+ CD133+CD45- cells. Twenty-three patients with T2DM were included in group 1, and 19 patients without metabolic disorders were included in group 2. Results. The levels of EPC in the peripheral blood of patients with T2DM before and after endovascular interventions were not significantly different. In the subgroup of patients without TDM2, the levels of CD34+VEGFR2 +CD45- cells increased after surgery to 55,5% (p

  3. Endothelial progenitor cells dysfunction and impaired tissue reparation: The missed link in diabetes mellitus development.

    Science.gov (United States)

    Berezin, Alexander E

    Diabetes mellitus (DM) is considered a leading cause of premature cardiovascular (CV) mortality and morbidity in general population and in individuals with known CV disease. Recent animal and clinical studies have shown that reduced number and weak function of endothelial progenitor cells (EPCs) may not only indicate to higher CV risk, but contribute to the impaired heart and vessels reparation in patients with DM. Moreover, EPCs having a protective impact on the vasculature may mediate the functioning of other organs and systems. Therefore, EPCs dysfunction is probably promising target for DM treatment strategy, while the role of restoring of EPCs number and functionality in CV risk diminish and reduce of DM-related complications is not fully clear. The aim of the review is summary of knowledge regarding EPCs dysfunction in DM patients. Copyright © 2016 Diabetes India. Published by Elsevier Ltd. All rights reserved.

  4. The association between circulating endothelial progenitor cells and coronary collateral formation.

    Science.gov (United States)

    Tokgözoğlu, Lale; Yorgun, Hikmet; Gürses, Kadri Murat; Canpolat, Uğur; Ateş, Ahmet Hakan; Tülümen, Erol; Kaya, Ergün Barış; Aytemir, Kudret; Kabakçı, Giray; Tuncer, Murat; Oto, Ali

    2011-12-01

    We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography. Circulating CD133(+)/34(+) and CD34(+)/KDR(+) EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133(+)/34(+) (p collateral formation (p collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037). In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Autophagy promotes degradation of polyethyleneimine–alginate nanoparticles in endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Wang GD

    2017-09-01

    Full Text Available Guo-dong Wang, Yu-zhen Tan, Hai-jie Wang, Pei Zhou Department of Anatomy, Histology and Embryology, Shanghai Medical School of Fudan University, Shanghai, China Abstract: Polyethyleneimine (PEI–alginate (Alg nanoparticle (NP is a safe and effective vector for delivery of siRNA or DNA. Recent studies suggest that autophagy is related to cytotoxicity of PEI NPs. However, contribution of autophagy to degradation of PEI–Alg NPs remains unknown. CD34+VEGFR-3+ endothelial progenitor cells isolated from rat bone marrow were treated with 25 kDa branched PEI modified by Alg. After treatment with the NPs, morphological changes and distribution of the NPs in the cells were examined with scanning and transmission electron microscopies. Cytotoxicity of the NPs was analyzed by reactive oxygen species (ROS production, lactate dehydrogenase leakage and induction of apoptosis. The level of autophagy was assessed with expression of Beclin-1 and LC3 and formation of autophagic structures and amphisomes. Colocalization of LC3-positive puncta and the NPs was determined by LC3–GFP tracing. Cytotoxicity of PEI NPs was reduced greatly after modification with Alg. PEI–Alg NPs were distributed in mitochondria, rough endoplasmic reticula and nuclei as well as cytoplasm. After phagocytosis of the NPs, expression of Beclin-1 mRNA and LC3 protein was upregulated, and the number of LC3-positive puncta, autophagic structures and amphisomes increased significantly. The number of lysosomes also increased obviously. There were LC3-positive puncta in nuclei, and some puncta were colocalized with the NPs. These results demonstrate that the activated autophagy promotes degradation of PEI–Alg NPs via multiple pathways. Keywords: polyethyleneimine, alginate, nanoparticles, endothelial progenitor cells, autophagy

  6. Pinocembrin ex vivo preconditioning improves the therapeutic efficacy of endothelial progenitor cells in monocrotaline-induced pulmonary hypertension in rats.

    Science.gov (United States)

    Ahmed, Lamiaa A; Rizk, Sherine M; El-Maraghy, Shohda A

    2017-08-15

    Pulmonary hypertension is still not curable and the available current therapies can only alleviate symptoms without hindering the progression of disease. The present study was directed to investigate the possible modulatory effect of pinocembrin on endothelial progenitor cells transplanted in monocrotaline-induced pulmonary hypertension in rats. Pulmonary hypertension was induced by a single subcutaneous injection of monocrotaline (60mg/kg). Endothelial progenitor cells were in vitro preconditioned with pinocembrin (25mg/L) for 30min before being i.v. injected into rats 2weeks after monocrotaline administration. Four weeks after monocrotaline administration, blood pressure, electrocardiography and right ventricular systolic pressure were recorded. Rats were sacrificed and serum was separated for determination of endothelin-1 and asymmetric dimethylarginine levels. Right ventricles and lungs were isolated for estimation of tumor necrosis factor-alpha and transforming growth factor-beta contents as well as caspase-3 activity. Moreover, protein expression of matrix metalloproteinase-9 and endothelial nitric oxide synthase in addition to myocardial connexin-43 was assessed. Finally, histological analysis of pulmonary arteries, cardiomyocyte cross-sectional area and right ventricular hypertrophy was performed and cryosections were done for estimation of cell homing. Preconditioning with pinocembrin provided a significant improvement in endothelial progenitor cells' effect towards reducing monocrotaline-induced elevation of inflammatory, fibrogenic and apoptotic markers. Furthermore, preconditioned cells induced a significant amelioration of endothelial markers and cell homing and prevented monocrotaline-induced changes in right ventricular function and histological analysis compared with native cells alone. In conclusion, pinocembrin significantly improves the therapeutic efficacy of endothelial progenitor cells in monocrotaline-induced pulmonary hypertension in rats

  7. Physiologic ischaemic training induces endothelial progenitor cell mobilization and myocardial angiogenesis via endothelial nitric oxide synthase related pathway in rabbits.

    Science.gov (United States)

    Xiao, Mingyue; Lu, Xiao; Li, Jianan; Li, Ling; Li, Yongxue

    2014-04-01

    Ischaemia-induced angiogenesis promises to improve neovascularization by delivery of angiogenic factors or endothelial progenitor cells (EPCs) to cardiac ischaemic areas. In order to avoid the risk of excessive myocardial ischaemia, therefore, we hypothesized that physiological ischaemic training (PIT) of normal skeletal muscle might contribute to myocardial angiogenesis via nitric oxide mediated mobilization of EPCs from the bone marrow in the established rabbit model of controllable myocardial ischaemia. The rabbits were grouped by sham-operation, myocardial ischaemia without PIT, PIT and PIT with pretreatment with the endothelial nitric oxide synthase (eNOS) inhibitor L-nitroarginine methyl ester (L-NAME). Controlled myocardial ischaemia was modelled by a water balloon constrictor implanted on the left ventricular branch in a rabbit. The PIT procedure included three cycles of 3 min of cuff inflation followed by 5 min of deflation on hind limbs of the rabbits for 4 weeks. At the endpoints, circulating EPCs (CD34/Flk-1) were measured by fluorescence-activated cell sorter; capillary density, by immunohistochemistry; blood flow, by a microsphere technique; endothelial nitric oxide synthase (eNOS) mRNA and protein, by real-time reverse transcriptase (RT)-PCR and Western blotting. The mRNA levels of eNOS were significantly higher in the PIT and L-NAME groups than in the sham-operation group (P < 0.05). Phospho-eNOS protein expression was higher in the PIT group than in the sham-operation and myocardial ischaemia without PIT groups (P < 0.05), and the effect was inhibited by L-NAME pretreatment (P < 0.05). Compared with sham-operation and myocardial ischaemia without PIT groups, the PIT group had the highest EPC count (P < 0.001), and the increase of capillary density (P < 0.01) and collateral blood flow (P < 0.05) in the ischaemic myocardium was consistent with the finding of EPC count. These effects were also inhibited by pretreatment with

  8. Circulating endothelial progenitor cells, Th1/Th2/Th17-related cytokines, and endothelial dysfunction in resistant hypertension.

    Science.gov (United States)

    Magen, Eli; Feldman, Arie; Cohen, Ziona; Alon, Dora Ben; Minz, Evegeny; Chernyavsky, Alexey; Linov, Lina; Mishal, Joseph; Schlezinger, Menacham; Sthoeger, Zev

    2010-02-01

    A possible link between chronic vascular inflammation and arterial hypertension is now an object of intensive studies. To compare Th1/Th2/Th17 cells-related cytokines, circulating endothelial progenitor cells (EPC), and endothelial function in subjects with resistant arterial hypertension (RAH) and controlled arterial hypertension (CAH). Blood pressure was measured by electronic sphygmomanometer. EPC were identified as CD34+/CD133+/kinase insert domain receptor (KDR)+ cells by flow cytometry. Th1/Th2/Th17 cells-related cytokines were identified using the Human Th1/Th2/Th17 Cytokines MultiAnalyte ELISArray Kit. Endothelium-dependent (FMD) vasodilatation of brachial artery was measured by Doppler ultrasound scanning. RAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, lower blood levels of EPC number (42.4 +/- 16.7 cells/mL) and EPC% (0.19 +/- 0.08%) were observed than in the CAH group (93.1 +/- 88.7 cells/mL; P = 0.017; 0.27 +/- 0.17; P = 0.036) and control group (68.5 +/- 63.6 cells/mL; P < 0.001; 0.28 +/- 0.17%; P = 0.003), respectively. Plasma transforming growth factor-beta1 levels were significantly higher in the RAH group (1767 +/- 364 pg/mL) than in the CAH group (1292 +/- 349; P < 0.001) and in control group (1203 +/- 419 pg/mL; P < 0.001). In the RAH group, statistically significant negative correlation was observed between systolic blood pressure and EPC% (r = -0.72, P < 0.01). FMD in the RAH group was significantly lower (5.5 +/- 0.8%) than in the CAH group (9.2 +/- 1.4; P < 0.001) and in healthy controls (10.1 +/- 1.1%; P < 0.001). RAH is characterized by reduced circulating EPC, substantial endothelial dysfunction, and increased plasma transforming growth factor-beta1 levels.

  9. Influence of depression and anxiety on circulating endothelial progenitor cells in patients with acute coronary syndromes.

    Science.gov (United States)

    Felice, Francesca; Di Stefano, Rossella; Pini, Stefano; Mazzotta, Gianfranco; Bovenzi, Francesco M; Bertoli, Daniele; Abelli, Marianna; Borelli, Lucia; Cardini, Alessandra; Lari, Lisa; Gesi, Camilla; Michi, Paola; Morrone, Doralisa; Gnudi, Luigi; Balbarini, Alberto

    2015-05-01

    Circulating endothelial progenitor cells (EPCs) are related to endothelial function and progression of coronary artery disease. There is evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression. We investigated the relationships between the level of circulating EPCs and depression and anxiety in patients with acute coronary syndrome (ACS). Patients with ACS admitted to three Cardiology Intensive Care Units were evaluated by the SCID-I to determine the presence of lifetime and/or current mood and anxiety disorders according to DSM-IV criteria. The EPCs were defined as CD133(+) CD34(+) KDR(+) and evaluated by flow cytometry. All patients underwent standardized cardiological and psychopathological evaluations. Parametric and nonparametric statistical tests were performed where appropriate. Out of 111 ACS patients, 57 were found to have a DSM-IV lifetime or current mood or anxiety disorder at the time of the inclusion in the study. The ACS group with mood or anxiety disorders showed a significant decrease in circulating EPC number compared with ACS patients without affective disorders. In addition, EPC levels correlated negatively with severity of depression and anxiety at index ACS episode. The current study indicates that EPCs circulate in decreased numbers in ACS patients with depression or anxiety and, therefore, contribute to explore new perspectives in the pathophysiology of the association between cardiovascular disorders and affective disorders. Copyright © 2015 John Wiley & Sons, Ltd.

  10. Atrial Natriuretic Peptide Accelerates Human Endothelial Progenitor Cell-Stimulated Cutaneous Wound Healing and Angiogenesis.

    Science.gov (United States)

    Lee, Tae Wook; Kwon, Yang Woo; Park, Gyu Tae; Do, Eun Kyoung; Yoon, Jung Won; Kim, Seung-Chul; Ko, Hyun-Chang; Kim, Moon-Bum; Kim, Jae Ho

    2018-05-26

    Atrial natriuretic peptide (ANP) is a powerful vasodilating peptide secreted by cardiac muscle cells, and endothelial progenitor cells (EPCs) have been reported to stimulate cutaneous wound healing by mediating angiogenesis. To determine whether ANP can promote the EPC-mediated repair of injured tissues, we examined the effects of ANP on the angiogenic properties of EPCs and on cutaneous wound healing. In vitro, ANP treatment enhanced the migration, proliferation, and endothelial tube-forming abilities of EPCs. Furthermore, small interfering RNA-mediated silencing of natriuretic peptide receptor-1, which is a receptor for ANP, abrogated ANP-induced migration, tube formation, and proliferation of EPCs. In a murine cutaneous wound model, administration of either ANP or EPCs had no significant effect on cutaneous wound healing or angiogenesis in vivo, whereas the co-administration of ANP and EPCs synergistically potentiated wound healing and angiogenesis. In addition, ANP promoted the survival and incorporation of transplanted EPCs into newly formed blood vessels in wounds. These results suggest ANP accelerates EPC-mediated cutaneous wound healing by promoting the angiogenic properties and survival of transplanted EPCs. This article is protected by copyright. All rights reserved. © 2018 by the Wound Healing Society.

  11. Reversing resistance to vascular-disrupting agents by blocking late mobilization of circulating endothelial progenitor cells.

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    Taylor, Melissa; Billiot, Fanny; Marty, Virginie; Rouffiac, Valérie; Cohen, Patrick; Tournay, Elodie; Opolon, Paule; Louache, Fawzia; Vassal, Gilles; Laplace-Builhé, Corinne; Vielh, Philippe; Soria, Jean-Charles; Farace, Françoise

    2012-05-01

    The prevailing concept is that immediate mobilization of bone marrow-derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow-derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic "rebounds" that may be targeted to improve VDA-based strategies. Our findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization. © 2012 AACR

  12. Butein Inhibits Angiogenesis of Human Endothelial Progenitor Cells via the Translation Dependent Signaling Pathway

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    Ching-Hu Chung

    2013-01-01

    Full Text Available Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a “catalytic” role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF- induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay in vivo. In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.

  13. Effects of angiopoietin-1 on inflammatory injury in endothelial progenitor cells and blood vessels.

    Science.gov (United States)

    Wang, Yi-Qing; Song, Jing-Jin; Han, Xiao; Liu, Yi-Ye; Wang, Xi-Huang; Li, Zhi-Ming; Tzeng, Chi-Meng

    2014-01-01

    Endothelial progenitor cells (EPCs) and angiopoietin-1 (Ang-1) play important roles in vasculogenesis and angiogenesis, respectively. Thus, targeting both aspects of cardiovascular tissue regeneration may offer promising therapeutic options for cardiovascular disorders. To this end, we constructed a lentiviral vector (pNL) with the Ang-1 gene and transfected EPCs with it (Ang-1-EPCs) to investigate vasculogenesis in both cellular and animal models. Compared to controls, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) increased significantly in both untreated EPCs and in the pNL vector group. After Ang-1 transcription, ICAM-1 and VCAM-1 decreased considerably in those treatment groups. Ang-1-modified EPCs alleviated inflammatory responses induced by tumor-necrosis factor-α (TNF-α) in vitro. Moreover, Ang-1-EPC implantation inhibited neointimal hyperplasia after balloon catheter injury in rats, dramatically diminishing the intimal-media (I/M) ratio and decreasing the neointimal area. Proliferating cell nuclear antigen expression in the Ang-1-EPC group was lower than the EPC non-treatment group as well, suggesting that Ang-1-EPC improved cell survival during inflammation and promoted endothelialization in damaged blood vessels.

  14. Folic acid supplementation normalizes the endothelial progenitor cell transcriptome of patients with type 1 diabetes: a case-control pilot study

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    Stubbs Andrew

    2009-08-01

    Full Text Available Abstract Background Endothelial progenitor cells play an important role in vascular wall repair. Patients with type 1 diabetes have reduced levels of endothelial progenitor cells of which their functional capacity is impaired. Reduced nitric oxide bioavailability and increased oxidative stress play a role in endothelial progenitor cell dysfunction in these patients. Folic acid, a B-vitamin with anti-oxidant properties, may be able to improve endothelial progenitor cell function. In this study, we investigated the gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes compared to endothelial progenitor cells from healthy subjects. Furthermore, we studied the effect of folic acid on gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes. Methods We used microarray analysis to investigate the gene expression profiles of endothelial progenitor cells from type 1 diabetes patients before (n = 11 and after a four week period of folic acid supplementation (n = 10 compared to the gene expression profiles of endothelial progenitor cells from healthy subjects (n = 11. The probability of genes being differentially expressed among the classes was computed using a random-variance t-test. A multivariate permutation test was used to identify genes that were differentially expressed among the two classes. Functional classification of differentially expressed genes was performed using the biological process ontology in the Gene Ontology database. Results Type 1 diabetes significantly modulated the expression of 1591 genes compared to healthy controls. These genes were found to be involved in processes regulating development, cell communication, cell adhesion and localization. After folic acid treatment, endothelial progenitor cell gene expression profiles from diabetic patients were similar to those from healthy controls. Genes that were normalized by folic acid played a prominent role in

  15. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    Science.gov (United States)

    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  16. Kinetics of circulating endothelial progenitor cells in patients undergoing carotid artery surgery

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    Kalender G

    2016-12-01

    Full Text Available G Kalender,1 A Kornberger,2 M Lisy,1 Andres Beiras-Fernandez,2 UA Stock2 1Deparment of General, Thoracic and Vascular Surgery, Hoechst Hospital, 2Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt am Main, Germany Aim: Endothelial progenitor cells (EPCs are primitive cells found in the bone marrow and peripheral blood (PB. In particular, the potential of EPCs to differentiate into mature endothelial cells remains of high interest for clinical applications such as bio-functionalized patches for autologous seeding after implantation. The objective of this study was to determine EPCs’ kinetics in patients undergoing carotid artery thromboendarterectomy (CTEA and patch angioplasty. Methods: Twenty CTEA patients were included (15 male, mean age 76 years. PB samples were taken at 1 day preoperatively, and at 1, 3, and 5 days postoperatively. Flow cytometric analysis was performed for CD34, CD133, KDR, and CD45. Expression of KDR, SDF-1α, and G-CSF was analyzed by means of enzyme-linked immunosorbent assay. Results: Fluorescence-activated cell sorting analysis revealed 0.031%±0.016% (% of PB mononuclear cells KDR+ cells and 0.052%±0.022% CD45-/CD34+/CD133+ cells, preoperatively. A 33% decrease of CD45–/CD34+/CD133+ cells was observed at day 1 after surgery. However, a relative number (compared to initial preoperative values of CD45-/CD34+/CD133+ cells was found on day 3 (82% and on day 5 (94% postoperatively. More profound upregulated levels of CD45–CD34+/CD133+ cells were observed for diabetic (+47% compared to nondiabetic and male (+38% compared to female patients. No significant postoperative time-dependent differences were found in numbers of KDR+ cells and the concentrations of the cytokines KDR and G-CSF. However, the SDF-1α levels decreased significantly on day 1 postoperatively but returned to preoperative levels by day 3. Conclusion: CTEA results in short-term downregulation of circulating

  17. Intratracheal transplantation of endothelial progenitor cells attenuates smoking-induced COPD in mice

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    Shi Z

    2017-03-01

    Full Text Available Zhihui Shi,1 Yan Chen,1 Jun Cao,2 Huihui Zeng,1 Yue Yang,1 Ping Chen,1 Hong Luo,1 Hong Peng,1 Shan Cai,1 Chaxiang Guan3 1Department of Internal Medicine, Division of Respiratory Disease, The Second Xiangya Hospital, Central-South University, 2Department of Internal Medicine, Division of Respiratory Disease, The People’s Hospital of Hunan Province, 3Department of Physiology, Xiangya Medical School, Central-South University, Changsha, Hunan, People’s Republic of China Background: Endothelial progenitor cells (EPCs might play a protective role in COPD. The aim of this study was to investigate whether intratracheal allogeneic transplantation of bone-marrow-derived EPCs would attenuate the development of smoking-induced COPD in mice.Methods: Isolated mononuclear cells from the bone marrow of C57BL/6J mice were cultured in endothelial cell growth medium-2 for 10 days, yielding EPCs. A murine model of COPD was established by passive 90-day exposure of cigarette smoke. On day 30, EPCs or phosphate-buffered saline alone was administered into the trachea. On day 90, EPCs or 30 µL phosphate-buffered saline alone was administered into the trachea, and on day 120, inflammatory cells, antioxidant activity, apoptosis, matrix metalloproteinase (MMP-2, and MMP-9 were measured.Results: After EPC treatment, the lung function of the mice had improved compared with the untreated mice. Mean linear intercept and destructive index were reduced in the EPCs-treated group compared with the untreated group. In addition, the EPCs-treated mice exhibited less antioxidant activity in bronchoalveolar lavage fluid compared with the untreated mice. Moreover, decreased activities of MMP-2, MMP-9, and TUNEL-positive cells in lung tissues were detected in EPCs-treated mice.Conclusion: Intratracheal transplantation of EPCs attenuated the development of pulmonary emphysema and lung function disorder probably by alleviating inflammatory infiltration, decelerating apoptosis

  18. VEGF 165 Gene Therapy for Patients with Refractory Angina: Mobilization of Endothelial Progenitor Cells

    International Nuclear Information System (INIS)

    Rodrigues, Clarissa G.; Plentz, Rodrigo D.M.; Dipp, Thiago; Salles, Felipe B.; Giusti, Imarilde I.; Sant'Anna, Roberto T.; Eibel, Bruna; Nesralla, Ivo A.; Markoski, Melissa; Beyer, Nance N.; Kalil, Renato A. K.

    2013-01-01

    Vascular endothelial growth factor (VEGF) induces mobilization of endothelial progenitor cells (EPCs) with the capacity for proliferation and differentiation into mature endothelial cells, thus contributing to the angiogenic process. We sought to assess the behavior of EPCs in patients with ischemic heart disease and refractory angina who received an intramyocardial injections of 2000 µg of VEGF 165 as the sole therapy. The study was a subanalysis of a clinical trial. Patients with advanced ischemic heart disease and refractory angina were assessed for eligibility. Inclusion criteria were as follows: signs and symptoms of angina and/or heart failure despite maximum medical treatment and a myocardial ischemic area of at least 5% as assessed by single-photon emission computed tomography (SPECT). Exclusion criteria were as follows: age > 65 years, left ventricular ejection fraction < 25%, and a diagnosis of cancer. Patients whose EPC levels were assessed were included. The intervention was 2000 µg of VEGF 165 plasmid injected into the ischemic myocardium. The frequency of CD34+/KDR+ cells was analyzed by flow cytometry before and 3, 9, and 27 days after the intervention. A total of 9 patients were included, 8 males, mean age 59.4 years, mean left ventricular ejection fraction of 59.3% and predominant class III angina. The number of EPCs on day 3 was significantly higher than that at baseline (p = 0.03); however, that on days 9 th and 27 th was comparable to that at baseline. We identified a transient mobilization of EPCs, which peaked on the 3th day after VEGF 165 gene therapy in patients with refractory angina and returned to near baseline levels on 9 th and 27 th days

  19. MicroRNA 107 partly inhibits endothelial progenitor cells differentiation via HIF-1β.

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    Shu Meng

    Full Text Available Endothelial progenitor cells (EPCs play an important role in tissue repair after ischemic heart disease. In particular, the recovery of endothelial function is reliant on the ability and rate of EPCs differentiate into mature endothelial cells. The present study evaluated the effect of microRNA 107 (miR-107 on the mechanism of EPCs differentiation. EPCs were isolated from rats' bone marrow and miR-107 expression of EPCs in hypoxic and normoxic conditions were measured by real-time qualitative PCR. CD31 was analyzed by flow cytometry and eNOS was examined by real-time qualitative PCR and western blotting and these were used as markers of EPC differentiation. In order to reveal the mechanism, we used miR107 inhibitor and lentiviral vector expressing a short hairpin RNA (shRNA that targets miR-107 and hypoxia-inducible factor-1 β (HIF-1β to alter miR107 and HIF-1β expression. MiR-107 expression were increased in EPCs under hypoxic conditions. Up-regulation of miR-107 partly suppressed the EPCs differentiation induced in hypoxia, while down-regulation of miR-107 promoted EPC differentiation. HIF-1β was the target. This study indicated that miR-107 was up-regulated in hypoxia to prevent EPCs differentiation via its target HIF-1β. The physiological mechanisms of miR-107 must be evaluated if it is to be used as a potential anti-ischemia therapeutic regime.

  20. VEGF 165 Gene Therapy for Patients with Refractory Angina: Mobilization of Endothelial Progenitor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, Clarissa G. [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Duke University Medical Center, Durham, North Carolina (United States); Plentz, Rodrigo D.M. [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil); Dipp, Thiago [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Salles, Felipe B. [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil); Giusti, Imarilde I.; Sant' Anna, Roberto T.; Eibel, Bruna; Nesralla, Ivo A.; Markoski, Melissa [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Beyer, Nance N. [Instituto de Cardiologia/Fundação Universitária de Cardiologia - Programa de Pós Graduação em Ciências da Saúde: Cardiologia, Porto Alegre, RS (Brazil); Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Kalil, Renato A. K., E-mail: kalil.pesquisa@gmail.com [Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS (Brazil)

    2013-08-15

    Vascular endothelial growth factor (VEGF) induces mobilization of endothelial progenitor cells (EPCs) with the capacity for proliferation and differentiation into mature endothelial cells, thus contributing to the angiogenic process. We sought to assess the behavior of EPCs in patients with ischemic heart disease and refractory angina who received an intramyocardial injections of 2000 µg of VEGF 165 as the sole therapy. The study was a subanalysis of a clinical trial. Patients with advanced ischemic heart disease and refractory angina were assessed for eligibility. Inclusion criteria were as follows: signs and symptoms of angina and/or heart failure despite maximum medical treatment and a myocardial ischemic area of at least 5% as assessed by single-photon emission computed tomography (SPECT). Exclusion criteria were as follows: age > 65 years, left ventricular ejection fraction < 25%, and a diagnosis of cancer. Patients whose EPC levels were assessed were included. The intervention was 2000 µg of VEGF 165 plasmid injected into the ischemic myocardium. The frequency of CD34+/KDR+ cells was analyzed by flow cytometry before and 3, 9, and 27 days after the intervention. A total of 9 patients were included, 8 males, mean age 59.4 years, mean left ventricular ejection fraction of 59.3% and predominant class III angina. The number of EPCs on day 3 was significantly higher than that at baseline (p = 0.03); however, that on days 9{sup th} and 27{sup th} was comparable to that at baseline. We identified a transient mobilization of EPCs, which peaked on the 3th day after VEGF 165 gene therapy in patients with refractory angina and returned to near baseline levels on 9{sup th} and 27{sup th}days.

  1. HOIP Deficiency Causes Embryonic Lethality by Aberrant TNFR1-Mediated Endothelial Cell Death

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    Nieves Peltzer

    2014-10-01

    Full Text Available Summary: Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC, consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1 prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF and lymphotoxin-α (LT-α, and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP’s catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death. : HOIP is the main catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC, a crucial regulator of TNF and other immune signaling pathways. Peltzer et al. find that HOIP deficiency results in embryonic lethality at midgestation due to endothelial cell death mediated by TNFR1. Aberrant formation of a TNF-mediated cell-death-inducing complex in HOIP-deficient (but not -proficient cells underlies the phenotype, with the catalytic activity of HOIP required for the control of cell death in response to TNF.

  2. The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells

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    Prasuna Paluru

    2014-03-01

    Full Text Available The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41 + CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP. Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

  3. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  4. Transplantation of endothelial progenitor cells ameliorates vascular dysfunction and portal hypertension in carbon tetrachloride-induced rat liver cirrhotic model.

    Science.gov (United States)

    Sakamoto, Masaharu; Nakamura, Toru; Torimura, Takuji; Iwamoto, Hideki; Masuda, Hiroshi; Koga, Hironori; Abe, Mitsuhiko; Hashimoto, Osamu; Ueno, Takato; Sata, Michio

    2013-01-01

    In cirrhosis, sinusoidal endothelial cell injury results in increased endothelin-1 (ET-1) and decreased nitric oxide synthase (NOS) activity, leading to portal hypertension. However, the effects of transplanted endothelial progenitor cells (EPCs) on the cirrhotic liver have not yet been clarified. We investigated whether EPC transplantation reduces portal hypertension. Cirrhotic rats were created by the administration of carbon tetrachloride (CCl(4) ) twice weekly for 10 weeks. From week 7, rat bone marrow-derived EPCs were injected via the tail vein in this model once a week for 4 weeks. Endothelial NOS (eNOS), vascular endothelial growth factor (VEGF) and caveolin expressions were examined by Western blots. Hepatic tissue ET-1 was measured by a radioimmunoassay (RIA). Portal venous pressure, mean aortic pressure, and hepatic blood flow were measured. Endothelial progenitor cell transplantation reduced liver fibrosis, α-smooth muscle actin-positive cells, caveolin expression, ET-1 concentration and portal venous pressure. EPC transplantation increased hepatic blood flow, protein levels of eNOS and VEGF. Immunohistochemical analyses of eNOS and isolectin B4 demonstrated that the livers of EPC-transplanted animals had markedly increased vascular density, suggesting reconstitution of sinusoidal blood vessels with endothelium. Transplantation of EPCs ameliorates vascular dysfunction and portal hypertension, suggesting this treatment may provide a new approach in the therapy of portal hypertension with liver cirrhosis. © 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  5. Manipulating Endothelial Progenitor Cell Homing with Sphingosine-1-Phosphate for Terapeutic Angiogenesis

    Science.gov (United States)

    Williams, Priscilla Anne

    Ischemic vascular diseases are the main cause of mortality worldwide and yet current therapies only delay disease progression and improve quality of life without addressing the fundamental problem of tissue loss. Within the field of tissue engineering, therapeutic angiogenesis provides a promising approach to alternatively provide new blood vessel formation via spatiotemporally controlled delivery of proangiogenic agents. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid that is upregulated under ischemic conditions, has recently gained great enthusiasm as a potential mediator in neovascularization strategies given its essential roles in promoting both neovessel formation and stabilization, and cellular trafficking along highly regulated endogenous gradients. Herein, the governing hypothesis guiding this dissertation is that local biomaterial-controlled delivery of S1P may be used to enhance migration and recruitment of vascular progenitor cells for enhanced therapeutic angiogenesis within ischemic tissue. The initial work in this dissertation investigated the effect of hypoxia on the angiogenic response of both mature and progenitor endothelial cells to S1P stimulation in vitro. Outgrowth endothelial cells (OECs) were isolated from human umbilical cord blood to provide a clinically relevant source of vascular progenitor cells for the studies conducted within this dissertation. S1P stimulation promoted angiogenic activity of both ECs and OECs under both ambient and hypoxic (1%) oxygen tensions. Furthermore, dual therapy with the combination of S1P and vascular endothelial growth factor (VEGF) further enhanced cellular responses. Interestingly, hypoxia substantially augmented the functional response of OECs to S1P, resulting in 25-fold and 6.5-fold increases in directed migration and sprouting, respectively. Thus, these studies highlighted the potential for S1P as a therapeutic agent for treatment of ischemic diseases. An injectable biomaterial system

  6. Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

    Science.gov (United States)

    Lange, Alexander W.; Sridharan, Anusha; Xu, Yan; Stripp, Barry R.; Perl, Anne-Karina; Whitsett, Jeffrey A.

    2015-01-01

    The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. PMID:25480985

  7. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells.

    Science.gov (United States)

    Léguillier, Teddy; Vandormael-Pournin, Sandrine; Artus, Jérôme; Houlard, Martin; Picard, Christel; Bernex, Florence; Robine, Sylvie; Cohen-Tannoudji, Michel

    2012-07-15

    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  8. Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells

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    Teddy Léguillier

    2012-05-01

    Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

  9. {sup 13}C dynamic nuclear polarization for measuring metabolic flux in endothelial progenitor cells

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    Nielsen, Nathalie; Laustsen, Christoffer; Bertelsen, Lotte Bonde, E-mail: Lotte@clin.au.dk

    2016-11-15

    Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population of progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence. The metabolic breakdown of hyperpolarized [1-{sup 13}C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period. - Highlights: • Hyperpolarized 13C MRS detects EPCs metabolic changes associated with ageing and cultivating conditions. • Increased lactate production in EPC’s correlates positively with aging.

  10. Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions

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    Agnete Kirkeby

    2012-06-01

    Full Text Available To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.

  11. Treating Diet-Induced Diabetes and Obesity with Human Embryonic Stem Cell-Derived Pancreatic Progenitor Cells and Antidiabetic Drugs

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    Jennifer E. Bruin

    2015-04-01

    Full Text Available Human embryonic stem cell (hESC-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs.

  12. Erythropoietin Attenuates Pulmonary Vascular Remodeling in Experimental Pulmonary Arterial Hypertension through Interplay between Endothelial Progenitor Cells and Heme Oxygenase

    OpenAIRE

    van Loon, Rosa Laura E; Bartelds, Beatrijs; Wagener, Frank A D T G; Affara, Nada; Mohaupt, Saffloer; Wijnberg, Hans; Pennings, Sebastiaan W C; Takens, Janny; Berger, Rolf M F

    2015-01-01

    BACKGROUND: Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease with a high mortality, characterized by typical angio-proliferative lesions. Erythropoietin (EPO) attenuates pulmonary vascular remodeling in PAH. We postulated that EPO acts through mobilization of endothelial progenitor cells (EPCs) and activation of the cytoprotective enzyme heme oxygenase-1 (HO-1). METHODS: Rats with flow-associated PAH, resembling pediatric PAH, were treated with HO-1 inducer EPO in the pre...

  13. Erythropoietin Attenuates Pulmonary Vascular Remodeling in Experimental Pulmonary Arterial Hypertension through Interplay between Endothelial Progenitor Cells and Heme Oxygenase

    OpenAIRE

    van Loon, Rosa Laura E.; Bartelds, Beatrijs; Wagener, Frank A. D. T. G.; Affara, Nada; Mohaupt, Saffloer; Wijnberg, Hans; Pennings, Sebastiaan W. C.; Takens, Janny; Berger, Rolf M. F.

    2015-01-01

    Background Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease with a high mortality, characterized by typical angio-proliferative lesions. Erythropoietin (EPO) attenuates pulmonary vascular remodeling in PAH. We postulated that EPO acts through mobilization of endothelial progenitor cells (EPCs) and activation of the cytoprotective enzyme heme oxygenase-1 (HO-1). Methods Rats with flow-associated PAH, resembling pediatric PAH, were treated with HO-1 inducer EPO i...

  14. Association of endothelial progenitor cells and peptic ulcer treatment in patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Nie, Zhihong; Xu, Limin; Li, Chuanyuan; Tian, Tao; Xie, Pingping; Chen, Xia; Li, Bojing

    2016-05-01

    The present study aimed to investigate the association between endothelial progenitor cells (EPCs) and peptic ulcers in patients with or without type 2 diabetes mellitus (T2DM), in association with the efficiency of peptic ulcer treatment. The study recruited healthy subjects and peptic ulcer patients with or without T2DM. All the ulcer patients, including those with and without T2DM, were administered omeprazole for 8 weeks. Peptic ulcer patients with T2DM were additionally treated with glipizide and novolin. Blood samples were then obtained from the three groups following ulcer treatment. CD133 + cells were isolated from the blood samples using magnetic bead selection, and cultured in complete medium 199. Morphological and quantity changes in EPCs were observed by light and fluorescence microscopy. In addition, flow cytometric analysis was used to quantify the number of vascular endothelial cells. The treatment was partially effective in 7 of the 32 peptic ulcer patients without T2DM and 12 of the 32 peptic ulcer patients with T2DM. However, this treatment was ineffective in 20 of the 32 peptic ulcer patients with T2DM. Notably, 25 peptic ulcer patients without T2DM were defined as completely recovered following treatment. In addition, the number of circulating EPCs as well as their colony forming ability was significantly reduced (Ppeptic ulcer patients with T2DM following ulcer treatment, compared with the other groups. Circulating EPC counts were significantly increased in peptic ulcer patients without T2DM, as compared with the healthy controls. With regards to colony formation, peptic ulcer patients without T2DM did not exhibit improved colony formation ability. In conclusion, the number of circulating EPCs and their colony-forming ability was significantly reduced in peptic ulcer patients with T2DM following ulcer treatment when compared with the other groups. This suggests that the poor curative effect of peptic ulcer treatment in these patients is

  15. Panax Notoginseng Saponins Promote Endothelial Progenitor Cell Mobilization and Attenuate Atherosclerotic Lesions in Apolipoprotein E Knockout Mice

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    Ya Liu

    2013-09-01

    Full Text Available Background: Endothelial progenitor cells (EPCs derived from the bone marrow (BM play a key role in the homeostasis of vascular repair by enhanced reendothelialization. Panax notoginseng saponins (PNS, a highly valued traditional Chinese medicine, has been shown to reduce morbidity and mortality from coronary artery disease. The present research was designed to explore the contribution of progenitor cells to the progression of atherosclerotic plaques and the possible modulatory role of PNS in this process. Methods: PNS (60 or 120 mg/kg via intraperitoneal injection was administered over 8 weeks in apolipoprotein E knockout mice on an atherogenic diet. The sizes and histochemical alteration of atherosclerotic lesions and numbers of EPCs in BM and peripheral blood were analyzed. The expression of chemokine stromal cell-derived factor 1α (SDF-1α and its receptor, CXCR4, was monitored as well. Results: PNS significantly reduced the lesion area and intima-to-media ratio compared to vehicle treatment. PNS also augmented endothelialization and reduced the smooth muscle cell (SMCs content of the lesions. The number of c-kit and sca-1 double-positive progenitor cells and flk-1 and sca-1 double-positive progenitor cells were significantly increased in the BM and the peripheral blood of the PNS-treated groups. PNS treatment increased the plasma levels of SDF-1α and SCF as well as the BM levels of matrix metalloproteinase-9 (MMP-9. Moreover, the mRNA levels of SDF-1α and protein levels of CXCR4 were both increased in the BM of mice treated with PNS, while SDF-1α expression decreased. Conclusion: PNS reduce the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilization. SDF-1α-CXCR4 interactions and the possible modulatory role of PNS in this process may contribute to the increased progenitor cell mobilization.

  16. Comparison of endothelial progenitor cells in Parkinson's disease patients treated with levodopa and levodopa/COMT inhibitor.

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    Phil Hyu Lee

    Full Text Available BACKGROUND: Levodopa treatment in Parkinson's disease (PD increases in serum homocysteine levels due to its metabolism via catechol O-methyltransferase. Endothelial progenitor cells (EPCs have the capacity to differentiate into mature endothelial cells and are markers for endothelial functions and cardiovascular risks. Along with traditional vascular risk factors, hyperhomocysteinemia is known to decrease the level of EPCs. In the present study, we hypothesized that that levodopa-induced hyperhomocysteinemia leads to a change in EPC levels. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled PD patients who had been prescribed either levodopa/carbidopa (PD-L group, n = 28 or levodopa/carbidopa/COMT inhibitor (PD-LC group, n = 25 for more than 1 year. The number of circulating EPCs was measured by flow cytometry using dual staining of anti-CD34 and anti-KDR antibodies. The EPCs were divided into tertiles based on their distributions and a logistic regression analysis was used to estimate independent predictors of the highest tertile of EPCs. The number of endothelial progenitor cells was significantly decreased in PD-L patients (118±99/mL compared with either PD-LC patients (269±258/mL, p = 0.007 or controls (206±204/mL, p = 0.012. The level of homocysteine was significantly increased in PD-L patients (14.9±5.3 µmol/L compared with either PD-LC patients (11.9±3.0 µmol/L, p = 0.028 or controls (11.1±2.5 µmol/L, p = 0.012. The level of homocysteine was negatively correlated with endothelial progenitor cell levels (r = -0.252, p = 0.028 and was an independent predictor of the highest tertile of endothelial progenitor cell levels (OR; 0.749 [95% CI: 0.584-0.961]. CONCLUSIONS/SIGNIFICANCE: These data indicate that a higher consumption of EPC for restoration of endothelial damage may be associated with chronic levodopa treatment in PD patients.

  17. Excess thyroid hormone inhibits embryonic neural stem/progenitor cells proliferation and maintenance through STAT3 signalling pathway.

    Science.gov (United States)

    Chen, Chunhai; Zhou, Zhou; Zhong, Min; Li, Maoquan; Yang, Xuesen; Zhang, Yanwen; Wang, Yuan; Wei, Aimin; Qu, Mingyue; Zhang, Lei; Xu, Shangcheng; Chen, Shude; Yu, Zhengping

    2011-07-01

    Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

  18. Schlafen 1 inhibits the proliferation and tube formation of endothelial progenitor cells.

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    Chun-yan Kuang

    Full Text Available Endothelial progenitor cells (EPCs are the major source of cells that restore the endothelium during reendothelialization. This study was designed to investigate whether Schlafen 1 (Slfn1 has an effect on the proliferation and tube formation of EPCs in vivo. Slfn1 was expressed in rat EPCs. The overexpression of Slfn1 suppressed the proliferation and tube formation of EPCs; conversely, the knockdown of Slfn1 by shRNA promoted the proliferation and tube formation of EPCs. Furthermore, when Slfn1 was overexpressed, the EPCs were arrested in the G1 phase of the cell cycle. In contrast, when Slfn1 was knocked down, the EPCs progressed into the S phase of the cell cycle. Additionally, the overexpression of Slfn1 decreased the expression of Cyclin D1, whereas the knockdown of Slfn1 increased the expression of Cyclin D1; these findings suggest that Cyclin D1 is downstream of Slfn1 in Slfn1-mediated EPC proliferation. Taken together, these results indicate a key role for Slfn1 in the regulation of EPC biological behavior, which may provide a new target for the use of EPCs during reendothelialization.

  19. Impact of age and gender interaction on circulating endothelial progenitor cells in healthy subjects.

    Science.gov (United States)

    Rousseau, Alexandra; Ayoubi, Fida; Deveaux, Christel; Charbit, Beny; Delmau, Catherine; Christin-Maitre, Sophie; Jaillon, Patrice; Uzan, Georges; Simon, Tabassome

    2010-02-01

    To assess the level of circulating endothelial progenitor cells (CEPC) in cycling women compared with men and menopausal women. Controlled clinical study. Healthy, nonsmoking volunteers. Twelve women, aged 18-40 years, with regular menstrual cycles, 12 menopausal women, and two groups of 12 age-matched men were recruited. Women did not receive any hormone therapy. Collection of 20 mL of peripheral blood. The number of CEPC, defined as (Lin-/7AAD-/CD34+/CD133+/KDR+) cells per 10(6) mononuclear cells (MNC), was measured by flow cytometry. The number of CEPC was significantly higher in cycling women than in age-matched men and menopausal women (26.5 per 10(6) MNC vs. 10.5 per 10(6) MNC vs. 10 per 10(6) MNC, respectively). The number of CEPC was similar in menopausal women, age-matched, and young men. The number of CEPC is influenced by an age-gender interaction. This phenomenon may explain in part the better vascular repair and relative cardiovascular protection in younger women as compared with age-matched men. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Obesity suppresses circulating level and function of endothelial progenitor cells and heart function

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    Tsai Tzu-Hsien

    2012-07-01

    Full Text Available Abstract Background and aim This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs and left ventricular ejection fraction (LVEF. Methods High fat diet (45 Kcal% fat was given to 8-week-old C57BL/6 J mice (n = 8 for 20 weeks to induce obesity (group 1. Another age-matched group (n = 8 were fed with control diet for 20 weeks as controls (group 2. The animals were sacrificed at the end of 20 weeks after obesity induction. Results By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p Conclusions Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

  1. Hydrogel-embedded endothelial progenitor cells evade LPS and mitigate endotoxemia.

    Science.gov (United States)

    Ghaly, Tammer; Rabadi, May M; Weber, Mia; Rabadi, Seham M; Bank, Michael; Grom, John M; Fallon, John T; Goligorsky, Michael S; Ratliff, Brian B

    2011-10-01

    Sepsis and its complications are associated with poor clinical outcomes. The circulatory system is a well-known target of lipopolysaccharide (LPS). Recently, several clinical studies documented mobilization of endothelial progenitor cells (EPCs) during endotoxemia, with the probability of patients' survival correlating with the rise in circulating EPCs. This fact combined with endotoxemia-induced vascular injury led us to hypothesize that the developing functional EPC incompetence could impede vascular repair and that adoptive transfer of EPCs could improve hemodynamics in endotoxemia. We used LPS injection to model endotoxemia. EPCs isolated from endotoxemic mice exhibited impaired clonogenic potential and LPS exerted Toll-like receptor 4-mediated cytotoxic effects toward EPCs, which was mitigated by embedding them in hyaluronic acid (HA) hydrogels. Therefore, intact EPCs were either delivered intravenously or embedded within pronectin-coated HA hydrogels. Adoptive transfer of EPCs in LPS-injected mice improved control of blood pressure and reduced hepatocellular and renal dysfunction. Specifically, EPC treatment was associated with the restoration of renal microcirculation and improved renal function. EPC therapy was most efficient when cells were delivered embedded in HA hydrogel. These findings establish major therapeutic benefits of adoptive transfer of EPCs, especially when embedded in HA hydrogels, in mice with LPS-induced endotoxemia, and they argue that hemodynamic and renal abnormalities of endotoxemia are in significant part due to developing incompetence of endogenous EPCs.

  2. Amlodipine Ameliorates Ischemia-Induced Neovascularization in Diabetic Rats through Endothelial Progenitor Cell Mobilization

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    Jiayin Sun

    2016-01-01

    Full Text Available Objectives. We investigated whether amlodipine could improve angiogenic responses in a diabetic rat model of acute myocardial infarction (AMI through improving bone marrow endothelial progenitor cell (EPC mobilization, in the same way as angiotensin converting enzyme inhibitors. Methods. After induction of AMI by coronary artery ligation, diabetic rats were randomly assigned to receive perindopril (2 mgkg−1 day−1, amlodipine (2.5 mgkg−1 day−1, or vehicle by gavage (n=20 per group. Circulating EPC counts before ligation and on days 1, 3, 5, 7, 14, and 28 after AMI were measured in each group. Microvessel density, cardiac function, and cardiac remodeling were assessed 4 weeks after treatment. The signaling pathway related to EPC mobilization was also measured. Results. Circulating EPC count in amlodipine- and perindopril-treated rats peaked at day 7, to an obvious higher level than the control group peak which was reached earlier (at day 5. Rats treated with amlodipine showed improved postischemia neovascularization and cardiac function, together with reduced cardiac remodeling, decreased interstitial fibrosis, and cardiomyocyte apoptosis. Amlodipine treatment also increased cardiac SDF-1/CXCR4 expression and gave rise to activation of VEGF/Akt/eNOS signaling in bone marrow. Conclusions. Amlodipine promotes neovascularization by improving EPC mobilization from bone marrow in diabetic rats after AMI, and activation of VEGF/Akt/eNOS signaling may in part contribute to this.

  3. Depletion of NAD pool contributes to impairment of endothelial progenitor cell mobilization in diabetes.

    Science.gov (United States)

    Wang, Pei; Yang, Xi; Zhang, Zheng; Song, Jie; Guan, Yun-Feng; Zou, Da-Jin; Miao, Chao-Yu

    2016-06-01

    The impaired mobilization of endothelial progenitor cells (EPCs) from bone marrow (BM) critically contributes to the diabetes-associated vascular complications. Here, we investigated the relationship between the nicotinamide phosphoribosyltransferase (NAMPT)-controlled nicotinamide adenine dinucleotide (NAD) metabolism and the impaired mobilization of BM-derived EPCs in diabetic condition. The NAMPT-NAD pool in BM and BM-derived EPCs in wild-type (WT) and diabetic db/db mice was determined. Nicotinamide, a natural substrate for NAD biosynthesis, was administrated for 2weeks in db/db mice to examine the influence of enhancing NAD pool on BM and blood EPCs number. The modulations of stromal cell-derived factor-1α (SDF-1α) and endothelial nitric oxide synthase (eNOS) protein in BM were measured using immunoblotting. The EPCs intracellular NAMPT level and NAD concentration, as well as the blood EPCs number, were compared between 9 healthy people and 16 patients with type 2 diabetes mellitus (T2DM). The T2DM patients were treated with nicotinamide for two weeks and then the blood EPCs number was determined. Moreover, the association between blood EPCs numbers and EPCs intracellular NAD(+)/NAMPT protein levels in 21 healthy individuals was determined. We found that NAD concentration and NAMPT expression in BM and BM-derived EPCs of db/db mice were significantly lower than those in WT mice BM. Enhancing NAD pool not only increased the EPCs intracellular NAD concentration and blood EPCs number, but also improved post-ischemic wound healing and blood reperfusion in db/db mice with hind-limb ischemia model. Enhancing NAD pool rescued the impaired modulations of stromal cell-derived factor-1α (SDF-1α) and endothelial nitric oxide synthase (eNOS) protein levels in db/db mice BM upon hind-limb ischemia. In addition, enhancing NAD pool significantly inhibited PARP and caspase-3 activates in db/db mice BM. The intracellular NAMPT-NAD pool was positively associated with blood

  4. Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.

    Science.gov (United States)

    Bruin, Jennifer E; Rezania, Alireza; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

    2013-09-01

    Islet transplantation is a promising cell therapy for patients with diabetes, but it is currently limited by the reliance upon cadaveric donor tissue. We previously demonstrated that human embryonic stem cell (hESC)-derived pancreatic progenitor cells matured under the kidney capsule in a mouse model of diabetes into glucose-responsive insulin-secreting cells capable of reversing diabetes. However, the formation of cells resembling bone and cartilage was a major limitation of that study. Therefore, we developed an improved differentiation protocol that aimed to prevent the formation of off-target mesoderm tissue following transplantation. We also examined how variation within the complex host environment influenced the development of pancreatic progenitors in vivo. The hESCs were differentiated for 14 days into pancreatic progenitor cells and transplanted either under the kidney capsule or within Theracyte (TheraCyte, Laguna Hills, CA, USA) devices into diabetic mice. Our revised differentiation protocol successfully eliminated the formation of non-endodermal cell populations in 99% of transplanted mice and generated grafts containing >80% endocrine cells. Progenitor cells developed efficiently into pancreatic endocrine tissue within macroencapsulation devices, despite lacking direct contact with the host environment, and reversed diabetes within 3 months. The preparation of cell aggregates pre-transplant was critical for the formation of insulin-producing cells in vivo and endocrine cell development was accelerated within a diabetic host environment compared with healthy mice. Neither insulin nor exendin-4 therapy post-transplant affected the maturation of macroencapsulated cells. Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

  5. Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration.

    Science.gov (United States)

    Zordan, P; Rigamonti, E; Freudenberg, K; Conti, V; Azzoni, E; Rovere-Querini, P; Brunelli, S

    2014-01-30

    The damage of the skeletal muscle prompts a complex and coordinated response that involves the interactions of many different cell populations and promotes inflammation, vascular remodeling and finally muscle regeneration. Muscle disorders exist in which the irreversible loss of tissue integrity and function is linked to defective neo-angiogenesis with persistence of tissue necrosis and inflammation. Here we show that macrophages (MPs) are necessary for efficient vascular remodeling in the injured muscle. In particular, MPs sustain the differentiation of endothelial-derived progenitors to contribute to neo-capillary formation, by secreting pro-angiogenic growth factors. When phagocyte infiltration is compromised endothelial-derived progenitors undergo a significant endothelial to mesenchymal transition (EndoMT), possibly triggered by the activation of transforming growth factor-β/bone morphogenetic protein signaling, collagen accumulates and the muscle is replaced by fibrotic tissue. Our findings provide new insights in EndoMT in the adult skeletal muscle, and suggest that endothelial cells in the skeletal muscle may represent a new target for therapeutic intervention in fibrotic diseases.

  6. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

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    Marjorie Pick

    Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

  7. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  8. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.

    Science.gov (United States)

    Keirstead, Hans S; Nistor, Gabriel; Bernal, Giovanna; Totoiu, Minodora; Cloutier, Frank; Sharp, Kelly; Steward, Oswald

    2005-05-11

    Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.

  9. Quantification of circulating endothelial progenitor cells using the modified ISHAGE protocol.

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    Caroline Schmidt-Lucke

    2010-11-01

    Full Text Available Circulating endothelial progenitor cells (EPC, involved in endothelial regeneration, neovascularisation, and determination of prognosis in cardiovascular disease can be characterised with functional assays or using immunofluorescence and flow cytometry. Combinations of markers, including CD34+KDR+ or CD133+KDR+, are used. This approach, however may not consider all characteristics of EPC. The lack of a standardised protocol with regards to reagents and gating strategies may account for the widespread inter-laboratory variations in quantification of EPC. We, therefore developed a novel protocol adapted from the standardised so-called ISHAGE protocol for enumeration of haematopoietic stem cells to enable comparison of clinical and laboratory data.In 25 control subjects, 65 patients with coronary artery disease (CAD; 40 stable CAD, 25 acute coronary syndrome/acute myocardial infarction (ACS, EPC were quantified using the following approach: Whole blood was incubated with CD45, KDR, and CD34. The ISHAGE sequential strategy was used, and finally, CD45(dimCD34(+ cells were quantified for KDR. A minimum of 100 CD34(+ events were collected. For comparison, CD45(+CD34(+ and CD45(-CD34(+ were analysed simultaneously. The number of CD45(dimCD34(+KDR(+ cells only were significantly higher in healthy controls compared to patients with CAD or ACS (p = 0.005 each, p<0.001 for trend. An inverse correlation of CD45(dimCD34(+KDR(+ with disease activity (r = -0.475, p<0.001 was confirmed. Only CD45(dimCD34(+KDR(+ correlated inversely with the number of diseased coronaries (r = -0.344; p<0.005. In a second study, a 4-week de-novo treatment of atorvastatin in stable CAD evoked an increase only of CD45(dimCD34(+KDR(+ EPC (p<0.05. CD45(+CD34(+KDR(+ and CD45(-CD34(+KDR(+ were indifferent between the three groups.Our newly established protocol adopted from the standardised ISHAGE protocol achieved higher accuracy in EPC enumeration confirming previous

  10. Transcriptional profiling of MEF2-regulated genes in human neural progenitor cells derived from embryonic stem cells

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    Shing Fai Chan

    2015-03-01

    Full Text Available The myocyte enhancer factor 2 (MEF2 family of transcription factors is highly expressed in the brain and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA, which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184. By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were up-regulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

  11. Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape.

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    Kirk, Kaitlyn; Hao, Ergeng; Lahmy, Reyhaneh; Itkin-Ansari, Pamela

    2014-05-01

    There are several challenges to successful implementation of a cell therapy for insulin dependent diabetes derived from human embryonic stem cells (hESC). Among these are development of functional insulin producing cells, a clinical delivery method that eliminates the need for chronic immunosuppression, and assurance that hESC derived tumors do not form in the patient. We and others have shown that encapsulation of cells in a bilaminar device (TheraCyte) provides immunoprotection in rodents and primates. Here we monitored human insulin secretion and employed bioluminescent imaging (BLI) to evaluate the maturation, growth, and containment of encapsulated islet progenitors derived from CyT49 hESC, transplanted into mice. Human insulin was detectable by 7 weeks post-transplant and increased 17-fold over the course of 8 weeks, yet during this period the biomass of encapsulated cells remained constant. Remarkably, by 20 weeks post-transplant encapsulated cells secreted sufficient levels of human insulin to ameliorate alloxan induced diabetes. Further, bioluminescent imaging revealed for the first time that hESCs remained fully contained in encapsulation devices for up to 150 days, the longest period tested. Collectively, the data suggest that encapsulated hESC derived islet progenitors hold great promise as an effective and safe cell replacement therapy for insulin dependent diabetes. Copyright © 2014. Published by Elsevier B.V.

  12. Efficient MRI labeling of endothelial progenitor cells: design of thiolated surface stabilized superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Shahnaz, Gul; Kremser, Christian; Reinisch, Andreas; Vetter, Anja; Laffleur, Flavia; Rahmat, Deni; Iqbal, Javed; Dünnhaupt, Sarah; Salvenmoser, Willi; Tessadri, Richard; Griesser, Ulrich; Bernkop-Schnürch, Andreas

    2013-11-01

    The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan-thioglycolic acid (chitosan-TGA) hydrogel and iron ions (Fe(2+)/Fe(3+)). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r1 and r2), and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1 ± 0.02 nm, a hydrodynamic diameter of 94 ± 20 nm, a zeta potential of +21 ± 5 mV, and an iron content of 3.6 ± 0.9 mg/mL. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than threefold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With twofold lower incubation iron concentration of TSS-SPIONs, more than threefold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24h. The molar MR relaxivities (r2) value at 1.5 T was threefold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity, and high MRI sensitivity seem to be promising for cell tracking. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Experimental study of an endothelial progenitor cell coated stent in transjugular intrahepatic portosystemic shunt

    International Nuclear Information System (INIS)

    Shi Hongjian; Teng Gaojun; Cao Aihong; Chen Jun; Deng Gang

    2009-01-01

    Objective: To evaluate the efficacy of a self-expandable metal stent coated with autologous endothelial progenitor cells (EPCs) for prevention of restenosis in transjugular intrahepatic portosystemic shunt (TIPS) in a swine model. Methods: EPCs were coated on the metal stents using fibrin gel before TIPS procedure. TIPS was performed in 15 young adult pigs, using an autologous EPC-seeded stent (treatment group, n=9) or a conventional bare metal stent (control group, n=6). All pigs were sacrificed at 2 weeks after TIPS procedure. Portography was performed immediately before the euthanasia. Gross and microscopic pathological exams and immunohistochemical exams of the TIPS track specimens were performed. Fisher test and t test were used to analyse the data. Results: TIPS was performed successfully in all the 15 swine. On day 14 of follow-up, direct portography and necropsy demonstrated that 5 shunts remained patent, 2 shunts stenosed, and the remaining 2 shunts occluded in the treatment group (n=9); while 5 shunts were occluded and one shunt was stenotic in the control group (n=6). The patency rate was 56% vs 0 (P=0.03) between the two groups. Histological analyses showed a greater pseudo-intimal hyperplasia in the TIPS track of the control group than that of the treatment group (pseudointimal thickness at hepatic vein, hepatic parenchyma and portal vein site was (1.2±0.4), (1.3±0.5), (1.5±0.4) mm vs (1.0±0.6), (0.9±0.5), (1.0±0.4) mm respectively (P<0.05). Conclusion: The EPC-coated metal stent is feasibly constructed in vitro and improves the patency in TIPS in a porcine model. (authors)

  14. Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus

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    Brenner Benjamin

    2009-10-01

    Full Text Available Abstract Background The function of endothelial progenitor cells (EPCs, which are key cells in vascular repair, is impaired in diabetes mellitus. Nitric oxide (NO and reactive oxygen species can regulate EPC functions. EPCs tolerate oxidative stress by upregulating superoxide dismutase (SOD, the enzyme that neutralizes superoxide anion (O2-. Therefore, we investigated the roles of NO and SOD in glucose-stressed EPCs. Methods The functions of circulating EPCs from patients with type 2 diabetes were compared to those from healthy individuals. Healthy EPCs were glucose-stressed, and then treated with insulin and/or SOD. We assessed O2- generation, NO production, SOD activity, and their ability to form colonies. Results EPCs from diabetic patients generated more O2-, had higher NAD(PH oxidase and SOD activity, but lower NO bioavailability, and expressed higher mRNA and protein levels of p22-phox, and manganese SOD and copper/zinc SOD than those from the healthy individuals. Plasma glucose and HbA1c levels in the diabetic patients were correlated negatively with the NO production from their EPCs. SOD treatment of glucose-stressed EPCs attenuated O2- generation, restored NO production, and partially restored their ability to form colonies. Insulin treatment of glucose-stressed EPCs increased NO production, but did not change O2- generation and their ability to form colonies. However, their ability to produce NO and to form colonies was fully restored after combined SOD and insulin treatment. Conclusion Our data provide evidence that SOD may play an essential role in EPCs, and emphasize the important role of antioxidant therapy in type 2 diabetic patients.

  15. Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus.

    Science.gov (United States)

    Hamed, Saher; Brenner, Benjamin; Aharon, Anat; Daoud, Deeb; Roguin, Ariel

    2009-10-30

    The function of endothelial progenitor cells (EPCs), which are key cells in vascular repair, is impaired in diabetes mellitus. Nitric oxide (NO) and reactive oxygen species can regulate EPC functions. EPCs tolerate oxidative stress by upregulating superoxide dismutase (SOD), the enzyme that neutralizes superoxide anion (O2-). Therefore, we investigated the roles of NO and SOD in glucose-stressed EPCs. The functions of circulating EPCs from patients with type 2 diabetes were compared to those from healthy individuals. Healthy EPCs were glucose-stressed, and then treated with insulin and/or SOD. We assessed O2- generation, NO production, SOD activity, and their ability to form colonies. EPCs from diabetic patients generated more O2-, had higher NAD(P)H oxidase and SOD activity, but lower NO bioavailability, and expressed higher mRNA and protein levels of p22-phox, and manganese SOD and copper/zinc SOD than those from the healthy individuals. Plasma glucose and HbA1c levels in the diabetic patients were correlated negatively with the NO production from their EPCs. SOD treatment of glucose-stressed EPCs attenuated O2- generation, restored NO production, and partially restored their ability to form colonies. Insulin treatment of glucose-stressed EPCs increased NO production, but did not change O2- generation and their ability to form colonies. However, their ability to produce NO and to form colonies was fully restored after combined SOD and insulin treatment. Our data provide evidence that SOD may play an essential role in EPCs, and emphasize the important role of antioxidant therapy in type 2 diabetic patients.

  16. Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers.

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    Francesco Tecilazich

    Full Text Available To evaluate changes in endothelial progenitor cells (EPCs and cytokines in patients with diabetic foot ulceration (DFU in association with wound healing.We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.All EPC phenotypes except the kinase insert domain receptor (KDR(+CD133(+ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1 and stem cell factor (SCF were increased in DFU patients. DFU patients who healed their ulcers had lower CD34(+KDR(+ count at visits 3 and 4, serum c-reactive protein (CRP and granulocyte-macrophage colony-stimulating factor (GM-CSF at visit 1, interleukin-1 (IL-1 at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34(+KDR(+ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.

  17. Endothelial Progenitor Cell Mobilization in Preterm Infants With Sepsis Is Associated With Improved Survival.

    Science.gov (United States)

    Siavashi, Vahid; Asadian, Simin; Taheri-Asl, Masoud; Keshavarz, Samaneh; Zamani-Ahmadmahmudi, Mohamad; Nassiri, Seyed Mahdi

    2017-10-01

    Microvascular dysfunction plays a key role in the pathology of sepsis, leading to multi-organ failure, and death. Circulating endothelial progenitor cells (cEPCs) are critically involved in the maintenance of the vascular homeostasis in both physiological and pathological contexts. In this study, concentration of cEPCs in preterm infants with sepsis was determined to recognize whether the EPC mobilization would affect the clinical outcome of infantile sepsis. One hundred and thirty-three preterm infants (81 with sepsis and 52 without sepsis) were enrolled in this study. The release of EPCs in circulation was first quantified. Thereafter, these cells were cultivated and biological features of these cells such as, proliferation and colony forming efficiency were analyzed. The levels of chemoattractant cytokines were also measured in infants. In mouse models of sepsis, effects of VEGF and SDF-1 as well as anti-VEGF and anti-SDF-1 were evaluated in order to shed light upon the role which the EPC mobilization plays in the overall survival of septic animals. Circulating EPCs were significantly higher in preterm infants with sepsis than in the non-sepsis group. Serum levels of VEGF, SDF-1, and Angiopoietin-2 were also higher in preterm infants with sepsis than in control non-sepsis. In the animal experiments, injection of VEGF and SDF-1 prompted the mobilization of EPCs, leading to an improvement in survival whereas injection of anti-VEGF and anti-SDF-1 was associated with significant deterioration of survival. Overall, our results demonstrated the beneficial effects of EPC release in preterm infants with sepsis, with increased mobilization of these cells was associated with improved survival. J. Cell. Biochem. 118: 3299-3307, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

    Science.gov (United States)

    James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

    2010-01-01

    Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

  19. CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

    Science.gov (United States)

    Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

    2010-01-01

    Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

  20. The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

    Science.gov (United States)

    Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

    2012-01-01

    Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

  1. Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model.

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    Rie Kawabe-Yako

    Full Text Available BACKGROUND: Cilostazol(CLZ has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM-derived endothelial progenitor cell (EPC contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs. METHODOLOGY/PRINCIPAL FINDINGS: Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2 weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury. CONCLUSIONS/SIGNIFICANCE: CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for

  2. Influence on bionomics of endothelial progenitor cells labeling with magnetic nanoparticles

    International Nuclear Information System (INIS)

    Mai Xiaoli; Teng Gaojun; Ma Zhanlong; Sun Junhui; Zhang Yu; Gu Ning

    2008-01-01

    Objective: To explore the influence of home synthesize magnetic iron oxide (called Fe 2 O 3 -PLL) labeling on peripheral blood endothelial progenitor cells (EPCs) bionomics to provide experimental foundation for MR imaging ex and in vivo. Methods: Fe 2 O 3 was incubated with PLL for 2 hours to obtain a complex of Fe 2 O 3 -PLL. Rabbit peripheral blood mononuclear cells were isolated and EPCs were selected by adherence method. Fe 2 O 3 -PLL was used to label EPCs. Prussian blue stain and electron microscope was used for showing intracellular iron. MTF assay was assessed to evaluate the difference of growth curve between unlabeled and labeled with 25 mg/L Fe 2 O 3 -PLL. Flow cytometry was performed to analyze cell cycle, cell apoptosis and the expression of surface markers of labeled and unlabeled cells. Expressions of eNOS, KDR and vWF at mRNA levels among unlabeled and labeled EPCs were detected by real-time polymerase chain reaction. Calcium ion channel and membrane fluidity were observed and analyzed by laser confocal microscopy. Statistical analyses were used with ANOVA and t test. Results: Almost 100% cells were labeled by Fe 2 O 3 -PLL, iron-containing vesicles were intracytoplasma. There was no statistical difference in cells growth curve, cell life cycle [(93.74±3.52)%, (94.57±3.66)%] and cell apoptosis rate (12.89±1.81)%, (11.67±1.18)%) between labeling with Fe 2 O 3 -PLL at a concentration of 25 mg/L and unlabeled cells (t=0.283, P>0.05; t=0.977, P>0.05). There was also no statistical difference in relative amount of eNOS, KDR and vWF at mRNA levels and the expression of surface phenotypic markers (CD34, CD106, CD146 and KDR) between two groups (P>0.05). In addition, Labeling had little influence on calcium ion channel and didn't significantly alter cell membrane fluidity. Conclusions: The rabbit peripheral blood EPCs can be effective labeled with Fe 2 O 3 -PLL and without significant influence on cells bionomics at a low concentration of 25 mg

  3. Zoledronate inhibits ischemia-induced neovascularization by impairing the mobilization and function of endothelial progenitor cells.

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    Shih-Hung Tsai

    Full Text Available BACKGROUND: Bisphosphonates are a class of pharmacologic compounds that are commonly used to treat postmenopausal osteoporosis and malignant osteolytic processes. Studies have shown that bone marrow-derived endothelial progenitor cells (EPCs play a significant role in postnatal neovascularization. Whether the nitrogen-containing bisphosphonate zoledronate inhibits ischemia-induced neovascularization by modulating EPC functions remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Unilateral hindlimb ischemia was surgically induced in wild-type mice after 2 weeks of treatment with vehicle or zoledronate (low-dose: 30 μg/kg; high-dose: 100 μg/kg. Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio was significantly lower in wild-type mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in controls 4 weeks after ischemic surgery (control vs. low-dose vs. high-dose: 87±7% vs. *61±18% vs. **49±17%, *p<0.01, **p<0.005 compared to control. Capillary densities were also significantly lower in mice treated with low-dose zoledronate and in mice treated with high-dose zoledronate than in control mice. Flow cytometry analysis showed impaired mobilization of EPC-like cells (Sca-1(+/Flk-1(+ after surgical induction of ischemia in mice treated with zoledronate but normal levels of mobilization in mice treated with vehicle. In addition, ischemic tissue from mice that received zoledronate treatment exhibited significantly lower levels of the active form of MMP-9, lower levels of VEGF, and lower levels of phosphorylated eNOS and phosphorylated Akt than ischemic tissue from mice that received vehicle. Results of the in vitro studies showed that incubation with zoledronate inhibited the viability, migration, and tube-forming capacities of EPC. CONCLUSIONS/SIGNIFICANCE: Zoledronate inhibited ischemia-induced neovascularization by impairing EPC mobilization and angiogenic functions

  4. [Effect of simvastatin on inducing endothelial progenitor cells homing and promoting bone defect repair].

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    Song, Quansheng; Wang, Lingying; Zhu, Jinglin; Han, Xiaoguang; Li, Xu; Yang, Yanlin; Sun, Yan; Song, Chunli

    2010-09-01

    To investigate the effect of simvastatin on inducing endothelial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation. Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 x 10(6) cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins (2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site. All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% +/- 4.07% in experimental group and 59.45% +/- 5.43% in control group, showing significant difference (P < 0.05). Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs

  5. The effects of smoking on levels of endothelial progenitor cells and microparticles in the blood of healthy volunteers.

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    Fariborz Mobarrez

    Full Text Available BACKGROUND: Cigarette smoking, both active and passive, is one of the leading causes of morbidity and mortality in cardiovascular disease. To assess the impact of brief smoking on the vasculature, we determined levels of circulating endothelial progenitor cells (EPCs and circulating microparticles (MPs following the smoking of one cigarette by young, healthy intermittent smokers. MATERIALS AND METHODS: 12 healthy volunteers were randomized to either smoking or not smoking in a crossover fashion. Blood sampling was performed at baseline, 1, 4 and 24 hours following smoking/not smoking. The numbers of EPCs and MPs were determined by flow cytometry. MPs were measured from platelets, leukocytes and endothelial cells. Moreover, MPs were also labelled with anti-HMGB1 and SYTO 13 to assess the content of nuclear molecules. RESULTS: Active smoking of one cigarette caused an immediate and significant increase in the numbers of circulating EPCs and MPs of platelet-, endothelial- and leukocyte origin. Levels of MPs containing nuclear molecules were increased, of which the majority were positive for CD41 and CD45 (platelet- and leukocyte origin. CD144 (VE-cadherin or HMGB1 release did not significantly change during active smoking. CONCLUSION: Brief active smoking of one cigarette generated an acute release of EPC and MPs, of which the latter contained nuclear matter. Together, these results demonstrate acute effects of cigarette smoke on endothelial, platelet and leukocyte function as well as injury to the vascular wall.

  6. DGCR8 Promotes Neural Progenitor Expansion and Represses Neurogenesis in the Mouse Embryonic Neocortex

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    Nadin Hoffmann

    2018-04-01

    Full Text Available DGCR8 and DROSHA are the minimal functional core of the Microprocessor complex essential for biogenesis of canonical microRNAs and for the processing of other RNAs. Conditional deletion of Dgcr8 and Drosha in the murine telencephalon indicated that these proteins exert crucial functions in corticogenesis. The identification of mechanisms of DGCR8- or DROSHA-dependent regulation of gene expression in conditional knockout mice are often complicated by massive apoptosis. Here, to investigate DGCR8 functions on amplification/differentiation of neural progenitors cells (NPCs in corticogenesis, we overexpress Dgcr8 in the mouse telencephalon, by in utero electroporation (IUEp. We find that DGCR8 promotes the expansion of NPC pools and represses neurogenesis, in absence of apoptosis, thus overcoming the usual limitations of Dgcr8 knockout-based approach. Interestingly, DGCR8 selectively promotes basal progenitor amplification at later developmental stages, entailing intriguing implications for neocortical expansion in evolution. Finally, despite a 3- to 5-fold increase of DGCR8 level in the mouse telencephalon, the composition, target preference and function of the DROSHA-dependent Microprocessor complex remain unaltered. Thus, we propose that DGCR8-dependent modulation of gene expression in corticogenesis is more complex than previously known, and possibly DROSHA-independent.

  7. Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

    Science.gov (United States)

    Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

    2018-05-01

    Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

  8. High-intensity Interval training enhances mobilization/functionality of endothelial progenitor cells and depressed shedding of vascular endothelial cells undergoing hypoxia.

    Science.gov (United States)

    Tsai, Hsing-Hua; Lin, Chin-Pu; Lin, Yi-Hui; Hsu, Chih-Chin; Wang, Jong-Shyan

    2016-12-01

    Exercise training improves endothelium-dependent vasodilation, whereas hypoxic stress causes vascular endothelial dysfunction. Monocyte-derived endothelial progenitor cells (Mon-EPCs) contribute to vascular repair process by differentiating into endothelial cells. This study investigates how high-intensity interval (HIT) and moderate-intensity continuous (MCT) exercise training affect circulating Mon-EPC levels and EPC functionality under hypoxic condition. Sixty healthy sedentary males were randomized to engage in either HIT (3-min intervals at 40 and 80 % VO 2max for five repetitions, n = 20) or MCT (sustained 60 % VO 2max , n = 20) for 30 min/day, 5 days/week for 6 weeks, or to a control group (CTL) that did not received exercise intervention (n = 20). Mon-EPC characteristics and EPC functionality under hypoxic exercise (HE, 100 W under 12 % O 2 ) were determined before and after HIT, MCT, and CTL. The results demonstrated that after the intervention, the HIT group exhibited larger improvements in VO 2peak , estimated peak cardiac output (Q C ), and estimated peak perfusions of frontal cerebral lobe (Q FC ) and vastus lateralis (Q VL ) than the MCT group. Furthermore, HIT (a) increased circulating CD14 ++ /CD16 - /CD34 + /KDR + (Mon-1 EPC) and CD14 ++ /CD16 + /CD34 + /KDR + (Mon-2 EPC) cell counts, (b) promoted the migration and tube formation of EPCs, (c) diminished the shedding of endothelial (CD34 - /KDR + /phosphatidylserine + ) cells, and (d) elevated plasma nitrite plus nitrate, stromal cell-derived factor-1, matrix metalloproteinase-9, and vascular endothelial growth factor-A concentrations at rest or following HE, compared to those of MCT. In addition, Mon-1 and -2 EPC counts were directly related to VO 2peak and estimated peak Q C , Q FC , and Q VL . HIT is superior to MCT for improving hemodynamic adaptation and Mon-EPC production. Moreover, HIT effectively enhances EPC functionality and suppresses endothelial injury undergoing hypoxia.

  9. Paracrine effects of bone marrow-derived endothelial progenitor cells: cyclooxygenase-2/prostacyclin pathway in pulmonary arterial hypertension.

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    Dong-Mei Jiang

    Full Text Available BACKGROUND: Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH. Some paracrine factors secreted by bone marrow-derived endothelial progenitor cells (BMEPCs have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT-induced PAH via producing vasoprotective substances in a paracrine fashion. METHODS AND RESULTS: Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2 expression, prostacyclin (PGI2 and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS and its progenies PGI2/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture. CONCLUSIONS: Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI2 and level of cAMP.

  10. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

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    Karina Needham

    2014-01-01

    Full Text Available In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps, with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  11. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Science.gov (United States)

    Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

    2015-01-01

    Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

  12. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  13. [VEGF165 transfected endothelial progenitor cells mediated by lentivirus alleviated ALI in rats].

    Science.gov (United States)

    He, Zhaohui; He, Huiwei; Lu, Yuanhua; Chen, Zhi; Xu, Fanghua; Wang, Rongsheng; Yang, Chunli

    2017-11-01

    To investigate the protective effects of vascular endothelial growth factor-165 (VEGF165) transfected the endothelial progenitor cells (EPCs) mediated by lentivirus on acute lung injury (ALI) in rats. The mononuclear cells from the male Sprague-Dawley (SD) rats were isolated and cultured to get the EPCs for study. The lentivirus vector carrying the human VEGF165 gene was constructed. According to the random number table method, 90 male SD rats were divided into ALI model group, phosphate buffer solution (PBS) group, EPCs treatment group, none transfected EPCs treatment group and VEGF165 transfected EPCs treatment group, and the rats in each group were subdivided into 4, 12 and 48 hours subgroups, with 6 rats in each subgroup. The rat model of ALI was reproduced by intravenous injection of oleic acid (0.15 μL/g). Then each treatment group was given PBS, EPCs, none transfected EPCs and VEGF165 transfected EPCs respectively with the same volume of 0.2 mL. For the groups with cells, about 1×10 6 cells were contained. Abdominal aortic blood and lung tissue were harvested at 4, 12 and 48 hours. Arterial blood gas analysis was performed. The lung wet/dry weight ratio (W/D) was calculated. The expressions of induced nitric oxide synthase (iNOS), endothelin-1 (ET-1) and VEGF165 were determined by enzyme-linked immunosorbent assay (ELISA). After dyed with hematoxylin-eosin (HE), the lung tissue pathology was observed and the lung injury score was performed. Compared with the ALI model group, the arterial partial pressure of oxygen (PaO 2 ) in EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups was significantly increased from 4 hours, and lung W/D, expressions of iNOS and ET-1 were significantly decreased, and VEGF165 expression was significantly increased. Compared with the EPCs treatment group, the increase in PaO 2 , the decrease in lung W/D and expressions of iNOS and ET-1, and the increase in VEGF165 expression in VEGF165 transfected EPCs

  14. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun; Wang, Jing [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Chen, Guian [Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Reproductive Medical Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Fan, Dongsheng, E-mail: dsfan@yahoo.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Deng, Min, E-mail: dengmin1706@yahoo.com.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China)

    2011-01-14

    Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  15. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    International Nuclear Information System (INIS)

    Zhang, Yun; Wang, Jing; Chen, Guian; Fan, Dongsheng; Deng, Min

    2011-01-01

    Research highlights: → Nicotinamide inhibit Sirt1. → MASH1 and Ngn2 activation. → Increase the expression of HB9. → Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and βIII-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 μM) or inhibitor nicotinamide (100 μM). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  16. Bone engineering in dog mandible: Coculturing mesenchymal stem cells with endothelial progenitor cells in a composite scaffold containing vascular endothelial growth factor.

    Science.gov (United States)

    Khojasteh, Arash; Fahimipour, Farahnaz; Jafarian, Mohammad; Sharifi, Davoud; Jahangir, Shahrbanoo; Khayyatan, Fahimeh; Baghaban Eslaminejad, Mohamadreza

    2017-10-01

    We sought to assess the effects of coculturing mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) in the repair of dog mandible bone defects. The cells were delivered in β-tricalcium phosphate scaffolds coated with poly lactic co-glycolic acid microspheres that gradually release vascular endothelial growth factor (VEGF). The complete scaffold and five partial scaffolds were implanted in bilateral mandibular body defects in eight beagles. The scaffolds were examined histologically and morphometrically 8 weeks after implantation. Histologic staining of the decalcified scaffolds demonstrated that bone formation was greatest in the VEGF/MSC scaffold (63.42 ± 1.67), followed by the VEGF/MSC/EPC (47.8 ± 1.87) and MSC/EPC (45.21 ± 1.6) scaffolds, the MSC scaffold (34.59 ± 1.49), the VEGF scaffold (20.03 ± 1.29), and the untreated scaffold (7.24 ± 0.08). Hence, the rate of new bone regeneration was highest in scaffolds containing MSC, either mixed with EPC or incorporating VEGF. Adding both EPC and VEGF with the MSC was not necessary. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1767-1777, 2017. © 2016 Wiley Periodicals, Inc.

  17. The C. elegans embryonic fate specification factor EGL-18 (GATA) is reutilized downstream of Wnt signaling to maintain a population of larval progenitor cells.

    Science.gov (United States)

    Gorrepati, Lakshmi; Eisenmann, David M

    2015-01-01

    In metazoans, stem cells in developing and adult tissues can divide asymmetrically to give rise to a daughter that differentiates and a daughter that retains the progenitor fate. Although the short-lived nematode C. elegans does not possess adult somatic stem cells, the lateral hypodermal seam cells behave in a similar manner: they divide once per larval stage to generate an anterior daughter that adopts a non-dividing differentiated fate and a posterior daughter that retains the seam fate and the ability to divide further. Wnt signaling pathway is known to regulate the asymmetry of these divisions and maintain the progenitor cell fate in one daughter, but how activation of the Wnt pathway accomplished this was unknown. We describe here our recent work that identified the GATA transcription factor EGL-18 as a downstream target of Wnt signaling necessary for maintenance of a progenitor population of larval seam cells. EGL-18 was previously shown to act in the initial specification of the seam cells in the embryo. Thus the acquisition of a Wnt-responsive cis-regulatory module allows an embryonic fate specification factor to be reutilized later in life downstream of a different regulator (Wnt signaling) to maintain a progenitor cell population. These results support the use of seam cell development in C. elegans as a simple model system for studying stem and progenitor cell biology.

  18. Impact of an endothelial progenitor cell capturing stent on coronary microvascular function: comparison with drug-eluting stents.

    Science.gov (United States)

    Choi, Woong Gil; Kim, Soo Hyun; Yoon, Hyung Seok; Lee, Eun Joo; Kim, Dong Woon

    2015-01-01

    Although drug-eluting stents (DESs) effectively reduce restenosis following percutaneous coronary intervention (PCI), they also delay re-endothelialization and impair microvascular function, resulting in adverse clinical outcomes. Endothelial progenitor cell (EPC) capturing stents, by providing a functional endothelial layer on the stent, have beneficial effects on microvascular function. However, data on coronary microvascular function in patients with EPC stents versus DESs are lacking. Seventy-four patients who previously underwent PCI were enrolled in this study. Microvascular function was evaluated 6 months after PCI based on the index of microvascular resistance (IMR) and the coronary flow reserve (CFR). IMR was calculated as the ratio of the mean distal coronary pressure at maximal hyperemia to the inverse of the hyperemic mean transit time (hTmn). The CFR was calculated by dividing the hTmn by the baseline mean transit time. Twenty-one patients (age, 67.2 ± 9.6 years; male:female, 15:6) with an EPC stent and 53 patients (age, 61.5 ± 14.7 years; male:female, 40:13) with second-generation DESs were included in the study. There were no significant differences in the baseline clinical and angiographic characteristics of the two groups. Angiography performed 6 months postoperatively did not show significant differences in their CFR values. However, patients with the EPC stent had a significantly lower IMR than patients with second-generation DESs (median, 25.5 [interquartile range, 12.85 to 28.18] vs. 29.0 [interquartile range, 15.42 to 39.23]; p = 0.043). Microvascular dysfunction was significantly improved after 6 months in patients with EPC stents compared to those with DESs. The complete re-endothelialization achieved with the EPC stent may provide clinical benefits over DESs, especially in patients with microvascular dysfunction.

  19. In vivo endothelization of tubular vascular grafts through in situ recruitment of endothelial and endothelial progenitor cells by RGD-fused mussel adhesive proteins

    International Nuclear Information System (INIS)

    Kang, Tae-Yun; Lee, Jung Ho; Kang, Jo-A; Rhie, Jong-Won; Kim, Bum Jin; Cha, Hyung Joon; Hong, Jung Min; Kim, Byoung Soo; Cho, Dong-Woo

    2015-01-01

    The use of tissue mimics in vivo, including patterned vascular networks, is expected to facilitate the regeneration of functional tissues and organs with large volumes. Maintaining patency of channels in contact with blood is an important issue in the development of a functional vascular network. Endothelium is the only known completely non-thrombogenic material; however, results from treatments to induce endothelialization are inconclusive. The present study was designed to evaluate the clinical applicability of in situ recruitment of endothelial cells/endothelial progenitor cells (EC/EPC) and pre-endothelization using a recombinant mussel adhesive protein fused with arginine–glycine–aspartic acid peptide (MAP-RGD) coating in a model of vascular graft implantation. Microporous polycaprolactone (PCL) scaffolds were fabricated with salt leaching methods and their surfaces were modified with collagen and MAP-RGD. We then evaluated their anti-thrombogenicity with an in vitro hemocompatibility assessment and a 4-week implantation in the rabbit carotid artery. We observed that MAP-RGD coating reduced the possibility of early in vivo graft failure and enhanced re-endothelization by in situ recruitment of EC/EPC (patency rate: 2/3), while endothelization prior to implantation aggravated the formation of thrombosis and/or IH (patency rate: 0/3). The results demonstrated that in situ recruitment of EC/EPC by MAP-RGD could be a promising strategy for vascular applications. In addition, it rules out several issues associated with pre-endothelization, such as cell source, purity, functional modulation and contamination. Further evaluation of long term performance and angiogenesis from the luminal surface may lead to the clinical use of MAP-RGD for tubular vascular grafts and regeneration of large-volume tissues with functional vascular networks. (paper)

  20. Increased circulating endothelial apoptotic microparticle to endothelial progenitor cell ratio is associated with subsequent decline in glomerular filtration rate in hypertensive patients.

    Directory of Open Access Journals (Sweden)

    Chien-Yi Hsu

    Full Text Available BACKGROUND: Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs and increased levels of endothelial apoptotic microparticles (EMP. However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR remains unclear. METHODS AND RESULTS: We enrolled totally 100 hypertensive out-patients with eGFR ≥ 30 mL/min/1.73 m(2. The mean annual rate of GFR decline (△GFR/y was -1.49 ± 3.26 mL/min/1.73 m(2 per year during the follow-up period (34 ± 6 months. Flow cytometry was used to assess circulating EPC (CD34(+/KDR(+ and EMP levels (CD31(+/annexin V(+ in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r= -0.465, p<0.001, microalbuminuria (r= -0.329, p=0.001, and the Framingham risk score (r= -0.245, p=0.013. When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08 ± 3.04 vs. -0.50 ± 2.84 vs. -1.25 ± 2.49 vs. -4.42 ± 2.82, p<0.001. Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y. CONCLUSIONS: An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.

  1. Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells

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    Xiugong Gao

    2017-12-01

    Full Text Available Induced pluripotent stem cells (iPSCs offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs derived from umbilical cord blood (CB or adult peripheral blood (PB afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5–7 days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications.

  2. Comparative transcriptomic analysis of endothelial progenitor cells derived from umbilical cord blood and adult peripheral blood: Implications for the generation of induced pluripotent stem cells.

    Science.gov (United States)

    Gao, Xiugong; Yourick, Jeffrey J; Sprando, Robert L

    2017-12-01

    Induced pluripotent stem cells (iPSCs) offer the potential to generate tissues with ethnic diversity enabling toxicity testing on selected populations. Recently, it has been reported that endothelial progenitor cells (EPCs) derived from umbilical cord blood (CB) or adult peripheral blood (PB) afford a practical and efficient cellular substrate for iPSC generation. However, differences between EPCs from different blood sources have rarely been studied. In the current study, we derived EPCs from blood mononuclear cells (MNCs) and reprogrammed EPCs into iPSCs. We also explored differences between CB-EPCs and PB-EPCs at the molecular and cellular levels through a combination of transcriptomic analysis and cell biology techniques. EPC colonies in CB-MNCs emerged 5-7days earlier, were 3-fold higher in number, and consistently larger in size than in PB-MNCs. Similarly, iPSC colonies generated from CB-EPCs was 2.5-fold higher in number than from PB-EPCs, indicating CB-EPCs have a higher reprogramming efficiency than PB-EPCs. Transcriptomic analysis using microarrays found a total of 1133 genes differentially expressed in CB-EPCs compared with PB-EPCs, with 675 genes upregulated and 458 downregulated. Several canonical pathways were impacted, among which the human embryonic stem cell pluripotency pathway was of particular interest. The differences in the gene expression pattern between CB-EPCs and PB-EPCs provide a molecular basis for the discrepancies seen in their derivation and reprogramming efficiencies, and highlight the advantages of using CB as the cellular source for the generation of iPSCs and their derivative tissues for ethnic-related toxicological applications. Published by Elsevier B.V.

  3. Impact of obesity control on circulating level of endothelial progenitor cells and angiogenesis in response to ischemic stimulation

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    Chen Yung-Lung

    2012-07-01

    Full Text Available Abstract Background and aim We tested the hypothesis that obesity reduced circulating number of endothelial progenitor cells (EPCs, angiogenic ability, and blood flow in ischemic tissue that could be reversed after obesity control. Methods 8-week-old C57BL/6J mice (n = 27 were equally divided into group 1 (fed with 22-week control diet, group 2 (22-week high fat diet, and group 3 (14-week high fat diet, followed by 8-week control diet. Critical limb ischemia (CLI was induced at week 20 in groups 2 and 3. The animals were sacrificed at the end of 22 weeks. Results Heart weight, body weight, abdominal fat weight, serum total cholesterol level, and fasting blood sugar were highest in group 2 (all p  Conclusion Obesity suppressed abilities of angiogenesis and recovery from CLI that were reversed by obesity control.

  4. Endothelial progenitor cells in long-standing asymptomatic type 1 diabetic patients with or without diabetic nephropathy

    DEFF Research Database (Denmark)

    Reinhard, Henrik; Jacobsen, Peter Karl; Lajer, Maria

    2011-01-01

    with or without DN and to study the effect of CVD and medication on EPC numbers. Methods: We examined EPC numbers in 37 type 1 diabetic patients with DN and 35 type 1 diabetic patients with long-standing normoalbuminuria. Patients were without symptoms of CVD and the prevalence of CVD was previously shown......A decrease in the number and dysfunction of endothelial progenitor cells (EPC) may increase the risk for progression of cardiovascular disease (CVD) in type 1 diabetic patients with diabetic nephropathy (DN). Our aim was to evaluate EPC numbers in asymptomatic CVD type 1 diabetic patients...... with CVD (p > 0.05). Conventional risk factors were significantly higher in patients with DN and they received more CVD-preventive treatment. All patients receiving simvastatin or calcium-channel blockers had higher numbers of EPC compared to patients not treated with these drugs. Conclusions: Asymptomatic...

  5. Resveratrol Improves Tube Formation in AGE-Induced Late Endothelial Progenitor Cells by Suppressing Syndecan-4 Shedding

    Directory of Open Access Journals (Sweden)

    Han Wu

    2018-01-01

    Full Text Available Dysfunction of endothelial progenitor cells (EPCs contributes to cardiovascular complications in diabetes, and resveratrol has been shown to improve EPC functions. Syndecan-4 (Synd4, a cell surface heparin sulfate proteoglycan, has been shown to promote neovascularization. Thus, the present study was performed to determine whether resveratrol promoted angiogenesis of EPCs by regulating Synd4. Late EPCs were isolated from human peripheral blood and stimulated with AGEs. Western blot showed that AGEs induced Synd4 shedding in a dose- and time-dependent manner. AGE-induced Synd4 shedding was partly reversed by NAC or resveratrol, along with normalized ROS production. Overexpression of Synd4 or pretreatment of resveratrol reversed AGE-impaired tube formation of EPCs and regulated the Akt/eNOS pathway. Furthermore, resveratrol suppressed Synd4 shedding via the inhibition of oxidative stress and improved tube formation of late EPCs via the regulation of the Synd4/Akt/eNOS pathway.

  6. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

    Energy Technology Data Exchange (ETDEWEB)

    Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Medicina, São Paulo, SP, Brasil, Departamento de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2014-04-15

    It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3endothelial function (T2>T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation.

  7. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

    International Nuclear Information System (INIS)

    Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H.

    2014-01-01

    It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3endothelial function (T2>T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation

  8. Effect of prepro-calcitonin gene-related peptide-expressing endothelial progenitor cells on pulmonary hypertension.

    Science.gov (United States)

    Zhao, Qiang; Liu, Zixiong; Wang, Zhe; Yang, Cheng; Liu, Jun; Lu, Jun

    2007-08-01

    Calcitonin gene-related peptide (CGRP) is a potent smooth muscle cell proliferation inhibitor and vasodilator. It is now believed that CGRP plays an important role in maintaining a low pulmonary vascular resistance. We evaluated the therapeutic effect of intravenously administered CGRP-expressing endothelial progenitor cells (EPCs) on left-to-right shunt-induced pulmonary hypertension in rats. Endothelial progenitor cells were obtained from cultured human peripheral blood mononuclear cells. The genetic sequence for CGRP was subcloned into cultured EPCs by human expression plasmid. Pulmonary hypertension was established in immunodeficient rats with an abdominal aorta to inferior vena cava shunt operation. The transfected EPCs were injected through the left jugular vein at 10 weeks after the shunt operation. Mean pulmonary artery pressure and total pulmonary vascular resistance were detected with right cardiac catheterization at 4 weeks. The distribution of EPCs in the lung tissue was examined with immunofluorescence technique. Histopathologic changes in the structure of the pulmonary arteries was observed with electron microscopy and subjected to computerized image analysis. The lungs of rats transplanted with CGRP-expressing EPCs demonstrated a decrease in both mean pulmonary artery pressure (17.64 +/- 0.79 versus 22.08 +/- 0.95 mm Hg; p = 0.018) and total pulmonary vascular resistance (1.26 +/- 0.07 versus 2.45 +/- 0.18 mm Hg x min/mL; p = 0.037) at 4 weeks. Immunofluorescence revealed that intravenously administered cells were incorporated into the pulmonary vasculature. Pulmonary vascular remodeling was remarkably attenuated with the administration of CGRP-expressing EPCs. The transplantation of CGRP-expressing EPCs may effectively attenuate established pulmonary hypertension and exert reversal effects on pulmonary vascular remodeling. Our findings suggest that the therapy based on the combination of both CGRP transfection and EPCs may be a potentially useful

  9. Valsartan reduces AT1-AA-induced apoptosis through suppression oxidative stress mediated ER stress in endothelial progenitor cells.

    Science.gov (United States)

    Wang, Z-C; Qi, J; Liu, L-M; Li, J; Xu, H-Y; Liang, B; Li, B

    2017-03-01

    Valsartan has been reported to have the function of treating hypertension and improving the prognosis of patients. Many studies indicated that valsartan can also increase angiotensin II, andosterone and plasma renin activity (PRA). Autoantibodies against the angiotensin II type 1 receptor (AT1-AA) have been showed to increase reactive oxygen species (ROS) and calcium (Ca2+) and result in apoptosis in vascular smooth muscle cells. In this study, we attempted to explore the effect of valsartan on AT1-AA-induced apoptosis in endothelial progenitor cells. Endothelial progenitor cells (EPCs) were cultured. The cytotoxicity was determined by MTT assay. EPCs apoptosis was determined by DAPI staining and flow cytometry. Reactive oxygen species, intracellular calcium concentration and calpain activity were measured using Fluostar Omega Spectrofluorimeter. The expression of p-ERK, p-eIF-2a, CHOP, Bcl-2 and caspase-3 were detected by Western blot. MTT assays showed valsartan significantly inhibited AT1-AA- induced decline of the viability of EPCs. DAPI staining and flow cytometry results indicated valsartan inhibited AT1-AA-induced decline of the viability of EPCs via inhibiting AT1-AA-induced apoptosis. Furthermore, the increasing of reactive oxygen species, intracellular calcium and calpain activity induced by AT1-AA in EPCs were also recovered after pre-treated with valsartan. Meanwhile, the upregulation of p-ERK, p-eIF-2a and CHOP, downregulation of Bcl-2, and activation of Caspase-3 caused by AT1-AA were reversed after pre-incubated with valsartan. Valsartan could inhibit AT1-AA-induced apoptosis through inhibiting oxidative stress mediated ER stress in EPCs.

  10. Mechanism of endothelial progenitor cell recruitment into neo-vessels in adjacent non-tumor tissues in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Yu, De-cai; Chen, Jun; Sun, Xi-tai; Zhuang, Lin-yuan; Jiang, Chun-ping; Ding, Yi-tao

    2010-01-01

    We investigated the distribution and clinical significance of mobilized endothelial progenitor cells (EPCs) in hepatocellular carcinoma (HCC). We found that many more EPCs were recruited to nonmalignant liver tissue (especially into adjacent non-tumor tissues (AT)) than to tumor vessels. These results suggest that the mechanism underlying the recruitment of EPCs into microvessels in AT merits further investigation Angiogenic factors were detected in three tissue microarrays comprising normal liver, paired tumor tissue (TT) and AT from 105 patients (who had undergone hepatectomy for HCC) using immunohistochemistry. Also, the number of EPCs (positive for Sca-1, Flk-1 and c-Kit) in the blood and liver of cirrhotic mice were determined by flow cytometry and immunohistochemistry. The distribution of these labeled EPCs in tumor and non-tumor tissues was then studied. The results from the tissue microarrays showed that the expression levels of VEGF-A, bFGF, TGF-β, MCP-1, TSP-1, MMP-9, TIMP-2, and endostatin were significantly higher in AT than in either normal liver or TT (p < 0.05), but no significant difference was found in the expression levels of COX-2 and NOS-2 between AT and TT. The expression of VEGF-A, bFGF, TGF-β, MCP-1, TSP-1, MMP-9, TIMP-2, endostatin, COX-2, and NOS-2 in normal liver tissue was weaker than that in AT or TT. In cirrhotic mice, the number of circulating endothelial progenitor cells gradually increased, before decreasing again. In this mouse model, increased numbers of EPCs were recruited and homed specifically to the cirrhotic liver. Both liver cirrhosis and HCC led to increased expression of pro-angiogenic factors, which resulted in the recruitment of EPCs into AT. Also, EPCs were mobilized, recruited and homed to cirrhotic liver. The unique pathology of HCC coupled with liver cirrhosis may, therefore, be associated with the distribution and function of EPCs

  11. Neurotrophins promote revascularization by local recruitment of TrkB+ endothelial cells and systemic mobilization of hematopoietic progenitors

    Science.gov (United States)

    Kermani, Pouneh; Rafii, Dahlia; Jin, David K.; Whitlock, Paul; Schaffer, Wendy; Chiang, Anne; Vincent, Loic; Friedrich, Matthias; Shido, Koji; Hackett, Neil R.; Crystal, Ronald G.; Rafii, Shahin; Hempstead, Barbara L.

    2005-01-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels. PMID:15765148

  12. Circulating endothelial progenitor cell numbers are not associated with donor organ age or allograft vasculopathy in cardiac transplant recipients.

    Science.gov (United States)

    Thomas, H E; Parry, G; Dark, J H; Arthur, H M; Keavney, B D

    2009-02-01

    Increasing age is associated with reduced numbers of circulating endothelial progenitor cells (EPCs). It is unclear whether this relates to depletion or impairment of bone marrow progenitors, or to deficient mobilization signals from aging tissues. In cardiac transplant patients, one previous study has reported an association between circulating EPCs and the risk of cardiac allograft vasculopathy (CAV). We investigated whether increased donor heart age, a strong risk factor for CAV, was associated with reduced circulating EPC numbers in a group of cardiac transplant recipients matched for factors which influence EPC numbers, but with maximally discordant donor heart ages. We identified 32 patient pairs, matched for factors known to influence EPC numbers, but who had discordant donor heart ages by at least 20 years. EPCs were quantified using flow cytometry for absolute counts of cells expressing all the combinations of CD45, CD34, CD133 and the kinase domain receptor (KDR). There were no significant differences in the numbers of circulating EPCs between patients with old or young donor heart age. There was no association between the presence of CAV and circulating EPC numbers. We suggest that the increased susceptibility to CAV of older donor hearts is not mediated via circulating EPCs. Our results are consistent with the theory that the normal age-related decline in EPC numbers relates to bone marrow aging rather than failure of target tissues to induce EPC mobilization.

  13. Mobilization of endogenous bone marrow derived endothelial progenitor cells and therapeutic potential of parathyroid hormone after ischemic stroke in mice.

    Directory of Open Access Journals (Sweden)

    Li-Li Wang

    Full Text Available Stroke is a major neurovascular disorder threatening human life and health. Very limited clinical treatments are currently available for stroke patients. Stem cell transplantation has shown promising potential as a regenerative treatment after ischemic stroke. The present investigation explores a new concept of mobilizing endogenous stem cells/progenitor cells from the bone marrow using a parathyroid hormone (PTH therapy after ischemic stroke in adult mice. PTH 1-34 (80 µg/kg, i.p. was administered 1 hour after focal ischemia and then daily for 6 consecutive days. After 6 days of PTH treatment, there was a significant increase in bone marrow derived CD-34/Fetal liver kinase-1 (Flk-1 positive endothelial progenitor cells (EPCs in the peripheral blood. PTH treatment significantly increased the expression of trophic/regenerative factors including VEGF, SDF-1, BDNF and Tie-1 in the brain peri-infarct region. Angiogenesis, assessed by co-labeled Glut-1 and BrdU vessels, was significantly increased in PTH-treated ischemic brain compared to vehicle controls. PTH treatment also promoted neuroblast migration from the subventricular zone (SVZ and increased the number of newly formed neurons in the peri-infarct cortex. PTH-treated mice showed significantly better sensorimotor functional recovery compared to stroke controls. Our data suggests that PTH therapy improves endogenous repair mechanisms after ischemic stroke with functional benefits. Mobilizing endogenous bone marrow-derived stem cells/progenitor cells using PTH and other mobilizers appears an effective and feasible regenerative treatment after ischemic stroke.

  14. Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Hiroko Shimada

    Full Text Available The common marmoset (Callithrix jacchus is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs derived from mouse and human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

  15. Lhx2 expression promotes self-renewal of a distinct multipotential hematopoietic progenitor cell in embryonic stem cell-derived embryoid bodies.

    Directory of Open Access Journals (Sweden)

    Lina Dahl

    Full Text Available The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2(-/- mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES cell-derived embryoid bodies (EBs can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

  16. The CMV early enhancer/chicken beta actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors

    DEFF Research Database (Denmark)

    Alexopoulou, Annika N; Couchman, John R; Whiteford, James

    2008-01-01

    BACKGROUND: Mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. The differentiation mimics early developmental events, including vasculogenesis and early angiogenesis and several differentiation systems are being...... used to identify factors that are important during the formation of the vascular system. Embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult....... RESULTS: CCE mouse embryonic stem cells were differentiated on collagen type IV for 4-5 days, Flk1+ mesodermal cells were sorted and replated either on collagen type IV in the presence of VEGFA to give rise to endothelial cells and smooth muscle cells or in collagen type I gels for the formation...

  17. Circulating endothelial progenitor cells: a new approach to anti-aging medicine?

    Directory of Open Access Journals (Sweden)

    Patel Amit N

    2009-12-01

    Full Text Available Abstract Endothelial dysfunction is associated with major causes of morbidity and mortality, as well as numerous age-related conditions. The possibility of preserving or even rejuvenating endothelial function offers a potent means of preventing/treating some of the most fearful aspects of aging such as loss of mental, cardiovascular, and sexual function. Endothelial precursor cells (EPC provide a continual source of replenishment for damaged or senescent blood vessels. In this review we discuss the biological relevance of circulating EPC in a variety of pathologies in order to build the case that these cells act as an endogenous mechanism of regeneration. Factors controlling EPC mobilization, migration, and function, as well as therapeutic interventions based on mobilization of EPC will be reviewed. We conclude by discussing several clinically-relevant approaches to EPC mobilization and provide preliminary data on a food supplement, Stem-Kine, which enhanced EPC mobilization in human subjects.

  18. Topical application of ex vivo expanded endothelial progenitor cells promotes vascularisation and wound healing in diabetic mice.

    Science.gov (United States)

    Asai, Jun; Takenaka, Hideya; Ii, Masaaki; Asahi, Michio; Kishimoto, Saburo; Katoh, Norito; Losordo, Douglas W

    2013-10-01

    Impaired wound healing leading to skin ulceration is a serious complication of diabetes and may be caused by defective angiogenesis. Endothelial progenitor cells (EPCs) can augment neovascularisation in the ischaemic tissue. Experiments were performed to test the hypothesis that locally administered EPCs can promote wound healing in diabetes. Full-thickness skin wounds were created on the dorsum of diabetic mice. EPCs were obtained from bone marrow mononuclear cells (BMMNCs) and applied topically to the wound immediately after surgery. Vehicle and non-selective BMMNCs were used as controls. Wound size was measured on days 5, 10 and 14 after treatment, followed by resection, histological analysis and quantification of vascularity. Topical application of EPCs significantly promoted wound healing, as assessed by closure rate and wound vascularity. Immunostaining revealed that transplanted EPCs induced increased expression of vascular endothelial growth factor and basic fibroblast growth factor. Few EPCs were observed in the neovasculature based on in vivo staining of the functional vasculature. Ex vivo expanded EPCs promote wound healing in diabetic mice via mechanisms involving increased local cytokine expression and enhanced neovascularisation of the wound. This strategy exploiting the therapeutic capacity of autologously derived EPCs may be a novel approach to skin repair in diabetes. © 2012 The Authors. International Wound Journal © 2012 John Wiley & Sons Ltd and Medicalhelplines.com Inc.

  19. Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

    International Nuclear Information System (INIS)

    Chen Qin; Dong Li; Wang Lian; Kang Lina; Xu Biao

    2009-01-01

    Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.

  20. Indoxyl Sulfate Impairs Endothelial Progenitor Cells and Might Contribute to Vascular Dysfunction in Patients with Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Cheng-Jui Lin

    2016-12-01

    Full Text Available Background/Aims: Indoxyl sulfate (IS is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD. We explored the effect of IS on human early endothelial progenitor cells (EPCs and analyzed the correlation between serum IS levels and parameters of vascular function, including endothelial function in a CKD-based cohort. Methods: A cross-sectional study with 128 stable CKD patients was conducted. Flow-mediated dilation (FMD, pulse wave velocity (PWV, ankle brachial index, serum IS and other biochemical parameters were measured and analyzed. In parallel, the activity of early EPCs was also evaluated after exposure to IS. Results: In human EPCs, a concentration-dependent inhibitory effect of IS on chemotactic motility and colony formation was observed. Additionally, serum IS levels were significantly correlated with CKD stages. The total IS (T-IS and free IS (F-IS were strongly associated with age, hypertension, cardiovascular disease, blood pressure, PWV, blood urea nitrogen, creatine and phosphate but negatively correlated with FMD, the estimated glomerular filtration rate (eGFR, hemoglobin, hematocrit, and calcium. A multivariate linear regression analysis also showed that FMD was significantly associated with IS after adjusting for other confounding factors. Conclusions: In humans, IS impairs early EPCs and was strongly correlated with vascular dysfunction. Thus, we speculate that this adverse effect of IS may partly result from the inhibition of early EPCs.

  1. Intradialytic aerobic cycling exercise alleviates inflammation and improves endothelial progenitor cell count and bone density in hemodialysis patients.

    Science.gov (United States)

    Liao, Min-Tser; Liu, Wen-Chih; Lin, Fu-Huang; Huang, Ching-Feng; Chen, Shao-Yuan; Liu, Chuan-Chieh; Lin, Shih-Hua; Lu, Kuo-Cheng; Wu, Chia-Chao

    2016-07-01

    Inflammation, endothelial dysfunction, and mineral bone disease are critical factors contributing to morbidity and mortality in hemodialysis (HD) patients. Physical exercise alleviates inflammation and increases bone density. Here, we investigated the effects of intradialytic aerobic cycling exercise on HD patients. Forty end-stage renal disease patients undergoing HD were randomly assigned to either an exercise or control group. The patients in the exercise group performed a cycling program consisting of a 5-minute warm-up, 20 minutes of cycling at the desired workload, and a 5-minute cool down during 3 HD sessions per week for 3 months. Biochemical markers, inflammatory cytokines, nutritional status, the serum endothelial progenitor cell (EPC) count, bone mineral density, and functional capacity were analyzed. After 3 months of exercise, the patients in the exercise group showed significant improvements in serum albumin levels, the body mass index, inflammatory cytokine levels, and the number of cells positive for CD133, CD34, and kinase insert domain-conjugating receptor. Compared with the exercise group, the patients in the control group showed a loss of bone density at the femoral neck and no increases in EPCs. The patients in the exercise group also had a significantly greater 6-minute walk distance after completing the exercise program. Furthermore, the number of EPCs significantly correlated with the 6-minute walk distance both before and after the 3-month program. Intradialytic aerobic cycling exercise programs can effectively alleviate inflammation and improve nutrition, bone mineral density, and exercise tolerance in HD patients.

  2. Role of integrin-linked kinase for functional capacity of endothelial progenitor cells in patients with stable coronary artery disease

    International Nuclear Information System (INIS)

    Werner, Christian; Boehm, Michael; Friedrich, Erik B.

    2008-01-01

    Number and function of endothelial progenitor cells (EPCs) are down-regulated in patients with coronary artery disease (CAD). Integrin-linked kinase (ILK) is a signal and adaptor protein that regulates survival of mature endothelial cells and vascular development. Here we show that EPC dysfunction in patients with CAD is paralleled by down-regulation of ILK while restoration of ILK expression rescues the migratory defect of CAD-EPCs. Human EPCs transduced with dominant-negative ILK (DN-ILK) display significantly reduced expression of CD34 + /VEGFR-2 + , DiI-Ac-LDL uptake, and Ulex europaeus lectin binding. Mechanistically, DN-ILK-transfected EPCs are characterized by decreased proliferation, while proliferation is increased in wild-type ILK-transfected EPCs. These effects are paralleled by changes in cyclin D1 expression, colony forming units, and cytoskeletal rearrangement. Functionally, ILK is necessary and sufficient for SDF-1-triggered migration and adhesion in EPCs. These data extend current knowledge about the role of ILK in EPC biology and implicate ILK as a therapeutic target in CAD.

  3. A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Richard Longeras

    2012-01-01

    Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

  4. Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Fabian Duttenhoefer

    2015-01-01

    Full Text Available In bone tissue engineering (TE endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs are a rich source of mesenchymal stem cells (MSCs able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34+ and CD133+ were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex+ or medium containing platelet lysate (PL. MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex+ medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs.

  5. Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells.

    Science.gov (United States)

    You, Jinzhi; Sun, Jiacheng; Ma, Teng; Yang, Ziying; Wang, Xu; Zhang, Zhiwei; Li, Jingjing; Wang, Longgang; Ii, Masaaki; Yang, Junjie; Shen, Zhenya

    2017-08-03

    Neovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs. Streptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs. Curcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment. Curcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

  6. Effect of Low Level Ionizing Radiation on Endothelial Progenitor Cells in Atherosclerotic Patients with Lower Limb Ischemia

    International Nuclear Information System (INIS)

    Taha, E.F.S.

    2013-01-01

    Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality throughout the developed world (Williamson et al., 2012). Coronary artery disease (CAD) or atherosclerotic heart disease is a chronic life-threatening disease, which characterized by reducing blood supply to the heart as a result of the accumulation of atheromatous plaques within the walls of the arteries supplying the myocardium. Progressive atherosclerosis in the coronary arteries may lead to intimal thickening and eventual artery occlusion. Coronary artery occlusion can cause acute myocardial ischemia as a result of reduced oxygen supply or increased oxygen demand (Luthje and Andreas, 2008). Convincing evidence indicates that atherosclerosis is associated with endothelial dysfunction at the early stage of the disease process (Chiang et al., 2012). The endothelium is a dynamic cell layer that represents a physiological barrier between circulating blood and the surrounding tissues. Impaired endothelial function is a critical event in the initiation of atherosclerotic plaque development and thus may lead to vasoconstriction, vascular smooth muscle proliferation, hypercoagulability, thrombosis, and eventually, adverse cardiovascular events (Berger and Lavie, 2011). Asahara et al., (1997) described endothelial progenitor cells (EPC) in human peripheral blood. EPC are immature endothelial circulating cells mobilized from the bone marrow. These cells are involved in Introduction and aim of the work repairing the damaged endothelium and in facilitating neovascularization after ischemia (Rouhl et al., 2008). The role of EPC in health and disease is not understood completely. Most studies of healthy subjects and patients with coronary artery disease (CAD) report that the number and function of circulating EPC decrease with age and with the presence of classical vascular risk factors (Fadini et al., 2007). Recent studies suggested that EPCs play an important role in the risk of vascular

  7. DPP4 inhibitors promote biological functions of human endothelial progenitor cells by targeting the SDF-1/CXCR4 signaling pathway

    Directory of Open Access Journals (Sweden)

    Liu Feng

    2016-01-01

    Full Text Available Dipeptidyl peptidase 4 (DPP4 inhibitors(oral hypoglycemic agentshave beneficial effects during the early stages of diabetes. In this study, we evaluated the role of DPP4inhibitorsonthe biological functions of cultured human endothelial progenitor cells (EPCs. After treating EPCs with the DPP4 inhibitors sitagliptin and vildagliptin, we examined the mRNA expression of DPP4, vascular endothelial growth factor (VEGF,VEGF receptor 2 (VEGFR-2,endothelial nitric oxide synthase (eNOS, caspase-3,stromal cell-derived factor-1 (SDF-1, chemokine (C-X-C motif receptor 4 (CXCR4 were measured by RT-PCR. The protein expression of SDF-1 and CXCR4 was determined by Western blot; cell proliferation was tested by the MTT method, and DPP4 activity was determined by a DPP4 assay. Our results revealed that DPP4 expression and activity were inhibited following the treatment with various doses of DPP4 inhibitors. Cell proliferation and the expression of VEGF, VEGFR-2andeNOS were up regulated, while cell apoptosis was inhibited by DPP4 inhibitors in a dose-dependent manner. DPP4 inhibitors activated the SDF-1/CXCR4 signaling pathway, shown by the elevated expression of SDF-1/CXCR4. This further proved that after the SDF-1/CXCR4 signaling pathway was blocked by its inhibitor ADM3100, the effects of DPP4 inhibitors on the proliferation and apoptosis, and the expression of VEGF, VEGFR-2and eNOS of EPCs were significantly reduced. These findings suggest that DPP4 inhibitors promote the biological functions of human EPCs by up regulating the SDF-1/CXCR4 signaling pathway.

  8. Alcohol consumption negates estrogen-mediated myocardial repair in ovariectomized mice by inhibiting endothelial progenitor cell mobilization and function.

    Science.gov (United States)

    Mackie, Alexander R; Krishnamurthy, Prasanna; Verma, Suresh K; Thorne, Tina; Ramirez, Veronica; Qin, Gangjian; Abramova, Tatiana; Hamada, Hiromichi; Losordo, Douglas W; Kishore, Raj

    2013-06-21

    We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.

  9. Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

    2017-06-01

    Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

  10. Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

    Science.gov (United States)

    Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

    2016-04-01

    The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by

  11. Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/Activin/Nodal signaling using SB-431542

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Schrøder, Henrik Daa

    2010-01-01

    Directing differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a prerequisite for the clinical use of hESC in regenerative medicine procedures. Here, we report a protocol for directing the differentiation of hESC into mesenchymal...... in vivo. Interestingly, SB-OG cells cultured in 10% fetal bovine serum (FBS) developed into a homogeneous population of mesenchymal progenitors that expressed CD markers characteristic of mesenchymal stem cells (MSC): CD44(+) (100%), CD73(+) (98%), CD146(+) (96%) and CD166(+) (88%) with the ability...... progenitor cells. We demonstrate that inhibition of TGF-beta/Activin/Nodal signaling during embryoid bodies (EB) formation using SB-431542 (SB) in serum free medium, markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), and several myogenic developmental markers including early myogenic...

  12. Circulating endothelial progenitor cells do not contribute to regeneration of endothelium after murine arterial injury

    DEFF Research Database (Denmark)

    Hagensen, Mette; Raarup, Merete Krog; Mortensen, Martin Bødtker

    2012-01-01

    into endothelial cells (ECs). We tested this theory in a murine arterial injury model using carotid artery transplants and fluorescent reporter mice. METHODS AND RESULTS: Wire-injured carotid artery segments from wild-type mice were transplanted into TIE2-GFP transgenic mice expressing green fluorescent protein...... (GFP) in ECs. We found that the endothelium regenerated with GFP(+) ECs as a function of time, evolving from the anastomosis sites towards the centre of the transplant. A migration front of ECs at Day 7 was verified by scanning electron microscopy and by bright-field microscopy using recipient TIE2-lac......Z mice with endothelial β-galactosidase expression. These experiments indicated migration of flanking ECs rather than homing of circulating cells as the underlying mechanism. To confirm this, we interposed non-injured wild-type carotid artery segments between the denuded transplant and the TIE2-GFP...

  13. Fetal exposure to a diabetic intrauterine environment resulted in a failure of cord blood endothelial progenitor cell adaptation against chronic hypoxia

    Directory of Open Access Journals (Sweden)

    Dincer UD

    2014-12-01

    Full Text Available U Deniz Dincer Department of Basic and Clinical Pharmacology, School of Medicine, Bezmialem Vakif University (BAVU, Fatih/Istanbul, Turkey Abstract: Gestational diabetes mellitus (GDM has long-term health consequences, and fetal exposure to a diabetic intrauterine environment increases cardiovascular risk for her adult offspring. Some part of this could be related to their endothelial progenitor cells (EPCs. Understanding the vessel-forming ability of human umbilical cord blood (HUCB-derived endothelial colony-forming cells (ECFCs against pathological stress such as GDM response to hypoxia could generate new therapeutic strategies. This study aims to investigate the role of chronic hypoxia in EPCs functional and vessel-forming ability in GDM subjects. Each ECFC was expressed in endothelial and pro-angiogenic specific markers, namely endothelial nitric oxide synthase (eNOS, platelet (PECAM-1 endothelial cell adhesion molecule 1, vascular endothelial-cadherin CdH5 (Ca-dependent cell adhesion molecule, vascular endothelial growth factor A, (VEGFA and insulin-like growth factor 1 (IGF1. Chronic hypoxia did not affect CdH5, but PECAM1 MRNA expressions were increased in control and GDM subjects. Control hypoxic and GDM normoxic VEGFA MRNA expressions and hypoxia-inducible factor 1-alpha (HIF1α protein expressions were significantly increased in HUCB ECFCs. GDM resulted in most failure of HUCB ECFC adaptation and eNOS protein expressions against chronic hypoxia. Chronic hypoxia resulted in an overall decline in HUCB ECFCs' proliferative ability due to reduction of clonogenic capacity and diminished vessel formation. Furthermore, GDM also resulted in most failure of cord blood ECFC adaptation against chronic hypoxic environment. Keywords: endothelial progenitor cells, gestational diabetes mellitus, chronic hypoxia, human cord blood

  14. Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis.

    Science.gov (United States)

    Chillà, Anastasia; Margheri, Francesca; Biagioni, Alessio; Del Rosso, Mario; Fibbi, Gabriella; Laurenzana, Anna

    2018-04-03

    Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular

  15. GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, impairs neovascularization by decreasing endothelial progenitor cell function.

    Directory of Open Access Journals (Sweden)

    Yi-Wen Lin

    Full Text Available The number and function of endothelial progenitor cells (EPCs are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4 mutation mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

  16. Taurine and magnesium supplementation enhances the function of endothelial progenitor cells through antioxidation in healthy men and spontaneously hypertensive rats.

    Science.gov (United States)

    Katakawa, Mayumi; Fukuda, Noboru; Tsunemi, Akiko; Mori, Mari; Maruyama, Takashi; Matsumoto, Taro; Abe, Masanori; Yamori, Yukio

    2016-12-01

    Endothelial damage is repaired by endothelial progenitor cells (EPCs), which are pivotal in preventing cardiovascular diseases and prolonging lifespan. The WHO Cardiovascular Diseases and Alimentary Comparison Study demonstrated that dietary taurine and magnesium (Mg) intake suppresses cardiovascular diseases. We herein evaluate the effects of taurine and Mg supplementation on EPC function and oxidative stress in healthy men and spontaneously hypertensive rats (SHRs). Healthy men received taurine (3 g per day) or Mg (340 mg per day) for 2 weeks. SHRs and Wistar-Kyoto (WKY) rats were housed with high-salt drinking water (1% NaCl). The SHRs received 3% taurine solution and/or a high-Mg (600 mg per 100 g) diet for 4 weeks. Their peripheral blood mononuclear cells were separated to quantify EPC colony formation. Oxidative stress markers in their peripheral blood were evaluated using a free radical analytical system and a thiobarbituric acid reactive substance (TBARS) assay. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased free radical levels and TBARS scores in healthy men. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased TBARS scores and free radical levels in SHRs. Nicotinamide adenine dinucleotide phosphate oxidase component mRNA expression was significantly higher in the renal cortex of salt-loaded SHRs than in WKY rats, in which it was suppressed by taurine and Mg supplementation. Taurine and Mg supplementation increased EPC colony formation in healthy men and improved impaired EPC function in SHRs through antioxidation, indicating that the dietary intake of taurine and Mg may prolong lifespan by preventing the progression of cardiovascular diseases.

  17. Hyperglycemia and oxidized-LDL exert a deleterious effect on endothelial progenitor cell migration in type 2 diabetes mellitus.

    Science.gov (United States)

    Hamed, Saher; Brenner, Benjamin; Abassi, Zaid; Aharon, Anat; Daoud, Deeb; Roguin, Ariel

    2010-09-01

    Type 2 diabetes mellitus (DM) patients with coronary artery disease (CAD) have elevated plasma oxidized-LDL (OxLDL) levels and impaired neovascularization. Hyperglycemia and hyperlipidemia impair endothelial progenitor cell (EPC) migration, and endothelial nitric oxide (NO) bioavailability and NO synthase (NOS) activity are essential for EPC migration. Stromal-derived factor-1alpha (SDF1alpha) contributes to EPC mobilization and homing by stimulating the CXC receptor-4 (CXCR4) on the EPC plasmalemma to activate the Pi3K/Akt/eNOS signaling pathway. Therefore, we investigated the effect of high glucose (HG) and OxLDL on the migration and NO bioavailability of EPCs from healthy individuals, and then correlated the findings with those of EPCs from type 2 DM patients with and without CAD. EPCs from 15 healthy and 55 patients were exposed to HG, OxLDL, or both before evaluating EPC count, migration and NO production, and expression of CXCR4 and members of Pi3K/Akt/eNOS signaling cascade. Counts, migration, CXCR4 expression, and NO production were significantly reduced in EPCs from DM and CAD patients compared with that obtained in EPCs from healthy, and were further reduced in DM patients with CAD. The expression of CXCR4 and activation of Pi3K/Akt/eNOS signaling cascade were suppressed in OxLDL- and HG-treated EPCs, and this suppression was exacerbated when EPCs were treated simultaneously with HG and OxLDL. Hyperglycemia and elevated circulating OxLDL in DM patients with CAD severely impair EPC migration. These results suggest that the underlying mechanism for this impaired EPC migration is linked to the CXCR4/Pi3K/Akt/eNOS signaling pathway. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  18. F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

    Science.gov (United States)

    Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

    2016-01-01

    Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

  19. Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

    Science.gov (United States)

    Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

    2017-08-01

    The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

  20. Effect of ambient temperature on the proliferation of brown adipocyte progenitors and endothelial cells during postnatal BAT development in Syrian hamsters.

    Science.gov (United States)

    Nagaya, Kazuki; Okamatsu-Ogura, Yuko; Nio-Kobayashi, Junko; Nakagiri, Shohei; Tsubota, Ayumi; Kimura, Kazuhiro

    2018-04-02

    In Syrian hamsters, brown adipose tissue (BAT) develops postnatally through the proliferation and differentiation of brown adipocyte progenitors. In the study reported here, we investigated how ambient temperature influenced BAT formation in neonatal hamsters. In both hamsters raised at 23 or 30 °C, the interscapular fat changed from white to brown coloration in an age-dependent manner and acquired the typical morphological features of BAT by day 16. However, the expression of uncoupling protein 1, a brown adipocyte marker, and of vascular endothelial growth factor α were lower in the group raised at 30 °C than in that raised at 23 °C. Immunofluorescent staining revealed that the proportion of Ki67-expressing progenitors and endothelial cells was lower in the 30 °C group than in the 23 °C group. These results indicate that warm ambient temperature suppresses the proliferation of brown adipocyte progenitors and endothelial cells and negatively affects the postnatal development of BAT in Syrian hamsters.

  1. Ca2+ signalling in endothelial progenitor cells: a novel means to improve cell-based therapy and impair tumour vascularisation.

    Science.gov (United States)

    Moccia, Francesco; Lodola, Francesco; Dragoni, Silvia; Bonetti, Elisa; Bottino, Cinzia; Guerra, Germano; Laforenza, Umberto; Rosti, Vittorio; Tanzi, Franco

    2014-01-01

    Endothelial progenitor cells (EPCs) have recently been employed in cell-based therapy (CBT) to promote regeneration of ischemic organs, such as heart and limbs. Furthermore, EPCs may sustain tumour vascularisation and provide an additional target for anticancer therapies. CBT is limited by the paucity of cells harvested from peripheral blood and suffers from several pitfalls, including the low rate of engrafted EPCs, whereas classic antiangiogenic treatments manifest a number of side effects and may induce resistance into the patients. CBT will benefit of a better understanding of the signal transduction pathway(s) which drive(s) EPC proliferation, trafficking, and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to impair tumor neovascularisation and improve the therapeutic outcome of antiangiogenic strategies. An increase in intracellular Ca(2+) concentration is the key signal in the regulation of cellular replication, migration, and differentiation. In particular, Ca(2+) signalling may regulate cellcycle progression, due to the Ca(2+)-sensitivity of a number of cycline-dependent kinases, and gene expression, owing to the Ca(2+)-dependence of several transcription factors. Recent work has outlined the role of the so-called store-operated Ca(2+) entry in driving EPC proliferation and migration. Unravelling the mechanisms guiding EPC engraftment into neovessels might supply the biological bases required to improve CBT and anticancer treatments. For example, genetic manipulation of the Ca(2+) signalling machinery could provide a novel approach to increase the extent of limb regeneration or preventing tumour vascularisation by EPCs.

  2. Exercise training-induced different improvement profile of endothelial progenitor cells function in mice with or without myocardial infarction.

    Science.gov (United States)

    Guo, Yuan; Peng, Ran; Liu, Qiong; Xu, Danyan

    2016-10-15

    Neovascularization in response to ischemia after myocardial infarction (MI) has been widely considered as being initiated by endothelial progenitor cells (EPCs). Well-documented evidences in recent years have proved exercise training (ET) improving EPC function. However, whether ET-induced improvement of EPC function under or without ischemic state is different has not been reported. Mice performed ET following an exercise prescription 1week after MI or non-MI surgery respectively. Bone marrow-derived EPCs were isolated at 0day, 3days, 1week, 2weeks, 4weeks, and 8weeks of ET. After 7days cultivation, EPC functions including proliferation, adhesion, migration, and in vitro angiogenesis were measured. AKT/glycogen synthase kinase 3β (GSK3β) signaling pathway was tested by western blotting. EPC function in mice underwent non-MI surgery was attenuated overtime, while ET ameliorated this tendency. EPC function was peaked at 4weeks ET in non-MI surgery mice and maintained with an extended exercise time. Besides, simple ischemia was sufficient to enhanced EPC function, with a maximum at 2weeks of MI surgery. In MI mice, ET further improved EPC function and achieved peak at 2weeks exercise. Furthermore, AKT/GSK3β signaling pathway activation was consistent with EPC function change after ischemia, which was further promoted by 4weeks exercise. ET significantly increased EPC function in mice both with and without MI, but the time points of peak function were different. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Endothelial progenitor cells in mothers of low-birthweight infants: a link between defective placental vascularization and increased cardiovascular risk?

    Science.gov (United States)

    King, Thomas F J; Bergin, David A; Kent, Etaoin M; Manning, Fiona; Reeves, Emer P; Dicker, Patrick; McElvaney, Noel G; Sreenan, Seamus; Malone, Fergal D; McDermott, John H

    2013-01-01

    Offspring birthweight is inversely associated with future maternal cardiovascular mortality, a relationship that has yet to be fully elucidated. Endothelial progenitor cells (EPCs) are thought to play a key role in vasculogenesis, and EPC numbers reflect cardiovascular risk. Our objective was to ascertain whether EPC number or function was reduced in mothers of low-birthweight infants. This was a prospective cohort study in a general antenatal department of a university maternity hospital. Twenty-three mothers of small for gestational age (SGA) infants (birthweight mothers of appropriate for gestational age (AGA) infants (birthweight ≥ 10th centile) were recruited. Maternal EPC number and function, conventional cardiovascular risk markers, and cord blood adiponectin were measured. Median EPC count was lower (294 vs. 367, P = 0.005) and EPC migration was reduced (0.91 vs. 1.59, P < 0.001) in SGA compared with AGA infants, with no difference in EPC adhesion (0.221 vs. 0.284 fluorescence units, P = 0.257). Maternal triglyceride levels were higher in SGA than AGA infants (0.98 vs. 0.78 mmol/liter, P = 0.006), but there was no difference in cholesterol, glucose, insulin, glycosylated hemoglobin, adiponectin, or blood pressure. There was a moderate monotone (increasing) relationship between birthweight and umbilical cord blood adiponectin (r = 0.475, P = 0.005). Giving birth to an SGA infant was associated with lower maternal EPC number and reduced migratory function. Cord blood adiponectin was significantly correlated with birthweight.

  4. Multilayer Membranes of Glycosaminoglycans and Collagen I Biomaterials Modulate the Function and Microvesicle Release of Endothelial Progenitor Cells.

    Science.gov (United States)

    Dai, Bingyan; Pan, Qunwen; Li, Zhanghua; Zhao, Mingyan; Liao, Xiaorong; Wu, Keng; Ma, Xiaotang

    2016-01-01

    Multilayer composite membrane of biomaterials can increase the function of adipose stem cells or osteoprogenitor cells. Recent evidence indicates endothelial progenitor cells (EPCs) and EPCs released microvesicles (MVs) play important roles in angiogenesis and vascular repair. Here, we investigated the effects of biomaterial multilayer membranes of hyaluronic acid (HA) or chondroitin sulfate (CS) and Collagen I (Col I) on the functions and MVs release of EPCs. Layer-by-layer (LBL) technology was applied to construct the multilayer composite membranes. Four types of the membranes constructed by adsorbing either HA or CS and Col I alternatively with different top layers were studied. The results showed that all four types of multilayer composite membranes could promote EPCs proliferation and migration and inhibit cell senility, apoptosis, and the expression of activated caspase-3. Interestingly, these biomaterials increased the release and the miR-126 level of EPCs-MVs. Moreover, the CS-Col I membrane with CS on the top layer showed the most effects on promoting EPCs proliferation, EPCs-MV release, and miR-126 level in EPCs-MVs. In conclusion, HA/CS and Collagen I composed multilayer composite membranes can promote EPCs functions and release of miR-126 riched EPCs-MVs, which provides a novel strategy for tissue repair treatment.

  5. Impact of a cardiac rehabilitation program and inflammatory state on endothelial progenitor cells in acute coronary syndrome patients.

    Science.gov (United States)

    Cesari, Francesca; Marcucci, Rossella; Gori, Anna Maria; Burgisser, Costanza; Francini, Sara; Sofi, Francesco; Gensini, Gian Franco; Abbate, Rosanna; Fattirolli, Francesco

    2013-09-01

    Among the benefits of a cardiac rehabilitation (CR) program for patients after an acute coronary syndrome (ACS) is the mobilization of endothelial progenitor cells (EPCs). However not all patients respond to CR with an increase of EPC. We performed this study to identify the characteristics of patients who will not benefit from an increase of EPCs at the end of a CR program. 112 ACS patients were admitted to a four-week CR program. EPCs, high sensitivity C-reactive protein (hsCRP) and NT-ProBNP levels were determined at the beginning (T1) and at the end (T2) of the CR program. All patients performed a cardiopulmonary exercise test at T1 and at T2. EPCs were defined as CD34+KDR+, CD133+KDR+ and CD34+CD133+KDR+. hsCRP and NT-ProBNP were measured by nephelometric and immunometric method, respectively. At T2, we observed a significant increase of EPCs (p=0.001), VO2 peak, Watt max HDL-cholesterol (pprogram. A CR program determines an increase of EPCs with a decrease of CRP and NT-ProBNP. A different trend for EPCs can be detected among patients correlated to CRP levels and exercise tolerance. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  6. Erythropoietin-enhanced endothelial progenitor cell recruitment in peripheral blood and renal vessels during experimental acute kidney injury in rats.

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    Cakiroglu, Figen; Enders-Comberg, Sora Maria; Pagel, Horst; Rohwedel, Jürgen; Lehnert, Hendrik; Kramer, Jan

    2016-03-01

    Beneficial effects of erythropoietin (EPO) have been reported in acute kidney injury (AKI) when administered prior to induction of AKI. We studied the effects of EPO administration on renal function shortly after ischemic AKI. For this purpose, rats were subjected to renal ischemia for 30 min and EPO was administered at a concentration of 500 U/kg either i.v. as a single shot directly after ischemia or with an additional i.p. dose until 3 days after surgery. The results were compared with AKI rats without EPO application and a sham-operated group. Renal function was assessed by measurement of serum biochemical markers, histological grading, and using an isolated perfused kidney (IPK) model. Furthermore, we performed flow cytometry to analyze the concentration of endothelial progenitor cells (EPCs) in the peripheral blood and renal vessels. Following EPO application, there was only a statistically non-significant tendency of serum creatinine and urea to improve, particularly after daily EPO application. Renal vascular resistance and the renal perfusion rate were not significantly altered. In the histological analysis, acute tubular necrosis was only marginally ameliorated following EPO administration. In summary, we could not demonstrate a significant improvement in renal function when EPO was applied after AKI. Interestingly, however, EPO treatment resulted in a highly significant increase in CD133- and CD34-positive EPC both in the peripheral blood and renal vessels. © 2015 International Federation for Cell Biology.

  7. Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

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    Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

    2017-03-01

    Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  8. Endothelial Cells Promote Expansion of Long‐Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

    Science.gov (United States)

    Gori, Jennifer L.; Butler, Jason M.; Kunar, Balvir; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Norgaard, Zachary K.; Adair, Jennifer E.; Rafii, Shahin

    2016-01-01

    Abstract Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876 PMID:28297579

  9. Aerobic exercise modulation of mental stress-induced responses in cultured endothelial progenitor cells from healthy and metabolic syndrome subjects.

    Science.gov (United States)

    Rocha, Natalia G; Sales, Allan R K; Miranda, Renan L; Silva, Mayra S; Silva, Jemima F R; Silva, Bruno M; Santos, Aline A; Nóbrega, Antonio C L

    2015-02-15

    Numerous studies have demonstrated that exercise acutely prevents the reduction in flow-mediated dilation induced by mental stress in subjects with metabolic syndrome (MetS). However, it is unknown whether a similar effect occurs in endothelial progenitors cells (EPCs). This study investigated whether exercise protects from the deleterious effect of mental stress on cultured EPCs in healthy subjects and those with MetS. Ten healthy subjects (aged 31±2) and ten subjects with MetS (aged 36±2) were enrolled. Subjects underwent a mental stress test, followed immediately by either 40 min of leg cycling or rest across two randomized sessions: mental stress+non-exercise control (MS) and mental stress+exercise (MS+EXE). The Stroop Color-Word Test was used to elicit mental stress. Blood samples were drawn at baseline and following sessions to isolate mononuclear cells. These cells were cultured in fibronectin-coated plates for seven days, and EPCs were identified by immunofluorescence (acLDL(+)/ UEA-I Lectin(+)). All subjects presented similar increases in mean blood pressure and heart rate during the mental stress test (P0.05). The EPC response to MS and MS+EXE was increased in healthy subjects, whereas it was decreased in subjects with MetS (Pexercise session increased EPCs in healthy subjects but did not prevent the EPC reduction induced by mental stress among subjects with MetS. © 2015.

  10. Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.

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    Silvia Dragoni

    Full Text Available BACKGROUND: An increase in the frequency of circulating endothelial colony forming cells (ECFCs, the only subset of endothelial progenitor cells (EPCs truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF. Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs and subjects suffering from renal cellular carcinoma (RCC-ECFCs. SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1. METHODOLOGY/PRINCIPAL FINDINGS: We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs. SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA or physiological (i.e. ATP stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective. CONCLUSIONS: Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3

  11. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

    Science.gov (United States)

    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  12. Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease: an in vitro study.

    Science.gov (United States)

    Kaur, Savneet; Kumar, T R Santhosh; Uruno, Akira; Sugawara, Akira; Jayakumar, Karunakaran; Kartha, Chandrasekharan Cheranellore

    2009-11-01

    Recent studies have reported a marked impairment in the number and functions of endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). In view of an important role of eNOS in angiogenesis, in the present study, we evaluated the effects of eNOS gene transfer in ex vivo expanded EPCs isolated from patients with CAD. The expanded EPCs were transfected with mammalian expression vector pcDNA3.1-eNOS containing the full-length human eNOS gene using lipofectamine. About 35-40% of the eNOS-EPCs had higher expression of eNOS as compared to untransfected EPCs. EPCs transfected with pcDNA3.0-EGFP, the plasmid vector expressing green fluorescent protein (GFP) were used as control. The untransfected, GFP-transfected and eNOS-transfected EPCs were compared in terms of important functional attributes of angiogenesis such as proliferation, migration, differentiation and adhesion/integration into tube-like structures in vitro. Functional studies revealed that in the presence of defined growth conditions, compared to the untransfected and GFP-transfected cells, eNOS-EPCs from patients with CAD have a significant increase in [3H] thymidine-labeled DNA (P < 0.01), migration (14.6 +/- 1.8 and 16.5 +/- 1.9 vs. 23.5 +/- 3.4 cells/field, P < 0.01), ability to differentiate into endothelial-like spindle-shaped cells (46 +/- 4.5 and 56.5 +/- 2.1 vs. 93.2 +/- 6.6 cells/field, P < 0.001) and also incorporation into tube-like structures on the matrigel (GFP-EPCs: 21.25 +/- 2.9 vs. GFP-eNOS-EPCs: 34.5 +/- 5.5 cells/field, P < 0.05). We conclude that eNOS gene transfection is a valuable approach to augment angiogenic properties of ex vivo expanded EPCs and eNOS-modified EPCs may offer significant advantages than EPCs alone in terms of their clinical use in patients with myocardial ischemia.

  13. Higher O-GlcNAc Levels Are Associated with Defects in Progenitor Proliferation and Premature Neuronal Differentiation during in-Vitro Human Embryonic Cortical Neurogenesis

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    Shama Parween

    2017-12-01

    Full Text Available The nutrient responsive O-GlcNAcylation is a dynamic post-translational protein modification found on several nucleocytoplasmic proteins. Previous studies have suggested that hyperglycemia induces the levels of total O-GlcNAcylation inside the cells. Hyperglycemia mediated increase in protein O-GlcNAcylation has been shown to be responsible for various pathologies including insulin resistance and Alzheimer's disease. Since maternal hyperglycemia during pregnancy is associated with adverse neurodevelopmental outcomes in the offspring, it is intriguing to identify the effect of increased protein O-GlcNAcylation on embryonic neurogenesis. Herein using human embryonic stem cells (hESCs as model, we show that increased levels of total O-GlcNAc is associated with decreased neural progenitor proliferation and premature differentiation of cortical neurons, reduced AKT phosphorylation, increased apoptosis and defects in the expression of various regulators of embryonic corticogenesis. As defects in proliferation and differentiation during neurodevelopment are common features of various neurodevelopmental disorders, increased O-GlcNAcylation could be one mechanism responsible for defective neurodevelopmental outcomes in metabolically compromised pregnancies such as diabetes.

  14. Endothelial Progenitor Cell Dysfunction in Myelodysplastic Syndromes: Possible Contribution of a Defective Vascular Niche to Myelodysplasia

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    Luciana Teofili

    2015-05-01

    Full Text Available We set a model to replicate the vascular bone marrow niche by using endothelial colony forming cells (ECFCs, and we used it to explore the vascular niche function in patients with low-risk myelodysplastic syndromes (MDS. Overall, we investigated 56 patients and we observed higher levels of ECFCs in MDS than in healthy controls; moreover, MDS ECFCs were found variably hypermethylated for p15INK4b DAPK1, CDH1, or SOCS1. MDS ECFCs exhibited a marked adhesive capacity to normal mononuclear cells. When normal CD34+ cells were co-cultured with MDS ECFCs, they generated significant lower amounts of CD11b+ and CD41+ cells than in co-culture with normal ECFCs. At gene expression profile, several genes involved in cell adhesion were upregulated in MDS ECFCs, while several members of the Wingless and int (Wnt pathways were underexpressed. Furthermore, at miRNA expression profile, MDS ECFCs hypo-expressed various miRNAs involved in Wnt pathway regulation. The addition of Wnt3A reduced the expression of intercellular cell adhesion molecule-1 on MDS ECFCs and restored the defective expression of markers of differentiation. Overall, our data demonstrate that in low-risk MDS, ECFCs exhibit various primary abnormalities, including putative MDS signatures, and suggest the possible contribution of the vascular niche dysfunction to myelodysplasia.

  15. Aromatic Hydrocarbon Receptor Suppresses Prostate Cancer Bone Metastasis Cells-Induced Vasculogenesis of Endothelial Progenitor Cells under Hypoxia

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    Shuai Huang

    2016-07-01

    Full Text Available Background/Aims: Hypoxia leads to the development of neovascularization in solid tumor by regulating VEGF expression. Aromatic hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with hypoxia-inducible factors 1β (HIF-1β and inhibits the secretion of vascular endothelial growth factor (VEGF. The purpose of this study was to explore whether AHR can suppress hypoxia-induced VEGF production in prostate bone metastasis cells and repress neovascularization in endothelial progenitor cells (EPCs, and, if so, through what mechanisms. Methods: PC-3 or LNCaP cells induced angiogenesis was detected by Matrigel-based tube formation assay, mRNA expression levels was measured by qRT-PCR, VEGF secretion level was determined by ELISA assay, respectively. Results: AHR activation inhibits hypoxia-induced adhesiveness and vasculogenesis of EPCs induced by PC-3 or LNCaP cells under hypoxia. Moreover, AHR activation suppressed hypoxia-induced VEGF production in PC-3 and LNCaP cells (48 ± 14% in PC-3, p = 0.000; 41 ± 14% in LNCaP, p = 0.000 by attenuating HIF-1α and HIF-1β level that in turn diminished the angiogenic ability of EPCs in vitro. Furthermore, we found the mRNA level of hypoxia-inducible factors 1α (HIF-1α (1.54 ± 0.13 fold in PC-3, p = 0.002, 1.62 ± 0.12 fold in LNCaP, p = 0.001 and HIF-1β (1.67 ± 0.23 fold in PC-3, p = 0.007; 1.75 ± 0.26 fold in LNCaP, p=0.008 were upregulated in prostate cancer bone metastasis PC-3 and LNCaP cell lines in response to hypoxia, and revealed that the regulation of VEGF by HIF-1α and HIF-1β was possibly mediated by the activation of phosphatidylinositol 3-kinase pathway. Conclusion: By providing a mechanistic insight into the modulation of neovascularization by AHR ligand, we suggest that AHR ligand has a strong potential of being a new therapeutic agent with applications in the field of bone metastatic prostate cancer.

  16. Data regarding association between serum osteoprotegerin level, numerous of circulating endothelial-derived and mononuclear-derived progenitor cells in patients with metabolic syndrome

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    Alexander E. Berezin

    2016-09-01

    Full Text Available Metabolic syndrome (MetS is defined as cluster of multiple metabolic and cardiovascular (CV abnormalities included abdominal obesity, high-normal blood pressure, dyslipidaemia, and impaired fasting glucose tolerance that exhibits has a growing prevalence worldwide. We investigated whether an elevated level of osteoprotegerin (OPG predicts imbalance between different phenotypes of circulating endothelial (EPCs and mononuclear (MPCs progenitor cells in MetS patients. We have analyzed data regarding dysmetabolic disorder subjects without known CV disease, as well as with known type two diabetes mellitus. All patients have given their informed written consent for participation in the study. This article contains data on the independent predictors of depletion in numerous of circulating EPCs and MPCs in MetS patients. The data are supplemental to our original research article describing detailed associations of elevated OPG level in MetS patients with numerous of EPCs and MPCs beyond traditional CV risk factors. Keywords: Metabolic syndrome, Osteoprotegerin, Circulating endothelial derived progenitor cells, Mononuclear-derived progenitor cells

  17. Pioglitazone Improves In Vitro Viability and Function of Endothelial Progenitor Cells from Individuals with Impaired Glucose Tolerance

    Science.gov (United States)

    Spigoni, Valentina; Picconi, Angela; Cito, Monia; Ridolfi, Valentina; Bonomini, Sabrina; Casali, Chiara; Zavaroni, Ivana; Gnudi, Luigi; Metra, Marco; Dei Cas, Alessandra

    2012-01-01

    Background Evidence suggests that the PPARγ-agonist insulin sensitizer pioglitazone, may provide potential beneficial cardiovascular (CV) effects beyond its anti-hyperglycaemic function. A reduced endothelial progenitor cell (EPC) number is associated with impaired glucose tolerance (IGT) or diabetes, conditions characterised by increased CV risk. Aim To evaluate whether pioglitazone can provide benefit in vitro in EPCs obtained from IGT subjects. Materials and Methods Early and late-outgrowth EPCs were obtained from peripheral blood mononuclear cells of 14 IGT subjects. The in vitro effect of pioglitazone (10 µM) with/without PPARγ-antagonist GW9662 (1 µM) was assessed on EPC viability, apoptosis, ability to form tubular-like structures and pro-inflammatory molecule expression. Results Pioglitazone increased early and late-outgrowth EPC viability, with negligible effects on apoptosis. The capacity of EPCs to form tubular-like structures was improved by pioglitazone in early (mean increase 28%; p = 0.005) and late-outgrowth (mean increase 30%; p = 0.037) EPCs. Pioglitazone reduced ICAM-1 and VCAM-1 adhesion molecule expression in both early (p = 0.001 and p = 0.012 respectively) and late-outgrowth (p = 0.047 and p = 0.048, respectively) EPCs. Similarly, pioglitazone reduced TNFα gene and protein expression in both early (p = 0.034;p = 0.022) and late-outgrowth (p = 0.026;p = 0.017) EPCs compared to control. These effects were prevented by incubation with the PPARγ-antagonist GW9662. Conclusion Pioglitazone exerts beneficial effects in vitro on EPCs isolated from IGT subjects, supporting the potential implication of pioglitazone as a CV protective agents. PMID:23139771

  18. Insulin sensitizers prevent fine particulate matter-induced vascular insulin resistance and changes in endothelial progenitor cell homeostasis.

    Science.gov (United States)

    Haberzettl, Petra; McCracken, James P; Bhatnagar, Aruni; Conklin, Daniel J

    2016-06-01

    Exposure to fine particular matter (PM2.5) increases the risk of developing cardiovascular disease and Type 2 diabetes. Because blood vessels are sensitive targets of air pollutant exposure, we examined the effects of concentrated ambient PM2.5 (CAP) on vascular insulin sensitivity and circulating levels of endothelial progenitor cells (EPCs), which reflect cardiovascular health. We found that CAP exposure for 9 days decreased insulin-stimulated Akt phosphorylation in the aorta of mice maintained on control diet. This change was accompanied by the induction of IL-1β and increases in the abundance of cleaved IL-18 and p10 subunit of Casp-1, consistent with the activation of the inflammasome pathway. CAP exposure also suppressed circulating levels of EPCs (Flk-1(+)/Sca-1(+) cells), while enhancing the bone marrow abundance of these cells. Although similar changes in vascular insulin signaling and EPC levels were observed in mice fed high-fat diet, CAP exposure did not exacerbate diet-induced changes in vascular insulin resistance or EPC homeostasis. Treatment with an insulin sensitizer, metformin or rosiglitazone, prevented CAP-induced vascular insulin resistance and NF-κB and inflammasome activation and restored peripheral blood and bone marrow EPC levels. These findings suggest that PM2.5 exposure induces diet-independent vascular insulin resistance and inflammation and prevents EPC mobilization, and that this EPC mobilization defect could be mediated by vascular insulin resistance. Impaired vascular insulin sensitivity may be an important mechanism underlying PM2.5-induced vascular injury, and pharmacological sensitization to insulin action could potentially prevent deficits in vascular repair and mitigate vascular inflammation due to exposure to elevated levels of ambient air pollution. Copyright © 2016 the American Physiological Society.

  19. Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huiqing; Li, Xiaojing [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Zhao, Yuancong, E-mail: zhaoyc7320@163.com [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Li, Jingan; Chen, Jiang [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Yang, Ping, E-mail: yangping8@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Maitz, Manfred F. [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Max Bergmann Center of Biomaterials Dresden, Leibniz of Polymer Research Dresden, 01069 Dresden (Germany); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2015-08-30

    Graphical abstract: The phospholipid groups of PMMDP can inhibit platele adhesion, and the EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. The catechol groups of PMMDP play a critical role as molecular anchor for balancing the binding between the coating and the substrate. - Highlights: • The uniform coating of PMMDP can be constructed on titanium surface successfully through the catechol groups. • The phospholipid groups of PMMDP can inhibit platele adhesion, fibrinogen denaturation and improve the hydrophilicity of substrate. • The EPCs-specific peptide of the PMMDP showed special recognition and capture for EPCs. - Abstract: A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

  20. Impact of ADMA, endothelial progenitor cells and traditional cardiovascular risk factors on pulse wave velocity among prediabetic individuals

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    Protopsaltis Ioannis

    2012-11-01

    Full Text Available Abstract Background Central arterial stiffness represents a well-established predictor of cardiovascular disease. Decreased circulating endothelial progenitor cells (EPCs, increased asymmetric dimethyl-arginine (ADMA levels, traditional cardiovascular risk factors and insulin resistance have all been associated with increased arterial stiffness. The correlations of novel and traditional cardiovascular risk factors with central arterial stiffness in prediabetic individuals were investigated in the present study. Methods The study population consisted of 53 prediabetic individuals. Individuals were divided into groups of isolated impaired fasting glucose (IFG, isolated impaired glucose tolerance (IGT and combined IGT-IFG. Age, sex, family history of diabetes, smoking history, body mass index (BMI, waist to hip ratio (WHR, waist circumference (WC, blood pressure, lipid profile, levels of high sensitive C-reactive protein (hsCRP, glomerular filtration rate (GFR, and history of antihypertensive or statin therapy were obtained from all participants. Insulin resistance was evaluated using the Homeostatic Model Assessment (HOMA-IR. Carotid -femoral pulse wave velocity was used as an index of arterial stiffness. Circulating EPC count and ADMA serum levels were also determined. Results Among studied individuals 30 (56.6% subjects were diagnosed with isolated IFG, 9 (17% with isolated IGT (17% and 14 with combined IFG-IGT (26.4%. In univariate analysis age, mean blood pressure, fasting glucose, total cholesterol, LDL cholesterol, and ADMA levels positively correlated with pulse-wave velocity while exercise and GFR correlated negatively. EPC count did not correlate with PWV. In multivariate stepwise regression analysis PWV correlated independently and positively with LDL-Cholesterol (low density lipoprotein and ADMA levels and negatively with exercise. Conclusions Elevated ADMA and LDL-C levels are strongly associated with increased arterial stiffness among

  1. PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

    Science.gov (United States)

    Foubert, Philippe; Silvestre, Jean-Sébastien; Souttou, Boussad; Barateau, Véronique; Martin, Coralie; Ebrahimian, Téni G.; Leré-Déan, Carole; Contreres, Jean Olivier; Sulpice, Eric; Levy, Bernard I.; Plouët, Jean; Tobelem, Gérard; Le Ricousse-Roussanne, Sophie

    2007-01-01

    Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy. PMID:17510705

  2. Coenzyme Q10 Attenuates High Glucose-Induced Endothelial Progenitor Cell Dysfunction through AMP-Activated Protein Kinase Pathways

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    Hsiao-Ya Tsai

    2016-01-01

    Full Text Available Coenzyme Q10 (CoQ10, an antiapoptosis enzyme, is stored in the mitochondria of cells. We investigated whether CoQ10 can attenuate high glucose-induced endothelial progenitor cell (EPC apoptosis and clarified its mechanism. EPCs were incubated with normal glucose (5 mM or high glucose (25 mM enviroment for 3 days, followed by treatment with CoQ10 (10 μM for 24 hr. Cell proliferation, nitric oxide (NO production, and JC-1 assay were examined. The specific signal pathways of AMP-activated protein kinase (AMPK, eNOS/Akt, and heme oxygenase-1 (HO-1 were also assessed. High glucose reduced EPC functional activities, including proliferation and migration. Additionally, Akt/eNOS activity and NO production were downregulated in high glucose-stimulated EPCs. Administration of CoQ10 ameliorated high glucose-induced EPC apoptosis, including downregulation of caspase 3, upregulation of Bcl-2, and increase in mitochondrial membrane potential. Furthermore, treatment with CoQ10 reduced reactive oxygen species, enhanced eNOS/Akt activity, and increased HO-1 expression in high glucose-treated EPCs. These effects were negated by administration of AMPK inhibitor. Transplantation of CoQ10-treated EPCs under high glucose conditions into ischemic hindlimbs improved blood flow recovery. CoQ10 reduced high glucose-induced EPC apoptosis and dysfunction through upregulation of eNOS, HO-1 through the AMPK pathway. Our findings provide a potential treatment strategy targeting dysfunctional EPC in diabetic patients.

  3. Coenzyme Q10 Attenuates High Glucose-Induced Endothelial Progenitor Cell Dysfunction through AMP-Activated Protein Kinase Pathways

    Science.gov (United States)

    Tsai, Hsiao-Ya; Lin, Chih-Pei; Huang, Po-Hsun; Li, Szu-Yuan; Chen, Jia-Shiong; Lin, Feng-Yen; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Coenzyme Q10 (CoQ10), an antiapoptosis enzyme, is stored in the mitochondria of cells. We investigated whether CoQ10 can attenuate high glucose-induced endothelial progenitor cell (EPC) apoptosis and clarified its mechanism. EPCs were incubated with normal glucose (5 mM) or high glucose (25 mM) enviroment for 3 days, followed by treatment with CoQ10 (10 μM) for 24 hr. Cell proliferation, nitric oxide (NO) production, and JC-1 assay were examined. The specific signal pathways of AMP-activated protein kinase (AMPK), eNOS/Akt, and heme oxygenase-1 (HO-1) were also assessed. High glucose reduced EPC functional activities, including proliferation and migration. Additionally, Akt/eNOS activity and NO production were downregulated in high glucose-stimulated EPCs. Administration of CoQ10 ameliorated high glucose-induced EPC apoptosis, including downregulation of caspase 3, upregulation of Bcl-2, and increase in mitochondrial membrane potential. Furthermore, treatment with CoQ10 reduced reactive oxygen species, enhanced eNOS/Akt activity, and increased HO-1 expression in high glucose-treated EPCs. These effects were negated by administration of AMPK inhibitor. Transplantation of CoQ10-treated EPCs under high glucose conditions into ischemic hindlimbs improved blood flow recovery. CoQ10 reduced high glucose-induced EPC apoptosis and dysfunction through upregulation of eNOS, HO-1 through the AMPK pathway. Our findings provide a potential treatment strategy targeting dysfunctional EPC in diabetic patients. PMID:26682233

  4. Increased expression of microRNA-221 inhibits PAK1 in endothelial progenitor cells and impairs its function via c-Raf/MEK/ERK pathway

    International Nuclear Information System (INIS)

    Zhang, Xiaoping; Mao, Haian; Chen, Jin-yuan; Wen, Shengjun; Li, Dan; Ye, Meng; Lv, Zhongwei

    2013-01-01

    Highlights: ► MicroRNA-221 is upregulated in the endothelial progenitor cells of atherosclerosis patients. ► PAK1 is a direct target of microRNA-221. ► MicroRNA-221 inhibits EPCs proliferation through c-Raf/MEK/ERK pathway. -- Abstract: Coronary artery disease (CAD) is associated with high mortality and occurs via endothelial injury. Endothelial progenitor cells (EPCs) restore the integrity of the endothelium and protect it from atherosclerosis. In this study, we compared the expression of microRNAs (miRNAs) in EPCs in atherosclerosis patients and normal controls. We found that miR-221 expression was significantly up-regulated in patients compared with controls. We predicted and identified p21/Cdc42/Rac1-activated kinase 1 (PAK1) as a novel target of miR-221 in EPCs. We also demonstrated that miR-221 targeted a putative binding site in the 3′UTR of PAK1, and absence of this site was inversely associated with miR-221 expression in EPCs. We confirmed this relationship using a luciferase reporter assay. Furthermore, overexpression of miR-221 in EPCs significantly decreased EPC proliferation, in accordance with the inhibitory effects induced by decreased PAK1. Overall, these findings demonstrate that miR-221 affects the MEK/ERK pathway by targeting PAK1 to inhibit the proliferation of EPCs

  5. Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

    Science.gov (United States)

    Nih, Lina R; Deroide, Nicolas; Leré-Déan, Carole; Lerouet, Dominique; Soustrat, Mathieu; Levy, Bernard I; Silvestre, Jean-Sébastien; Merkulova-Rainon, Tatiana; Pocard, Marc; Margaill, Isabelle; Kubis, Nathalie

    2012-04-01

    Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  6. Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

    Science.gov (United States)

    Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

    2017-11-01

    Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p1). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p1) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p1). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The Postischemic Environment Differentially Impacts Teratoma or Tumor Formation After Transplantation of Human Embryonic Stem Cell-Derived Neural Progenitors

    Czech Academy of Sciences Publication Activity Database

    Seminatore, CH.; Polentes, J.; Ellman, D.; Kozubenko, Nataliya; Itier, V.; Tine, S.; Tritschler, L.; Brenot, M.; Guidou, E.; Blondeau, J.; Lhuillier, M.; Bugi, A.; Aubry, L.; Jendelová, Pavla; Syková, Eva; Perrier, A. L.; Finsen, B.; Onteniente, B.

    2010-01-01

    Roč. 41, č. 1 (2010), s. 153-159 ISSN 0039-2499 Institutional research plan: CEZ:AV0Z50390703 Keywords : brain transplantation * human embryonic stem cells * neural differentiation Subject RIV: FH - Neurology Impact factor: 5.756, year: 2010

  8. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery

    Czech Academy of Sciences Publication Activity Database

    Ratajczak, J.; Miekus, K.; Kucia, M.; Zhang, J.; Reca, R.; Dvořák, Petr; Rtajczak, M.Z.

    2006-01-01

    Roč. 20, - (2006), s. 847-856 ISSN 0887-6924 R&D Projects: GA ČR GA301/03/1122 Institutional research plan: CEZ:AV0Z50390512 Keywords : Microvesicles * Embryonic stem cells * Stem cell expansion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.146, year: 2006

  9. Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing.

    Science.gov (United States)

    Wang, Chenggui; Wang, Qingqing; Gao, Wendong; Zhang, Zengjie; Lou, Yiting; Jin, Haiming; Chen, Xiaofeng; Lei, Bo; Xu, Huazi; Mao, Cong

    2018-03-15

    Wound therapy with a rapid healing performance remains a critical clinical challenge. Cellular delivery is considered to be a promising approach to improve the efficiency of healing, yet problems such as compromised cell viability and functionality arise due to the inefficient delivery. Here, we report the efficient delivery of endothelial progenitor cells (EPCs) with a bioactive nanofibrous scaffold (composed of collagen and polycaprolactone and bioactive glass nanoparticles, CPB) for enhancing wound healing. Under the stimulation of CPB nanofibrous system, the viability and angiogenic ability of EPCs were significantly enhanced through the activation of Hif-1α/VEGF/SDF-1α signaling. In vivo, CPB/EPC constructs significantly enhanced the formation of high-density blood vessels by greatly upregulating the expressions of Hif-1α, VEGF, and SDF-1α. Moreover, owing to the increased local delivery of cells and fast neovascularization within the wound site, cell proliferative activity, granulation tissue formation, and collagen synthesis and deposition were greatly promoted by CPB/EPC constructs resulting in rapid re-epithelialization and regeneration of skin appendages. As a result, the synergistic enhancement of wound healing was observed from CPB/EPC constructs, which suggests the highly efficient delivery of EPCs. CPB/EPC constructs may become highly competitive cell-based therapeutic products for efficient impaired wound healing application. This study may also provide a novel strategy to develop bioactive cell therapy constructs for angiogenesis-related regenerative medicine. This paper reported a highly efficient local delivery of EPCs using bioactive glass-based CPB nanofibrous scaffold for enhancing angiogenesis and wound regeneration. In vitro study showed that CPB can promote the proliferation, migration, and tube formation of EPCs through upregulation of the Hif-1α/VEGF/SDF-1α signaling pathway, indicating that the bioactivity and angiogenic ability of

  10. Assessments of proliferation capacity and viability of New Zealand rabbit peripheral blood endothelial progenitor cells labeled with superparamagnetic particles.

    Science.gov (United States)

    Mai, Xiao-Li; Ma, Zhan-Long; Sun, Jun-Hui; Ju, Sheng-Hong; Ma, Ming; Teng, Gao-Jun

    2009-01-01

    Magnetic resonance imaging (MRI) has proven to be effective in tracking the distribution of transplanted stem cells to target organs by way of labeling cells with superparamagnetic iron oxide particles (SPIO). However, the effect of SPIO upon labeled cells is still unclear on a cellular level. With this study, the proliferation and viability of New Zealand rabbit peripheral blood endothelial progenitor cells (EPCs) labeled with SPIO were evaluated and in vitro images were obtained using a 1.5 T MR scanner. Mononuclear cells (MNCs) were isolated from peripheral blood of the adult New Zealand rabbit and cultured in fibronectin-coated culture flasks, in which EPCs were identified from cell morphology, outgrowth characteristics, and internalization of DiI-Ac-LDL and binding to FITC-UEA I. EPCs were incubated with the self-synthesized poly-L-lysine-conjugated SPIO (PLL-SPIO) particles in a range of concentrations. The prevalence of iron-containing vesicles or endosomes in the cytoplasm of labeled cells was confirmed with Prussian blue staining and transmission electron microscopy. Tetrazolium salt (MTT) assay, cell apoptosis, and cycle detection were assessed to evaluate proliferation and function of various concentrations, magnetically labeled EPCs. The quantity of iron per cell was determined by atomic absorption spectrometry. The cells underwent MRI with different sequences. The result showed that rabbit EPCs were efficiently labeled with the home synthesized PLL-SPIO. There was found to be no statistically significant difference in the MTT values of light absorption measured on the third and fifth days. Between labeled and unlabeled cells, there were also no aberrations found in the cell cycles, apoptosis, or growth curves. The atomic absorption spectrophotometer showed that the intracellular content of Fe decreased as more time elapsed after labeling. The labeled EPCs demonstrated a loss of MRI signal intensity (SI) when compared with the SI of unlabeled cells

  11. Lipid lowering and HDL raising gene transfer increase endothelial progenitor cells, enhance myocardial vascularity, and improve diastolic function.

    Directory of Open Access Journals (Sweden)

    Stephanie C Gordts

    Full Text Available BACKGROUND: Hypercholesterolemia and low high density lipoprotein (HDL cholesterol contribute to coronary heart disease but little is known about their direct effects on myocardial function. Low HDL and raised non-HDL cholesterol levels carried increased risk for heart failure development in the Framingham study, independent of any association with myocardial infarction. The objective of this study was to test the hypothesis that increased endothelial progenitor cell (EPC number and function after lipid lowering or HDL raising gene transfer in C57BL/6 low density lipoprotein receptor deficient (LDLr(-/- mice may be associated with an enhanced relative vascularity in the myocardium and an improved cardiac function. METHODOLOGY/PRINCIPAL FINDINGS: Lipid lowering and HDL raising gene transfer were performed using the E1E3E4-deleted LDLr expressing adenoviral vector AdLDLr and the human apolipoprotein A-I expressing vector AdA-I, respectively. AdLDLr transfer in C57BL/6 LDLr(-/- mice resulted in a 2.0-fold (p<0.05 increase of the circulating number of EPCs and in an improvement of EPC function as assessed by ex vivo EPC migration and EPC adhesion. Capillary density and relative vascularity in the myocardium were 28% (p<0.01 and 22% (p<0.05 higher, respectively, in AdLDLr mice compared to control mice. The peak rate of isovolumetric relaxation was increased by 12% (p<0.05 and the time constant of isovolumetric relaxation was decreased by 14% (p<0.05 after AdLDLr transfer. Similarly, HDL raising gene transfer increased EPC number and function and raised both capillary density and relative vascularity in the myocardium by 24% (p<0.05. The peak rate of isovolumetric relaxation was increased by 16% (p<0.05 in AdA-I mice compared to control mice. CONCLUSIONS/SIGNIFICANCE: Both lipid lowering and HDL raising gene transfer have beneficial effects on EPC biology, relative myocardial vascularity, and diastolic function. These findings raise concerns over the

  12. Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

    Directory of Open Access Journals (Sweden)

    Hui Shi

    2018-04-01

    Full Text Available AIM: To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells (EPCs in a mouse model of laser-induced retinal injury. METHODS: EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes: 5-(and-6-carboxyfluorescein diacetate succinimidyl ester (CFSE, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein (DiI-AcLDL, and green fluorescent protein (GFP. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo. RESULTS: EPCs labeled with CFSE and DiI-AcLDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and DiI-AcLDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina. CONCLUSION: The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and DiI-AcLDL are suitable for short-term EPC-labeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

  13. Rapid synthesis of water-dispersible superparamagnetic iron oxide nanoparticles by a microwave-assisted route for safe labeling of endothelial progenitor cells.

    Science.gov (United States)

    Carenza, Elisa; Barceló, Verónica; Morancho, Anna; Montaner, Joan; Rosell, Anna; Roig, Anna

    2014-08-01

    We synthesize highly crystalline citrate-coated iron oxide superparamagnetic nanoparticles that are stable and readily dispersible in water by an extremely fast microwave-assisted route and investigate the uptake of magnetic nanoparticles by endothelial cells. Nanoparticles form large aggregates when added to complete endothelial cell medium. The size of the aggregates was controlled by adjusting the ionic strength of the medium. The internalization of nanoparticles into endothelial cells was then investigated by transmission electron microscopy, magnetometry and chemical analysis, together with cell viability assays. Interestingly, a sevenfold more efficient uptake was found for systems with larger nanoparticle aggregates, which also showed significantly higher magnetic resonance imaging effectiveness without compromising cell viability and functionality. We are thus presenting an example of a straightforward microwave synthesis of citrate-coated iron oxide nanoparticles for safe endothelial progenitor cell labeling and good magnetic resonance cell imaging with potential application for magnetic cell guidance and in vivo cell tracking. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Endothelial progenitor cells (EPCs) in ageing and age-related diseases: How currently available treatment modalities affect EPC biology, atherosclerosis, and cardiovascular outcomes.

    Science.gov (United States)

    Altabas, Velimir; Altabas, Karmela; Kirigin, Lora

    2016-10-01

    Endothelial progenitor cells (EPCs) are mononuclear cells that circulate in the blood and are derived from different tissues, expressing cell surface markers that are similar to mature endothelial cells. The discovery of EPCs has lead to new insights in vascular repair and atherosclerosis and also a new theory for ageing. EPCs from the bone marrow and some other organs aid in vascular repair by migrating to distant vessels where they differentiate into mature endothelial cells and replace old and injured endothelial cells. The ability of EPCs to repair vascular damage depends on their number and functionality. Currently marketed drugs used in a variety of diseases can modulate these characteristics. In this review, the effect of currently available treatment options for cardiovascular and metabolic disorders on EPC biology will be discussed. The various EPC-based therapies that will be discussed include lipid-lowering agents, antihypertensive agents, antidiabetic drugs, phosphodiesteraze inhibitors, hormones, as well as EPC capturing stents. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

    2016-06-03

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  16. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    International Nuclear Information System (INIS)

    Kawaguchi, Manami; Kitajima, Kenji; Kanokoda, Mai; Suzuki, Hidenori; Miyashita, Kazuya; Nakajima, Marino; Nuriya, Hideko; Kasahara, Kohji; Hara, Takahiko

    2016-01-01

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit − Tie2 − CD41 + Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41 + CD42b + CD61 + platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit − Tie2 − CD41 + . •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  17. Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

    Science.gov (United States)

    Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

    2014-04-01

    The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

  18. Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

    2018-03-15

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically

  19. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

    Science.gov (United States)

    Rezania, Alireza; Bruin, Jennifer E; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

    2013-11-01

    Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm

  20. Revocation of European patent for neural progenitors highlights patent challenges for inventions relating to human embryonic stem cells.

    Science.gov (United States)

    Rigby, Barbara

    2013-11-01

    Cells derived from human embryonic stem cells have great therapeutic potential. Patents are key to allowing companies that develop methods of generating such cells to recuperate their investment. However, in Europe, inventions relating to the use of human embryos for commercial purposes are excluded from patentability on moral grounds. The scope of this morality exclusion was recently tested before Germany's highest court and before the European Patent Office (EPO), with diverging results. The decision by the EPO's Opposition Division to revoke EP1040185 relating to neural precursors and methods for their generation has received a mixed reception. The decision has very recently been appealed, and the outcome of this Appeal should provide more definitive guidance on the scope of the morality exclusion.

  1. Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

    Science.gov (United States)

    Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

    2010-10-01

    The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

  2. Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

    Directory of Open Access Journals (Sweden)

    Ingo Ahrens

    Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

  3. Vildagliptin, but not glibenclamide, increases circulating endothelial progenitor cell number: a 12-month randomized controlled trial in patients with type 2 diabetes.

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    Dei Cas, Alessandra; Spigoni, Valentina; Cito, Monia; Aldigeri, Raffaella; Ridolfi, Valentina; Marchesi, Elisabetta; Marina, Michela; Derlindati, Eleonora; Aloe, Rosalia; Bonadonna, Riccardo C; Zavaroni, Ivana

    2017-02-23

    Fewer circulating endothelial progenitor cells (EPCs) and increased plasma (C-term) stromal cell-derived factor 1α (SDF-1α), a substrate of DPP-4, are biomarkers, and perhaps mediators, of cardiovascular risk and mortality. Short-term/acute treatment with DPP-4 inhibitors improve EPC bioavailability; however, long-term effects of DPP-4i on EPCs bioavailability/plasma (C-term) SDF-1α are unknown. Randomized (2:1) open-label trial to compare the effects of vildagliptin (V) (100 mg/day) vs glibenclamide (G) (2.5 mg bid to a maximal dose of 5 mg bid) on circulating EPC levels at 4 and 12 months of treatment in 64 patients with type 2 diabetes in metformin failure. At baseline, and after 4 and 12 months, main clinical/biohumoral parameters, inflammatory biomarkers, concomitant therapies, EPC number (CD34 + /CD133 + /KDR + /10 6 cytometric events) and plasma (C-term) SDF-1α (R&D system) were assessed. Baseline characteristics were comparable in the two groups. V and G similarly and significantly (p < 0.0001) improved glucose control. At 12 months, V significantly increased EPC number (p < 0.05) and significantly reduced (C-term) SDF-1α plasma levels (p < 0.01) compared to G, with no differences in inflammatory biomarkers. V exerts a long-term favorable effect on EPC and (C-term) SDF-1α levels at glucose equipoise, thereby implying a putative beneficial effect on vascular integrity. Trial registration Clinical Trials number: NCT01822548; name: Effect of Vildagliptin vs. Glibenclamide on Circulating Endothelial Progenitor Cell Number Type 2 Diabetes. Registered 28 March, 2013.

  4. Transplantation of rat embryonic stem cell-derived retinal progenitor cells preserves the retinal structure and function in rat retinal degeneration.

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    Qu, Zepeng; Guan, Yuan; Cui, Lu; Song, Jian; Gu, Junjie; Zhao, Hanzhi; Xu, Lei; Lu, Lixia; Jin, Ying; Xu, Guo-Tong

    2015-11-09

    Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. We have successfully differentiated

  5. Surface modification of a polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer as a stent coating for enhanced capture of endothelial progenitor cells.

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    Tan, Aaron; Farhatnia, Yasmin; Goh, Debbie; G, Natasha; de Mel, Achala; Lim, Jing; Teoh, Swee-Hin; Malkovskiy, Andrey V; Chawla, Reema; Rajadas, Jayakumar; Cousins, Brian G; Hamblin, Michael R; Alavijeh, Mohammad S; Seifalian, Alexander M

    2013-12-01

    An unmet need exists for the development of next-generation multifunctional nanocomposite materials for biomedical applications, particularly in the field of cardiovascular regenerative biology. Herein, we describe the preparation and characterization of a novel polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) nanocomposite polymer with covalently attached anti-CD34 antibodies to enhance capture of circulating endothelial progenitor cells (EPC). This material may be used as a new coating for bare metal stents used after balloon angioplasty to improve re-endothelialization. Biophysical characterization techniques were used to assess POSS-PCU and its subsequent functionalization with anti-CD34 antibodies. Results indicated successful covalent attachment of anti-CD34 antibodies on the surface of POSS-PCU leading to an increased propensity for EPC capture, whilst maintaining in vitro biocompatibility and hemocompatibility. POSS-PCU has already been used in 3 first-in-man studies, as a bypass graft, lacrimal duct and a bioartificial trachea. We therefore postulate that its superior biocompatibility and unique biophysical properties would render it an ideal candidate for coating medical devices, with stents as a prime example. Taken together, anti-CD34 functionalized POSS-PCU could form the basis of a nano-inspired polymer platform for the next generation stent coatings.

  6. WISP-3 inhibition of miR-452 promotes VEGF-A expression in chondrosarcoma cells and induces endothelial progenitor cells angiogenesis.

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    Lin, Chih-Yang; Tzeng, Huey-En; Li, Te-Mao; Chen, Hsien-Te; Lee, Yi; Yang, Yi-Chen; Wang, Shih-Wei; Yang, Wei-Hung; Tang, Chih-Hsin

    2017-06-13

    Chondrosarcoma is the second most prevalent general primary tumor of bone following osteosarcoma. Chondrosarcoma development may be linked to angiogenesis, which is principally elicited by vascular endothelial growth factor-A (VEGF-A). VEGF-A level has been recognized as a prognostic marker in angiogenesis. WNT1-inducible signaling pathway protein-3 (WISP)-3/CCN6 belongs to the CCN family and is involved in regulating several cellular functions, including cell proliferation, differentiation, and migration. Nevertheless, the effect of WISP-3 on VEGF-A production and angiogenesis in human chondrosarcoma remains largely unknown. This current study shows that WISP-3 promoted VEGF-A production and induced angiogenesis of human endothelial progenitor cells. Moreover, WISP-3-enhanced VEGF-A expression and angiogenesis involved the c-Src and p38 signaling pathways, while miR-452 expression was negatively affected by WISP-3 via the c-Src and p38 pathways. Our results illustrate the clinical significance of WISP-3, VEGF-A and miR-452 in human chondrosarcoma patients. WISP-3 may illustrate a novel therapeutic target in the metastasis and angiogenesis of chondrosarcoma.

  7. MicroRNA-126 Priming Enhances Functions of Endothelial Progenitor Cells under Physiological and Hypoxic Conditions and Their Therapeutic Efficacy in Cerebral Ischemic Damage

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    Qunwen Pan

    2018-01-01

    Full Text Available Endothelial progenitor cells (EPCs have shown the potential for treating ischemic stroke (IS, while microRNA-126 (miR-126 is reported to have beneficial effects on endothelial function and angiogenesis. In this study, we investigated the effects of miR-126 overexpression on EPCs and explore the efficacy of miR-126-primed EPCs (EPCmiR-126 in treating IS. The effects of miR-126 overexpression on EPC proliferation, migratory, tube formation capacity, reactive oxygen species (ROS production, and nitric oxide (NO generation were determined. In in vivo study, the effects of EPCmiR-126 on the cerebral blood flow (CBF, neurological deficit score (NDS, infarct volume, cerebral microvascular density (cMVD, and angiogenesis were determined. Moreover, the levels of circulating EPCs (cEPCs and their contained miR-126 were measured. We found (1 miR-126 overexpression promoted the proliferation, migration, and tube formation abilities of EPCs; decreased ROS; and increased NO production of EPCs via activation of PI3K/Akt/eNOS pathway; (2 EPCmiR-126 was more effective than EPCs in attenuating infarct volume and NDS and enhancing cMVD, CBF, and angiogenesis; and (3 infusion of EPCmiR-126 increased the number and the level of miR-126 in cEPCs. Our data indicate that miR-126 overexpression enhanced the function of EPCs in vitro and in vivo.

  8. The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti implants.

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    Achneck, Hardean E; Jamiolkowski, Ryan M; Jantzen, Alexandra E; Haseltine, Justin M; Lane, Whitney O; Huang, Jessica K; Galinat, Lauren J; Serpe, Michael J; Lin, Fu-Hsiung; Li, Madison; Parikh, Amar; Ma, Liqiao; Chen, Tao; Sileshi, Bantayehu; Milano, Carmelo A; Wallace, Charles S; Stabler, Thomas V; Allen, Jason D; Truskey, George A; Lawson, Jeffrey H

    2011-01-01

    Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  9. Use of autologous blood-derived endothelial progenitor cells at point-of-care to protect against implant thrombosis in a large animal model.

    Science.gov (United States)

    Jantzen, Alexandra E; Lane, Whitney O; Gage, Shawn M; Jamiolkowski, Ryan M; Haseltine, Justin M; Galinat, Lauren J; Lin, Fu-Hsiung; Lawson, Jeffrey H; Truskey, George A; Achneck, Hardean E

    2011-11-01

    Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine (n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  11. Molecular functions of the LIM-homeobox transcription factor Lhx2 in hematopoietic progenitor cells derived from mouse embryonic stem cells.

    Science.gov (United States)

    Kitajima, Kenji; Kawaguchi, Manami; Iacovino, Michelina; Kyba, Michael; Hara, Takahiko

    2013-12-01

    We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells. © AlphaMed Press.

  12. microRNA 126 inhibits the transition of endothelial progenitor cells to mesenchymal cells via the PIK3R2-PI3K/Akt signalling pathway.

    Science.gov (United States)

    Zhang, Junfeng; Zhang, Zongqi; Zhang, David Y; Zhu, Jianbing; Zhang, Tiantian; Wang, Changqian

    2013-01-01

    Endothelial progenitor cells (EPCs) are capable of proliferating and differentiating into mature endothelial cells, and they have been considered as potential candidates for coronary heart disease therapy. However, the transition of EPCs to mesenchymal cells is not fully understood. This study aimed to explore the role of microRNA 126 (miR-126) in the endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor beta 1 (TGFβ1). EndMT of rat bone marrow-derived EPCs was induced by TGFβ1 (5 ng/mL) for 7 days. miR-126 expression was depressed in the process of EPC EndMT. The luciferase reporter assay showed that the PI3K regulatory subunit p85 beta (PIK3R2) was a direct target of miR-126 in EPCs. Overexpression of miR-126 by a lentiviral vector (lenti-miR-126) was found to downregulate the mRNA expression of mesenchymal cell markers (α-SMA, sm22-a, and myocardin) and to maintain the mRNA expression of progenitor cell markers (CD34, CD133). In the cellular process of EndMT, there was an increase in the protein expression of PIK3R2 and the nuclear transcription factors FoxO3 and Smad4; PI3K and phosphor-Akt expression decreased, a change that was reversed markedly by overexpression of miR-126. Furthermore, knockdown of PIK3R2 gene expression level showed reversed morphological changes of the EPCs treated with TGFβ1, thereby giving the evidence that PIK3R2 is the target gene of miR-126 during EndMT process. These results show that miR-126 targets PIK3R2 to inhibit EPC EndMT and that this process involves regulation of the PI3K/Akt signalling pathway. miR-126 has the potential to be used as a biomarker for the early diagnosis of intimal hyperplasia in cardiovascular disease and can even be a therapeutic tool for treating cardiovascular diseases mediated by the EndMT process.

  13. microRNA 126 inhibits the transition of endothelial progenitor cells to mesenchymal cells via the PIK3R2-PI3K/Akt signalling pathway.

    Directory of Open Access Journals (Sweden)

    Junfeng Zhang

    Full Text Available AIMS: Endothelial progenitor cells (EPCs are capable of proliferating and differentiating into mature endothelial cells, and they have been considered as potential candidates for coronary heart disease therapy. However, the transition of EPCs to mesenchymal cells is not fully understood. This study aimed to explore the role of microRNA 126 (miR-126 in the endothelial-to-mesenchymal transition (EndMT induced by transforming growth factor beta 1 (TGFβ1. METHODS AND RESULTS: EndMT of rat bone marrow-derived EPCs was induced by TGFβ1 (5 ng/mL for 7 days. miR-126 expression was depressed in the process of EPC EndMT. The luciferase reporter assay showed that the PI3K regulatory subunit p85 beta (PIK3R2 was a direct target of miR-126 in EPCs. Overexpression of miR-126 by a lentiviral vector (lenti-miR-126 was found to downregulate the mRNA expression of mesenchymal cell markers (α-SMA, sm22-a, and myocardin and to maintain the mRNA expression of progenitor cell markers (CD34, CD133. In the cellular process of EndMT, there was an increase in the protein expression of PIK3R2 and the nuclear transcription factors FoxO3 and Smad4; PI3K and phosphor-Akt expression decreased, a change that was reversed markedly by overexpression of miR-126. Furthermore, knockdown of PIK3R2 gene expression level showed reversed morphological changes of the EPCs treated with TGFβ1, thereby giving the evidence that PIK3R2 is the target gene of miR-126 during EndMT process. CONCLUSIONS: These results show that miR-126 targets PIK3R2 to inhibit EPC EndMT and that this process involves regulation of the PI3K/Akt signalling pathway. miR-126 has the potential to be used as a biomarker for the early diagnosis of intimal hyperplasia in cardiovascular disease and can even be a therapeutic tool for treating cardiovascular diseases mediated by the EndMT process.

  14. Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding.

    Science.gov (United States)

    Elsheikh, E; Andersson, E; Sylvén, C; Ericzon, B-G; Palmblad, J; Mints, M

    2014-01-01

    Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P Market Insurance. The authors have no conflict of interest to declare.

  15. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Effects of a 12-week alpine skiing intervention on endothelial progenitor cells, peripheral arterial tone and endothelial biomarkers in the elderly

    DEFF Research Database (Denmark)

    Niederseer, David; Steidle-Kloc, Eva; Mayr, Matthias

    2016-01-01

    : +0.18±0.76) and CG (-0.39±0.85; p=0.045), as did homocysteine (IG: -1.3±1.3μmol/l; CG: -0.4±1.4μmol/l; p=0.037) while other endothelial biomarkers remained essentially unchanged. CONCLUSIONS: This study shows that skiing induces several beneficial effects on markers of atherogenesis including EPCs......, peripheral arterial tone and homocysteine. Our findings suggest that recreational alpine skiing may serve as a further mode of preventive exercise training, which might result in improved compliance with current recommendations....

  17. LncRNA-AK131850 Sponges MiR-93-5p in Newborn and Mature Osteoclasts to Enhance the Secretion of Vascular Endothelial Growth Factor a Promoting Vasculogenesis of Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Hongyu Quan

    2018-03-01

    Full Text Available Background/Aims: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. Methods: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8 analysis, quantitative real-time polymerase chain reaction (qRT-PCR analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs, respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA, fluorescence in situ hybridization (FISH and luciferase reporter assay were carried out to dissect molecular mechanisms. Results: In this study, we found that newborn OCs (N-OC and mature OCs (M-OC during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs. Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa, were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. Conclusion: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in

  18. Effect of increased exercise in school children on physical fitness and endothelial progenitor cells: a prospective randomized trial.

    Science.gov (United States)

    Walther, Claudia; Gaede, Luise; Adams, Volker; Gelbrich, Götz; Leichtle, Alexander; Erbs, Sandra; Sonnabend, Melanie; Fikenzer, Kati; Körner, Antje; Kiess, Wieland; Bruegel, Mathias; Thiery, Joachim; Schuler, Gerhard

    2009-12-01

    The aim of this prospective, randomized study was to examine whether additional school exercise lessons would result in improved peak oxygen uptake (primary end point) and body mass index-standard deviation score, motor and coordinative abilities, circulating progenitor cells, and high-density lipoprotein cholesterol (major secondary end points). Seven sixth-grade classes (182 children, aged 11.1+/-0.7 years) were randomized to an intervention group (4 classes with 109 students) with daily school exercise lessons for 1 year and a control group (3 classes with 73 students) with regular school sports twice weekly. The significant effects of intervention estimated from ANCOVA adjusted for intraclass correlation were the following: increase of peak o(2) (3.7 mL/kg per minute; 95% confidence interval, 0.3 to 7.2) and increase of circulating progenitor cells evaluated by flow cytometry (97 cells per 1 x 10(6) leukocytes; 95% confidence interval, 13 to 181). No significant difference was seen for body mass index-standard deviation score (-0.08; 95% confidence interval, -0.28 to 0.13); however, there was a trend to reduction of the prevalence of overweight and obese children in the intervention group (from 12.8% to 7.3%). No treatment effect was seen for motor and coordinative abilities (4; 95% confidence interval, -1 to 8) and high-density lipoprotein cholesterol (0.03 mmol/L; 95% confidence interval, -0.08 to 0.14). Regular physical activity by means of daily school exercise lessons has a significant positive effect on physical fitness (o(2)max). Furthermore, the number of circulating progenitor cells can be increased, and there is a positive trend in body mass index-standard deviation score reduction and motor ability improvement. Therefore, we conclude that primary prevention by means of increasing physical activity should start in childhood. URL: http://www.clinicaltrials.gov. Identifier: NCT00176371.

  19. Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress.

    Science.gov (United States)

    Chen, Jianfei; Song, Minbao; Yu, Shiyong; Gao, Pan; Yu, Yang; Wang, Hong; Huang, Lan

    2010-02-01

    Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 microg/ml). A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the

  20. Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro.

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    Peters, Erica B; Liu, Betty; Christoforou, Nicolas; West, Jennifer L; Truskey, George A

    2015-10-01

    Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-β, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and similar effects as HVPs upon total EPC tubule length (p = 0.95) and number of branch points (p = 0.93). In a reductionist coculture system, MNCs served as pro-angiogenic mural cells by supporting EPC network formation to a significantly greater extent than HVP cocultures, by day 14 of coculture, as evidenced through EPC total tubule length (p < 0.0001) and number of branch points (p < 0.0001). Our findings are significant as we demonstrate mural cell progenitors can be isolated from umbilical cord blood and develop culture conditions to support their use in microvascular tissue engineering applications.

  1. p38 MAPK and JNK antagonistically control senescence and cytoplasmic p16INK4A expression in doxorubicin-treated endothelial progenitor cells.

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    Paolo Spallarossa

    Full Text Available Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A, suggests that doxorubicin-induced p16( INK4A does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

  2. Progenitor Epithelium

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    Marty-Santos, Leilani

    2015-01-01

    Insulin-producing β cells within the vertebrate fetal pancreas acquire their fate in a step-wise manner. Whereas the intrinsic factors dictating the transcriptional or epigenetic status of pancreatic lineages have been intensely examined, less is known about cell–cell interactions that might constitute a niche for the developing β cell lineage. It is becoming increasingly clear that understanding and recapitulating these steps may instruct in vitro differentiation of embryonic stem cells and/or therapeutic regeneration. Indeed, directed differentiation techniques have improved since transitioning from 2D to 3D cultures, suggesting that the 3D microenvironment in which β cells are born is critical. However, to date, it remains unknown whether the changing architecture of the pancreatic epithelium impacts the fate of cells therein. An emerging challenge in the field is to elucidate how progenitors are allocated during key events, such as the stratification and subsequent resolution of the pre-pancreatic epithelium, as well as the formation of lumens and branches. Here, we assess the progenitor epithelium and examine how it might influence the emergence of pancreatic multipotent progenitors (MPCs), which give rise to β cells and other pancreatic lineages. PMID:26216134

  3. Progress of stem/progenitor cell-based therapy for retinal degeneration.

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    Tang, Zhimin; Zhang, Yi; Wang, Yuyao; Zhang, Dandan; Shen, Bingqiao; Luo, Min; Gu, Ping

    2017-05-10

    Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.

  4. Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

    Science.gov (United States)

    Yoo, Chae Hwa; Na, Hee-Jun; Lee, Dong-Seol; Heo, Soon Chul; An, Yuri; Cha, Junghwa; Choi, Chulhee; Kim, Jae Ho; Park, Joo-Cheol; Cho, Yee Sook

    2013-11-01

    Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Shexiang Baoxin pills promotes angiogenesis in myocardial infarction rats via up-regulation of 20-HETE-mediated endothelial progenitor cells mobilization.

    Science.gov (United States)

    Huang, Feifei; Liu, Yang; Yang, Xia; Che, Di; Qiu, Kaifeng; Hammock, Bruce D; Wang, Jingfeng; Wang, Mong-Heng; Chen, Jie; Huang, Hui

    2017-08-01

    Therapeutic angiogenesis is a pivotal strategy for ischemic heart disease. The aim of the present study was to determine the effect and molecular mechanism of Shexiang Baoxin pills, a widely-used traditional Chinese medicine for ischemic heart disease, on angiogenesis in a rat model of myocardial infarction (MI). We used the occlusion of left anterior descending coronary artery of Sprague-Dawley rats as a model of MI. The MI rats were treated with distilled water, Shexiang Baoxin pills, or Shexiang Baoxin pills + HET0016 (a selective blocker of the biosynthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) at 10 mg/kg/day), respectively. Sham-operated rats were used as controls. Treatment with Shexiang Baoxin pills increases the level of serum 20-HETE in MI rats, which can be suppressed by HET0016 treatment. Shexiang Baoxin pills shows cardio-protective effects on MI rats, including improving cardiac function, decreasing infarction area, and promoting angiogenesis in peri-infarct area. The protective effects of Shexiang Baoxin pills are partly inhibited by HET0016. Furthermore, Shexiang Baoxin pills enhances the number of circulating endothelial progenitor cells (EPCs) and the expression of the vascular endothelial growth factor (VEGF), based on immunohistochemical analysis, in peri-infarct area of MI rats, which is partly suppressed by HET0016. Shexiang Baoxin pills may partially participate in angiogenesis in MI rats. The protective mechanism of Shexiang Baoxin pills may be mediated via up-regulation of 20-HETE, which promotes EPCs mobilization and VEGF expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting.

    Science.gov (United States)

    Lippross, Sebastian; Loibl, Markus; Hoppe, Sven; Meury, Thomas; Benneker, Lorin; Alini, Mauro; Verrier, Sophie

    2011-01-01

    Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell

  7. Establishing clonal cell lines with endothelial-like potential from CD9(hi, SSEA-1(- cells in embryonic stem cell-derived embryoid bodies.

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    Qizhou Lian

    Full Text Available BACKGROUND: Differentiation of embryonic stem cells (ESCs into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs. Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2 = 0.93 while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi, SSEA-1(- while ESCs are CD9(lo, SSEA-1(+. Isolation of CD9(hi, SSEA-1(- cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2 = 0.95 and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells

  8. YKL-40-Induced Inhibition of miR-590-3p Promotes Interleukin-18 Expression and Angiogenesis of Endothelial Progenitor Cells

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    Te-Mao Li

    2017-04-01

    Full Text Available YKL-40, also known as human cartilage glycoprotein-39 or chitinase-3-like-1, is a pro-inflammatory protein that is highly expressed in rheumatoid arthritis (RA patients. Angiogenesis is a critical step in the pathogenesis of RA, promoting the infiltration of inflammatory cells into joints and providing oxygen and nutrients to RA pannus. In this study, we examined the effects of YKL-40 in the production of the pro-inflammatory cytokine interleukin-18 (IL-18, and the stimulation of angiogenesis and accumulation of osteoblasts. We observed that YKL-40 induces IL-18 production in osteoblasts and thereby stimulates angiogenesis of endothelial progenitor cells (EPCs. We found that this process occurs through the suppression of miR-590-3p via the focal adhesion kinase (FAK/PI3K/Akt signaling pathway. YKL-40 inhibition reduced angiogenesis in in vivo models of angiogenesis: the chick embryo chorioallantoic membrane (CAM and Matrigel plug models. We report that YKL-40 stimulates IL-18 expression in osteoblasts and facilitates EPC angiogenesis.

  9. Notch-RBP-J signaling regulates the mobilization and function of endothelial progenitor cells by dynamic modulation of CXCR4 expression in mice.

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    Lin Wang

    Full Text Available Bone marrow (BM-derived endothelial progenitor cells (EPC have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

  10. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro

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    Markus Loibl

    2014-01-01

    Full Text Available Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs and endothelial progenitor cells (EPCs. We identified stabilising pericytes (PCs as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

  11. Diabetes-Induced Oxidative Stress in Endothelial Progenitor Cells May Be Sustained by a Positive Feedback Loop Involving High Mobility Group Box-1

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    Han Wu

    2016-01-01

    Full Text Available Oxidative stress is considered to be a critical factor in diabetes-induced endothelial progenitor cell (EPC dysfunction, although the underlying mechanisms are not fully understood. In this study, we investigated the role of high mobility group box-1 (HMGB-1 in diabetes-induced oxidative stress. HMGB-1 was upregulated in both serum and bone marrow-derived monocytes from diabetic mice compared with control mice. In vitro, advanced glycation end productions (AGEs induced, expression of HMGB-1 in EPCs and in cell culture supernatants in a dose-dependent manner. However, inhibition of oxidative stress with N-acetylcysteine (NAC partially inhibited the induction of HMGB-1 induced by AGEs. Furthermore, p66shc expression in EPCs induced by AGEs was abrogated by incubation with glycyrrhizin (Gly, while increased superoxide dismutase (SOD activity in cell culture supernatants was observed in the Gly treated group. Thus, HMGB-1 may play an important role in diabetes-induced oxidative stress in EPCs via a positive feedback loop involving the AGE/reactive oxygen species/HMGB-1 pathway.

  12. Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line.

    Science.gov (United States)

    Hunt, C P J; Pouton, C W; Haynes, J M

    2017-06-01

    In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32 WT/eGFP-AMP-LacZ ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Specialized mouse embryonic stem cells for studying vascular development.

    Science.gov (United States)

    Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

    2014-01-01

    Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

  14. Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.

    NARCIS (Netherlands)

    Johnson, C.E.; Crawford, B.E.; Stavridis, M.; Dam, G.B. ten; Wat, A.L.; Rushton, G.; Ward, C.M.; Wilson, V.; Kuppevelt, A.H.M.S.M. van; Esko, J.D.; Smith, A.; Gallagher, J.T.; Merry, C.L.

    2007-01-01

    Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe

  15. A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells

    OpenAIRE

    Geng, Yijie; Feng, Bradley

    2016-01-01

    The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the ...

  16. Hematopoietic Substrate-1-Associated Protein X-1 Regulates the Proliferation and Apoptosis of Endothelial Progenitor Cells Through Akt Pathway Modulation.

    Science.gov (United States)

    Guo, Xin-Bin; Deng, Xin; Wei, Ying

    2018-03-01

    Endothelial precursor cells (EPCs) are involved in vasculogenesis of various physiological and pathological processes. The proliferation and survival mechanism of EPCs needs to be explored further for the purpose of developing an effective glioma treatment. Hematopoietic substrate-1-associated protein X-1 (HAX-1) has been reported as an anti-apoptotic protein that plays an important role in several malignant tumors. However, the effect and mechanism of HAX-1 on EPCs remains unknown. This study aims to investigate the effect of HAX-1 on the proliferation and apoptosis of EPCs and explore its mechanism. According to our results, HAX-1 was overexpressed in EPCs. The results of clone formation and 5-ethynyl-2'-deoxyuridine proliferation assay showed that HAX-1 promoted multiplication of EPCs. Flow cytometry showed HAX-1 knockout cell cycle arrest mainly in G0/G1 phase. Apoptosis analysis showed that HAX-1 could protect EPCs from apoptosis in oxidative stress. Western blot assay indicated that HAX-1 could inhibit the activation of caspase cascade and reduce the expression of p21, Bcl-2-associated X protein, and p53. HAX-1 also enhanced the degradation rate and ubiquitination of p53 through the promotion of phosphorylation of proteins MDM-2 and Akt1. Co-immunoprecipitation and immunofluorescent colocalization assays were performed to test the influence of HAX-1 on the interaction between Akt1 and heat shock protein 90 (Hsp90), which is crucial for the activity of Akt1. In conclusion, this novel study suggests that HAX-1 could facilitate the Akt1 pathway through Hsp90, which led to a decline in the levels of p53, and finally promoted the proliferation and inhibited the apoptosis of EPCs. Stem Cells 2018;36:406-419. © 2017 AlphaMed Press.

  17. Analysis of CD45- [CD34+/KDR+] endothelial progenitor cells as juvenile protective factors in a rat model of ischemic-hemorrhagic stroke.

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    Julius L Decano

    Full Text Available Identification of juvenile protective factors (JPFs which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. A priori, if EPC aging plays a 'master-switch JPF-role' in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset.FACS analysis revealed that CD45- [CD34+/KDR+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[CD34+/KDR+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003. Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in CD45-[CD34+/KDR]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001, suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age-associated intimal hyperplasia.Altogether, the data

  18. Far infra-red therapy promotes ischemia-induced angiogenesis in diabetic mice and restores high glucose-suppressed endothelial progenitor cell functions

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    Huang Po-Hsun

    2012-08-01

    Full Text Available Abstract Background Far infra-red (IFR therapy was shown to exert beneficial effects in cardiovascular system, but effects of IFR on endothelial progenitor cell (EPC and EPC-related vasculogenesis remain unclear. We hypothesized that IFR radiation can restore blood flow recovery in ischemic hindlimb in diabetic mice by enhancement of EPCs functions and homing process. Materials and methods Starting at 4 weeks after the onset of diabetes, unilateral hindlimb ischemia was induced in streptozotocine (STZ-induced diabetic mice, which were divided into control and IFR therapy groups (n = 6 per group. The latter mice were placed in an IFR dry sauna at 34°C for 30 min once per day for 5 weeks. Results Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls, and significantly greater capillary density was seen in the IFR therapy group. Flow cytometry analysis showed impaired EPCs (Sca-1+/Flk-1+ mobilization after ischemia surgery in diabetic mice with or without IFR therapy (n = 6 per group. However, as compared to those in the control group, bone marrow-derived EPCs differentiated into endothelial cells defined as GFP+/CD31+ double-positive cells were significantly increased in ischemic tissue around the vessels in diabetic mice that received IFR radiation. In in-vitro studies, cultured EPCs treated with IFR radiation markedly augmented high glucose-impaired EPC functions, inhibited high glucose-induced EPC senescence and reduced H2O2 production. Nude mice received human EPCs treated with IFR in high glucose medium showed a significant improvement in blood flow recovery in ischemic limb compared to those without IFR therapy. IFR therapy promoted blood flow recovery and new vessel formation in STZ-induced diabetic mice. Conclusions Administration of IFR therapy promoted collateral flow recovery and new vessel formation in STZ

  19. Downregulation of microRNA-130a contributes to endothelial progenitor cell dysfunction in diabetic patients via its target Runx3.

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    Shu Meng

    Full Text Available Dysfunction of endothelial progenitor cells (EPCs contributes to diabetic vascular disease. MicroRNAs (miRs have emerged as key regulators of diverse cellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs from type II diabetes mellitus (DM patients, and downregulation of miR-126 impairs EPC function. The present study further explored whether dysregulated miR-130a were also related to EPC dysfunction. EPCs were cultured from peripheral blood mononuclear cells of diabetic patients and healthy controls. Assays on EPC function (proliferation, migration, differentiation, apoptosis, and colony and tubule formation were performed. Bioinformatics analyses were used to identify the potential targets of miR-130a in EPCs. Gene expression of miR-103a and Runx3 was measured by real-time PCR, and protein expression of Runx3, extracellular signal-regulated kinase (ERK, vascular endothelial growth factor (VEGF and Akt was measured by Western blotting. Runx3 promoter activity was measured by luciferase reporter assay. A miR-130a inhibitor or mimic and lentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targeting Runx3 were transfected into EPCs to manipulate miR-130a and Runx3 levels. MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibited whereas miR-130a overexpression promoted EPC function. miR-130a negatively regulated Runx3 (mRNA, protein and promoter activity in EPCs. Knockdown of Runx3 expression enhanced EPC function. MiR-130a also upregulated protein expression of ERK/VEGF and Akt in EPCs. In conclusion, miR-130a plays an important role in maintaining normal EPC function, and decreased miR-130a in EPCs from DM contributes to impaired EPC function, likely via its target Runx3 and through ERK/VEGF and Akt pathways.

  20. Effect of a Mediterranean diet on endothelial progenitor cells and carotid intima-media thickness in type 2 diabetes: Follow-up of a randomized trial.

    Science.gov (United States)

    Maiorino, Maria Ida; Bellastella, Giuseppe; Petrizzo, Michela; Gicchino, Maurizio; Caputo, Mariangela; Giugliano, Dario; Esposito, Katherine

    2017-03-01

    Background We assessed the long-term effects of a Mediterranean diet on circulating levels of endothelial progenitor cells (EPCs) and the carotid intima-media thickness (CIMT) in patients with type 2 diabetes. Design This was a parallel, two-arm, single-centre trial. Methods Two hundred and fifteen men and women with newly diagnosed type 2 diabetes were randomized to a Mediterranean diet ( n = 108) or a low-fat diet ( n = 107). The primary outcome measures were changes in the EPC count and the CIMT of the common carotid artery after the treatment period defined as the end of trial (EOT). Results At the EOT, both the CD34 + KDR + and CD34 + KDR + CD133 + counts had increased with the Mediterranean diet compared with the low-fat diet ( p Mediterranean diet. Compared with the low-fat diet, the rate of regression in the CIMT was higher in the Mediterranean diet group (51 vs. 26%), whereas the rate of progression was lower (25 vs. 50%) ( p = 0.032 for both). Changes in the CIMT were inversely correlated with the changes in EPC levels (CD34 + KDR + , r = -0.24, p = 0.020; CD34 + KDR + CD133 + , r = -0.28, p = 0.014). At the EOT, changes in levels of HbA1c, HOMA, total cholesterol, high-density lipoprotein cholesterol and systolic blood pressure were significantly greater with the Mediterranean diet than with the low-fat diet. Conclusion Compared with a low-fat diet, a long-term trial with Mediterranean diet was associated with an increase in circulating EPCs levels and prevention of the progression of subclinical atherosclerosis in patients with newly diagnosed type 2 diabetes.

  1. Impact of adipose tissue or umbilical cord derived mesenchymal stem cells on the immunogenicity of human cord blood derived endothelial progenitor cells.

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    Kefang Tan

    Full Text Available The application of autologous endothelial progenitor cell (EPC transplantation is a promising approach in therapeutic cardiovascular diseases and ischemic diseases. In this study, we compared the immunogenicity of EPCs, adipose tissue (AD-derived mesenchymal stem cells (MSCs and umbilical cord (UC-derived MSCs by flow cytometry and the mixed lymphocyte reaction. The impact of AD-MSCs and UC-MSCs on the immunogenicity of EPCs was analyzed by the mixed lymphocyte reaction and cytokine secretion in vitro and was further tested by allogenic peripheral blood mononuclear cell (PBMC induced immuno-rejection on a cell/matrigel graft in an SCID mouse model. EPCs and AD-MSCs express higher levels of MHC class I than UC-MSCs. All three kinds of cells are negative for MHC class II. UC-MSCs also express lower levels of IFN-γ receptor mRNA when compared with EPCs and AD-MSCs. EPCs can stimulate higher rates of proliferation of lymphocytes than AD-MSCs and UC-MSCs. Furthermore, AD-MSCs and UC-MSCs can modulate immune response and inhibit lymphocyte proliferation induced by EPCs, mainly through inhibition of the proliferation of CD8+ T cells. Compared with UC-MSCs, AD-MSCs can significantly improve vessel formation and maintain the integrity of neovascular structure in an EPC+MSC/matrigel graft in SCID mice, especially under allo-PBMC induced immuno-rejection. In conclusion, our study shows that AD-MSC is a powerful candidate to minimize immunological rejection and improve vessel formation in EPC transplantation treatment.

  2. The effect of angiotensin-2 receptor blocker valsartan on circulating level of endothelial progenitor cells in diabetic patients with asymptomatic coronary artery disease.

    Science.gov (United States)

    Berezin, Alexander E; Kremzer, Alexander A; Martovitskaya, Yulia V; Samura, Tatyana A

    2015-01-01

    Decreased circulating endothelial progenitor cells (EPCs) are considered as strong and robust biomarkers for the prediction of cardiovascular outcomes in diabetic populations. The perspectives for modulating EPCs levels in T2DM with known coronary artery disease (CAD) with different drugs, affected mechanisms of improving mobilization of EPCs from tissue, are not still understood. To evaluate an effect of angiotensin-2 receptor blocker valsartan on circulating level of EPCs in diabetic patients with asymptomatic CAD. The study population was structured retrospectively after determining the CAD by contrast-enhanced spiral computed tomography angiography in 126 asymptomatic subjects. All subjects were distributed into two cohorts depending on daily doses of valsartan given. Low (80-160 mg daily orally) and high doses (240-320 mg daily orally) of valsartan were used and they were adjusted depending on achieving BP level less than 140/80 mmHg. The change from baseline in CD34(+) subset cells (frequencies and absolute values) was not significantly different between treatment cohorts. We found a significant increase of circulating level of CD14(+)CD309(+) cells in two patient cohorts. But more prominent change of CD14(+)CD309(+) cells was verified in subjects who were given valsartan in high daily doses when compared with persons who were included into cohort with low daily doses of the drug (1.96% versus 2.59%, respectively; Pvalsartan only. We found positive influence of angiotensin-2 receptor blocker valsartan in escalation doses on bone marrow-derived EPCs phenotyped as CD14(+)CD309(+) and CD14(+)CD309(+)Tie(2+) in T2DM patients with known asymptomatic CAD. Copyright © 2014 Diabetes India. Published by Elsevier Ltd. All rights reserved.

  3. Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

    Science.gov (United States)

    Zhang, Shishuang; Zhi, Yongle; Li, Fei; Huang, Shan; Gao, Huabin; Han, Zhaoli; Ge, Xintong; Li, Dai; Chen, Fanglian; Kong, Xiaodong; Lei, Ping

    2018-04-15

    To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aβ in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-β (Aβ) transport, and chronic cerebral hypoperfusion causes Aβ deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aβ deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aβ senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aβ clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Chemokine receptors CXCR2 and CX3CR1 differentially regulate functional responses of bone-marrow endothelial progenitors during atherosclerotic plaque regression

    Science.gov (United States)

    Herlea-Pana, Oana; Yao, Longbiao; Heuser-Baker, Janet; Wang, Qiongxin; Wang, Qilong; Georgescu, Constantin; Zou, Ming-Hui; Barlic-Dicen, Jana

    2015-01-01

    Aims Atherosclerosis manifests itself as arterial plaques, which lead to heart attacks or stroke. Treatments supporting plaque regression are therefore aggressively pursued. Studies conducted in models in which hypercholesterolaemia is reversible, such as the Reversa mouse model we have employed in the current studies, will be instrumental for the development of such interventions. Using this model, we have shown that advanced atherosclerosis regression occurs when lipid lowering is used in combination with bone-marrow endothelial progenitor cell (EPC) treatment. However, it remains unclear how EPCs home to regressing plaques and how they augment atherosclerosis reversal. Here we identify molecules that support functional responses of EPCs during plaque resolution. Methods and results Chemokines CXCL1 and CX3CL1 were detected in the vascular wall of atheroregressing Reversa mice, and their cognate receptors CXCR2 and CX3CR1 were observed on adoptively transferred EPCs in circulation. We tested whether CXCL1–CXCR2 and CX3CL1–CX3CR1 axes regulate functional responses of EPCs during plaque reversal. We show that pharmacological inhibition of CXCR2 or CX3CR1, or genetic inactivation of these two chemokine receptors interfered with EPC-mediated advanced atherosclerosis regression. We also demonstrate that CXCR2 directs EPCs to regressing plaques while CX3CR1 controls a paracrine function(s) of these cells. Conclusion CXCR2 and CX3CR1 differentially regulate EPC functional responses during atheroregression. Our study improves understanding of how chemokines and chemokine receptors regulate plaque resolution, which could determine the effectiveness of interventions reducing complications of atherosclerosis. PMID:25765938

  5. Magnetic Resonance Tracking of Endothelial Progenitor Cells Labeled with Alkyl-Polyethylenimine 2 kDa/Superparamagnetic Iron Oxide in a Mouse Lung Carcinoma Xenograft Model

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    Cong Chen

    2014-11-01

    Full Text Available The potential of using endothelial progenitor cells (EPCs in novel anticancer therapy and the repair of vascular injury has been increasingly recognized. In the present study, EPCs were labeled with N-alkyl-polyethylenimine 2 kDa (PEI2k-stabilized superparamagnetic iron oxide (SPIO to facilitate magnetic resonance imaging (MRI of EPCs in a mouse lung carcinoma xenograft model. EPCs derived from human peripheral blood were labeled with alkyl-PEI2k/SPIO. The viability and activity of labeled cells were evaluated using proliferation, migration, and tubulogenesis assays. Alkyl-PEI2k/SPIO-labeled EPCs were injected intravenously (group 1 or mixed and injected together with A549 cells subcutaneously (group 2 into groups of six mice with severe combined immunodeficiency. The labeling efficiency with alkyl-PEI2k/SPIO at 7 mg Fe/mL concentration was approximately 100%. Quantitative analysis of cellular iron was 6.062 ± 0.050 pg/cell. No significant effects on EPC proliferation, migration, or tubulogenesis were seen after labeling. Seventesla micro-MRI showed the presence of schistic or linear hypointense regions at the tumor margins starting from days 7 to 8 after EPC administration. This gradually extended into the inner tumor layers in group 1. In group 2, tumor growth was accompanied by dispersion of low-signal intensity regions inside the tumor. Iron-positive cells identified by Prussian blue dye were seen at the sites identified using MRI. Human CD31-positive cells and mouse CD31-positive cells were present in both groups. Labeling EPCs with alkyl-PEI2k/SPIO allows noninvasive magnetic resonance investigation of EPC involvement in tumor neovasculature and is associated with excellent biocompatibility and MRI sensitivity.

  6. Tanshinone II A Attenuates TNF-α-Induced Expression of VCAM-1 and ICAM-1 in Endothelial Progenitor Cells by Blocking Activation of NF-κB

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    Jin-Xiu Yang

    2016-11-01

    Full Text Available Background/Aims: Tanshinone IIA (Tan IIA is effective in the treatment of inflammation and atherosclerosis. The adhesion of inflammatory cells to vascular endothelium plays important role in atherogenic processes. This study examined the effects of Tan IIA on expression of adhesion molecules in tumor necrosis factor-α (TNF-α-induced endothelial progenitor cells (EPCs. Methods: EPCs were pretreated with Tan IIA and stimulated with TNF-α. Mononuclear cell (MNC adhesion assay was performed to assess the effects of Tan IIA on TNF-α-induced MNC adhesion. Expression of vascular cell adhesion molecule-1 (VCAM-1/intracellular adhesion molecule-1 (ICAM-1 and activation of Nuclear factor κB (NF-κB signaling pathway were measured. Results: The results showed that the adhesion of MNCs to TNF-α-induced EPCs and expression of VCAM-1/ICAM-1 in EPCs were promoted by TNF-α, which were reduced by Tan IIA. TNF-α increased the amount of phosphorylation of NF-κB, IκB-α and IKKα/β in cytosolic fractions and NF-κB p65 in nucleus, while Tan IIA reduced its amount. Conclusion: This study demonstrated a novel mechanism for the anti-inflammatory/anti-atherosclerotic activity of Tan IIA, which may involve down-regulation of VCAM-1 and ICAM-1 through partial blockage of TNF-α-induced NF-κB activation and IκB-α phosphorylation by the inhibition of IKKα/β pathway in EPCs.

  7. Electroacupuncture modulates stromal cell-derived factor-1α expression and mobilization of bone marrow endothelial progenitor cells in focal cerebral ischemia/reperfusion model rats.

    Science.gov (United States)

    Xie, Chenchen; Gao, Xiang; Luo, Yong; Pang, Yueshan; Li, Man

    2016-10-01

    Stromal cell-derived factor-1α(SDF-1α) plays a crucial role in regulating the mobilization, migration and homing of endothelial progenitor cells(EPCs). Electroacupuncture(EA), a modern version of Traditional Chinese Medicine, can improve neurological recovery and angiogenesis in cerebral ischemic area. This study aimed to investigate the effects of electroacupuncture(EA) on the mobilization and migration of bone marrow EPCs and neurological functional recovery in rats model after focal cerebral ischemia/reperfusion and the potentially involved mechanisms. Sprague-Dawley rats received filament occlusion of the right middle cerebral artery for 2h followed by reperfusion for 12h, 1d, 2d, 3d, 7d respectively. Rats were randomly divided into sham group, model group and EA group. After 2h of the reperfusion, EA was given at the "Baihui" (GV 20)/Siguan ("Hegu" (LI 4)/"Taichong" (LR 3)) acupoints in the EA group. Modified neurological severity score (mNSS) was used to assess the neurological functional recovery. EPCs number and SDF-1α level in bone marrow(BM) and peripheral blood(PB) were detected by using fluorescence-activated cell sorting (FACS) analysis and quantitative real time polymerase chain reaction (qRT-PCR) respectively. An mNSS test showed that EA treatment significantly improved the neurological functional outcome. EPCs number in PB and BM were obviously increased in the EA group. After cerebral ischemia, the SDF-1α level was decreased in BM while it was increased in PB, which implied a gradient of SDF-1α among BM and PB after ischemia. It suggested that the forming of SDF-1α concentration gradient can induce the mobilization and homing of EPCs. Eletroacupuncture as a treatment can accelerate and increase the forming of SDF-1α concentration gradient to further induce the mobilization of EPCs and angiogenesis in ischemic brain and improve the neurological function recovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

    International Nuclear Information System (INIS)

    Aguirre, A.; Planell, J.A.; Engel, E.

    2010-01-01

    Research highlights: → BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. → Co-culture decreases proliferation by cellular self-regulatory mechanisms. → Co-cultured cells present an activated proangiogenic phenotype. → qRT-PCR and cluster analysis identify new target genes playing important roles. -- Abstract: Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

  9. Complications impaired endothelial progenitor cell function in Type 2 diabetic patients with or without critical leg ischaemia: implication for impaired neovascularization in diabetes.

    Science.gov (United States)

    Chen, M-C; Sheu, J-J; Wang, P-W; Chen, C-Y; Kuo, M-C; Hsieh, C-J; Chen, J-F; Chang, H-W

    2009-02-01

    This study tested the hypothesis that migratory function of endothelial progenitor cells (EPCs) is impaired in Type 2 diabetic patients with or without critical leg ischaemia. Seventy-four patients were classified into four groups: Type 2 diabetic (n = 21) and non-diabetic patients (n = 10) with critical leg ischaemia and Type 2 diabetic patients without lower extremity vascular disease (n = 30) and healthy subjects (n = 13). The number and functional activity of circulating and cultured EPCs were determined. The migratory function of cultured EPCs was significantly impaired in diabetic patients without (median, 48, interquartile range, 46, 49 count/view/well) and with (median, 51, interquartile range, 46, 60 count/view/well) critical leg ischaemia and non-diabetic patients with critical leg ischaemia (median, 49, interquartile range, 47, 55 count/view/well) compared with healthy subjects (median, 63, interquartile range, 57, 65 count/view/well) (P interquartile range, 1600, 6600/10(6) cytometric events) than Type 2 diabetic patients with critical leg ischaemia (median, 5300, interquartile range, 2400, 11,100/10(6) cytometric events), non-diabetic patients with critical leg ischaemia (median, 5550, interquartile range, 2000, 32,100/10(6) cytometric events) and healthy subjects (median, 5400, interquartile range, 2700, 8700/10(6) cytometric events) (P = 0.413). The migratory function of EPCs is impaired in patients with Type 2 diabetes, even in those without critical leg ischaemia. These findings present an important new insight into the pathogenesis of impaired neovascularization and critical limb ischaemia in diabetic patients and provide avenues of future clinical study.

  10. Transplantation of autologous endothelial progenitor cells in porous PLGA scaffolds create a microenvironment for the regeneration of hyaline cartilage in rabbits.

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    Chang, N-J; Lam, C-F; Lin, C-C; Chen, W-L; Li, C-F; Lin, Y-T; Yeh, M-L

    2013-10-01

    Repairing articular cartilage is clinically challenging. We investigated a simple, effective and clinically feasible cell-based therapeutic approach using a poly(lactide-co-glycolide) (PLGA) scaffold seeded with autologous endothelial progenitor cells (EPC) to repair a full-thickness osteochondral defect in rabbits using a one-step surgery. EPC obtained by purifying a small amount of peripheral blood from rabbits were seeded into a highly porous, biocompatible PLGA scaffold, namely, EPC-PLGA, and implanted into the osteochondral defect in the medial femoral condyle. Twenty two rabbits were randomized into one of three groups: the empty defect group (ED), the PLGA-only group or the EPC-PLGA group. The defect sites were evaluated 4 and 12 weeks after implantation. At the end of testing, only the EPC-PLGA group showed the development of new cartilage tissue with a smooth, transparent and integrated articular surface. Moreover, histological analysis showed obvious differences in cartilage regeneration. At week 4, the EPC-PLGA group showed considerably higher TGF-β2 and TGF-β3 expression, a greater amount of synthesized glycosaminoglycan (GAG) content, and a higher degree of osteochondral angiogenesis in repaired tissues. At week 12, the EPC-PLGA group showed enhanced hyaline cartilage regeneration with a normal columnar chondrocyte arrangement, higher SOX9 expression, and greater GAG and collagen type II (COLII) content. Moreover, the EPC-PLGA group showed organized osteochondral integration, the formation of vessel-rich tubercular bone and significantly higher bone volume per tissue volume and trabecular thickness (Tb.Th). The present EPC-PLGA cell delivery system generates a suitable in situ microenvironment for osteochondral regeneration without the supplement of exogenous growth factors. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  11. Visualisation of axolotl blastema cells and pig endothelial progenitor cells using very small super paramagnetic iron oxide particles in MRI: A technique with applications for non invasive visualisation of regenerative processes

    DEFF Research Database (Denmark)

    Lauridsen, Henrik; Kjær, N.B.; Bek, Maria

    oxide particles (VSOP) in animal cells enable non invasive cell tracking using magnetic resonance imaging (MRI) and can prove useful, when visualising regenerative processes. This study examines the possibility of labelling limited numbers of axolotl blastema cells (aBC) and pig endothelial progenitor...... implanted in live axolotl tail and dead porcine heart, respectively. Cellular iron uptake was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Results: T2*-weighted 2D gradient-echo sequences on samples of 10˄5 cells yielded at significant linear correlations between...

  12. Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

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    Francesco Lodola

    Full Text Available Endothelial progenitor cells (EPCs may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+ entry (SOCE, which is activated by a depletion of the intracellular Ca(2+ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+-sensor, Stim1, and the plasmalemmal Ca(2+ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.The present study employed Ca(2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs as compared to control EPCs (N-EPCs. SOCE, activated by either pharmacological (i.e. cyclopiazonic acid or physiological (i.e. ATP stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+ and Gd(3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI, blocked both SOCE and the intracellular Ca(2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1 at both mRNA and protein level The intracellular Ca(2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.SOCE is remodelled in EPCs

  13. Moderate-to-high-intensity training and a hypocaloric Mediterranean diet enhance endothelial progenitor cells and fitness in subjects with the metabolic syndrome.

    Science.gov (United States)

    Fernández, Juan Marcelo; Rosado-Álvarez, Daniel; Da Silva Grigoletto, Marzo Edir; Rangel-Zúñiga, Oriol Alberto; Landaeta-Díaz, Leslie Lorena; Caballero-Villarraso, Javier; López-Miranda, José; Pérez-Jiménez, Francisco; Fuentes-Jiménez, Francisco

    2012-09-01

    A reduction in EPC (endothelial progenitor cell) number could explain the development and progression of atherosclerosis in the MetS (metabolic syndrome). Although much research in recent years has focused on the Mediterranean dietary pattern and the MetS, the effect of this diet with/without moderate-to-high-intensity endurance training on EPCs levels and CrF (cardiorespiratory fitness) remains unclear. In the present study, the objective was to assess the effect of a Mediterranean diet hypocaloric model with and without moderate-to-high-intensity endurance training on EPC number and CrF of MetS patients. Thus 45 MetS patients (50-66 years) were randomized to a 12-week intervention with the hypocaloric MeD (Mediterranean diet) or the MeDE (MeD plus moderate-to-high-intensity endurance training). Training included two weekly supervised sessions [80% MaxHR (maximum heart rate); leg and arm pedalling] and one at-home session (65-75% MaxHR; walking controlled by heart rate monitors). Changes in: (i) EPC number [CD34(+)KDR(+) (kinase insert domain-containing receptor)], (ii) CrF variables and (iii) MetS components and IRH (ischaemic reactive hyperaemia) were determined at the end of the study. A total of 40 subjects completed all 12 weeks of the study, with 20 in each group. The MeDE led to a greater increase in EPC numbers and CrF than did the MeD intervention (P ≤ 0.001). In addition, a positive correlation was observed between the increase in EPCs and fitness in the MeDE group (r=0.72; r(2)=0.52; P ≤ 0.001). Body weight loss, insulin sensitivity, TAGs (triacylglycerols) and blood pressure showed a greater decrease in the MeDE than MeD groups. Furthermore, IRH was only improved after the MeDE intervention. In conclusion, compliance with moderate-to-high-intensity endurance training enhances the positive effects of a model of MeD on the regenerative capacity of endothelium and on the fitness of MetS patients.

  14. Comparative analysis of circulating endothelial progenitor cells in age-related macular degeneration patients using automated rare cell analysis (ARCA and fluorescence activated cell sorting (FACS.

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    Emil Anthony T Say

    Full Text Available BACKGROUND: Patients with age-related macular degeneration (ARMD begin with non-neovascular (NNV phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs, defined as CD34(+VEGR2(+ using traditional fluorescence activated cell sorting (FACS, are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA, a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion. METHODS: We obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and p<0.2 was considered indicative of a trend for this proof of concept study, while statistical significance was established at 0.05. RESULTS: We measured levels of CD34(+VEGFR2(+ EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17 using ARCA. Interestingly, CD34(+VEGR2(+ EPC analysis using FACS did not produce similar results (p = 0.94. CONCLUSIONS: CD34(+VEGR2(+ may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample

  15. Comparative analysis of circulating endothelial progenitor cells in age-related macular degeneration patients using automated rare cell analysis (ARCA) and fluorescence activated cell sorting (FACS).

    Science.gov (United States)

    Say, Emil Anthony T; Melamud, Alex; Esserman, Denise Ann; Povsic, Thomas J; Chavala, Sai H

    2013-01-01

    Patients with age-related macular degeneration (ARMD) begin with non-neovascular (NNV) phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV) ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs), defined as CD34(+)VEGR2(+) using traditional fluorescence activated cell sorting (FACS), are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA), a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion. We obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and ptrend for this proof of concept study, while statistical significance was established at 0.05. We measured levels of CD34(+)VEGFR2(+) EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17) using ARCA. Interestingly, CD34(+)VEGR2(+) EPC analysis using FACS did not produce similar results (p = 0.94). CD34(+)VEGR2(+) may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample size were suggestive of a trend in ARMD using ARCA but not FACS. ARCA could be a

  16. Transfusion of CXCR4-primed endothelial progenitor cells reduces cerebral ischemic damage and promotes repair in db/db diabetic mice.

    Directory of Open Access Journals (Sweden)

    Ji Chen

    Full Text Available This study investigated the role of stromal cell-derived factor-1α (SDF-1α/CXC chemokine receptor 4 (CXCR4 axis in brain and endothelial progenitor cells (EPCs, and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO. In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null or CXCR4 (Ad-CXCR4 followed with high glucose (HG treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1 The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2 The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3 Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4 Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4 Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do.

  17. Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

    2015-07-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

  18. The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

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    Ralf Kist

    2014-10-01

    Full Text Available In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

  19. The formation of endoderm-derived taste sensory organs requires a Pax9-dependent expansion of embryonic taste bud progenitor cells.

    Science.gov (United States)

    Kist, Ralf; Watson, Michelle; Crosier, Moira; Robinson, Max; Fuchs, Jennifer; Reichelt, Julia; Peters, Heiko

    2014-10-01

    In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.

  20. Endothelial-regenerating cells: an expanding universe.

    Science.gov (United States)

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

    Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

  1. An anti-CD34 antibody-functionalized clinical-grade POSS-PCU nanocomposite polymer for cardiovascular stent coating applications: a preliminary assessment of endothelial progenitor cell capture and hemocompatibility.

    Directory of Open Access Journals (Sweden)

    Aaron Tan

    Full Text Available In situ endothelialization of cardiovascular implants has emerged in recent years as an attractive means of targeting the persistent problems of thrombosis and intimal hyperplasia. This study aimed to investigate the efficacy of immobilizing anti-CD34 antibodies onto a POSS-PCU nanocomposite polymer surface to sequester endothelial progenitor cells (EPCs from human blood, and to characterize the surface properties and hemocompatibility of this surface. Amine-functionalized fumed silica was used to covalently conjugate anti-CD34 to the polymer surface. Water contact angle, fluorescence microscopy, and scanning electron microscopy were used for surface characterization. Peripheral blood mononuclear cells (PBMCs were seeded on modified and pristine POSS-PCU polymer films. After 7 days, adhered cells were immunostained for the expression of EPC and endothelial cell markers, and assessed for the formation of EPC colonies. Hemocompatibility was assessed by thromboelastography, and platelet activation and adhesion assays. The number of EPC colonies formed on anti-CD34-coated POSS-PCU surfaces was not significantly higher than that of POSS-PCU (5.0±1.0 vs. 1.7±0.6, p>0.05. However, antibody conjugation significantly improved hemocompatibility, as seen from the prolonged reaction and clotting times, decreased angle and maximum amplitude (p<0.05, as well as decreased platelet adhesion (76.8±7.8 vs. 8.4±0.7, p<0.05 and activation. Here, we demonstrate that POSS-PCU surface immobilized anti-CD34 antibodies selectively captured CD34+ cells from peripheral blood, although only a minority of these were EPCs. Nevertheless, antibody conjugation significantly improves the hemocompatibility of POSS-PCU, and should therefore continue to be explored in combination with other strategies to improve the specificity of EPC capture to promote in situ endothelialization.

  2. Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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    Der-I Kao

    2015-02-01

    Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

  3. How to utilize Ca²⁺ signals to rejuvenate the repairative phenotype of senescent endothelial progenitor cells in elderly patients affected by cardiovascular diseases: a useful therapeutic support of surgical approach?

    Science.gov (United States)

    Moccia, Francesco; Dragoni, Silvia; Cinelli, Mariapia; Montagnani, Stefania; Amato, Bruno; Rosti, Vittorio; Guerra, Germano; Tanzi, Franco

    2013-01-01

    Endothelial dysfunction or loss is the early event that leads to a host of severe cardiovascular diseases, such as atherosclerosis, hypertension, brain stroke, myocardial infarction, and peripheral artery disease. Ageing is regarded among the most detrimental risk factor for vascular endothelium and predisposes the subject to atheroscleorosis and inflammatory states even in absence of traditional comorbid conditions. Standard treatment to restore blood perfusion through stenotic arteries are surgical or endovascular revascularization. Unfortunately, ageing patients are not the most amenable candidates for such interventions, due to high operative risk or unfavourable vascular involvement. It has recently been suggested that the transplantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) might constitute an alternative and viable therapeutic option for these individuals. Albeit pre-clinical studies demonstrated the feasibility of EPC-based therapy to recapitulate the diseased vasculature of young and healthy animals, clinical studies provided less impressive results in old ischemic human patients. One hurdle associated to this kind of approach is the senescence of autologous EPCs, which are less abundant in peripheral blood and display a reduced pro-angiogenic activity. Conversely, umbilical cord blood (UCB)-derived EPCs are more suitable for cellular therapeutics due to their higher frequency and sensitivity to growth factors, such as vascular endothelial growth factor (VEGF). An increase in intracellular Ca(2+) concentration is central to EPC activation by VEGF. We have recently demonstrated that the Ca(2+) signalling machinery driving the oscillatory Ca(2+) response to this important growth factor is different in UCB-derived EPCs as compared to their peripheral counterparts. In particular, we focussed on the so-called endothelial colony forming cells (ECFCs), which are the only EPC population belonging to the endothelial lineage and able

  4. Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells.

    Science.gov (United States)

    Haigh, Jody J; Ema, Masatsugu; Haigh, Katharina; Gertsenstein, Marina; Greer, Peter; Rossant, Janet; Nagy, Andras; Wagner, Erwin F

    2004-02-01

    Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A-mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A-independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell-derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

  5. The non-alcoholic fraction of beer increases stromal cell derived factor 1 and the number of circulating endothelial progenitor cells in high cardiovascular risk subjects: a randomized clinical trial.

    Science.gov (United States)

    Chiva-Blanch, Gemma; Condines, Ximena; Magraner, Emma; Roth, Irene; Valderas-Martínez, Palmira; Arranz, Sara; Casas, Rosa; Martínez-Huélamo, Miriam; Vallverdú-Queralt, Anna; Quifer-Rada, Paola; Lamuela-Raventos, Rosa M; Estruch, Ramon

    2014-04-01

    Moderate alcohol consumption is associated with a decrease in cardiovascular risk, but fermented beverages seem to confer greater cardiovascular protection due to their polyphenolic content. Circulating endothelial progenitor cells (EPC) are bone-marrow-derived stem cells with the ability to repair and maintain endothelial integrity and function and are considered as a surrogate marker of vascular function and cumulative cardiovascular risk. Nevertheless, no study has been carried out on the effects of moderate beer consumption on the number of circulating EPC in high cardiovascular risk patients. To compare the effects of moderate consumption of beer, non-alcoholic beer and gin on the number of circulating EPC and EPC-mobilizing factors. In this crossover trial, 33 men at high cardiovascular risk were randomized to receive beer (30 g alcohol/d), the equivalent amount of polyphenols in the form of non-alcoholic beer, or gin (30 g alcohol/d) for 4 weeks. Diet and physical exercise were carefully monitored. The number of circulating EPC and EPC-mobilizing factors were determined at baseline and after each intervention. After the beer and non-alcoholic beer interventions, the number of circulating EPC significantly increased by 8 and 5 units, respectively, while no significant differences were observed after the gin period. In correlation, stromal cell derived factor 1 increased significantly after the non-alcoholic and the beer interventions. The non-alcoholic fraction of beer increases the number of circulating EPC in peripheral blood from high cardiovascular risk subjects. http://www.controlled-trials.com/ISRCTN95345245 ISRCTN95345245. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Black Tea Increases Circulating Endothelial Progenitor Cells and Improves Flow Mediated Dilatation Counteracting Deleterious Effects from a Fat Load in Hypertensive Patients: A Randomized Controlled Study

    Science.gov (United States)

    Grassi, Davide; Draijer, Richard; Schalkwijk, Casper; Desideri, Giovambattista; D’Angeli, Anatolia; Francavilla, Sandro; Mulder, Theo; Ferri, Claudio

    2016-01-01

    (1) Background: Endothelial dysfunction predicts cardiovascular events. Circulating angiogenic cells (CACs) maintain and repair the endothelium regulating its function. Tea flavonoids reduce cardiovascular risk. We investigated the effects of black tea on the number of CACs and on flow-mediated dilation (FMD) before and after an oral fat in hypertensives; (2) Methods: In a randomized, double-blind, controlled, cross-over study, 19 patients were assigned to black tea (150 mg polyphenols) or a placebo twice a day for eight days. Measurements were obtained in a fasted state and after consuming whipping cream, and FMD was measured at baseline and after consumption of the products; (3) Results: Compared with the placebo, black tea ingestion increased functionally active CACs (36 ± 22 vs. 56 ± 21 cells per high-power field; p = 0.006) and FMD (5.0% ± 0.3% vs. 6.6% ± 0.3%, p FMD 1, 2, 3, and 4 h after consumption, with maximal response 2 h after intake (p FMD, while tea consumption counteracted FMD impairment (p < 0.0001); (4) Conclusions: We demonstrated the vascular protective properties of black tea by increasing the number of CACs and preventing endothelial dysfunction induced by acute oral fat load in hypertensive patients. Considering that tea is the most consumed beverage after water, our findings are of clinical relevance and interest. PMID:27854314

  7. Black Tea Increases Circulating Endothelial Progenitor Cells and Improves Flow Mediated Dilatation Counteracting Deleterious Effects from a Fat Load in Hypertensive Patients: A Randomized Controlled Study

    Directory of Open Access Journals (Sweden)

    Davide Grassi

    2016-11-01

    Full Text Available (1 Background: Endothelial dysfunction predicts cardiovascular events. Circulating angiogenic cells (CACs maintain and repair the endothelium regulating its function. Tea flavonoids reduce cardiovascular risk. We investigated the effects of black tea on the number of CACs and on flow-mediated dilation (FMD before and after an oral fat in hypertensives; (2 Methods: In a randomized, double-blind, controlled, cross-over study, 19 patients were assigned to black tea (150 mg polyphenols or a placebo twice a day for eight days. Measurements were obtained in a fasted state and after consuming whipping cream, and FMD was measured at baseline and after consumption of the products; (3 Results: Compared with the placebo, black tea ingestion increased functionally active CACs (36 ± 22 vs. 56 ± 21 cells per high-power field; p = 0.006 and FMD (5.0% ± 0.3% vs. 6.6% ± 0.3%, p < 0.0001. Tea further increased FMD 1, 2, 3, and 4 h after consumption, with maximal response 2 h after intake (p < 0.0001. Fat challenge decreased FMD, while tea consumption counteracted FMD impairment (p < 0.0001; (4 Conclusions: We demonstrated the vascular protective properties of black tea by increasing the number of CACs and preventing endothelial dysfunction induced by acute oral fat load in hypertensive patients. Considering that tea is the most consumed beverage after water, our findings are of clinical relevance and interest.

  8. Differentiation state determines neural effects on microvascular endothelial cells

    International Nuclear Information System (INIS)

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-01-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. ► Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. ► Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell production of nitric oxide. ► Neural progenitor cells and dorsal root

  9. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

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    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  10. Progenitor cells in pulmonary vascular remodeling

    Science.gov (United States)

    Yeager, Michael E.; Frid, Maria G.; Stenmark, Kurt R.

    2011-01-01

    Pulmonary hypertension is characterized by cellular and structural changes in the walls of pulmonary arteries. Intimal thickening and fibrosis, medial hypertrophy and fibroproliferative changes in the adventitia are commonly observed, as is the extension of smooth muscle into the previously non-muscularized vessels. A majority of these changes are associated with the enhanced presence of α-SM-actin+ cells and inflammatory cells. Atypical abundances of functionally distinct endothelial cells, particularly in the intima (plexiform lesions), and also in the perivascular regions, are also described. At present, neither the origin(s) of these cells nor the molecular mechanisms responsible for their accumulation, in any of the three compartments of the vessel wall, have been fully elucidated. The possibility that they arise from either resident vascular progenitors or bone marrow–derived progenitor cells is now well established. Resident vascular progenitor cells have been demonstrated to exist within the vessel wall, and in response to certain stimuli, to expand and express myofibroblastic, endothelial or even hematopoietic markers. Bone marrow–derived or circulating progenitor cells have also been shown to be recruited to sites of vascular injury and to assume both endothelial and SM-like phenotypes. Here, we review the data supporting the contributory role of vascular progenitors (including endothelial progenitor cells, smooth muscle progenitor cells, pericytes, and fibrocytes) in vascular remodeling. A more complete understanding of the processes by which progenitor cells modulate pulmonary vascular remodeling will undoubtedly herald a renaissance of therapies extending beyond the control of vascular tonicity and reduction of pulmonary artery pressure. PMID:22034593

  11. HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

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    Parisa Imanirad

    2014-01-01

    Full Text Available Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α, a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver, and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.

  12. Endothelial marker-expressing stromal cells are critical for kidney formation.

    Science.gov (United States)

    Mukherjee, Elina; Maringer, Katherine; Papke, Emily; Bushnell, Daniel; Schaefer, Caitlin; Kramann, Rafael; Ho, Jacqueline; Humphreys, Benjamin D; Bates, Carlton; Sims-Lucas, Sunder

    2017-09-01

    Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice ( Flk1 fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1 fl/fl ( Flk1 ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1 ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1 ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1 ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1 ST-/- kidneys vs. Juvenile Flk1 ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1 ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages. Copyright © 2017 the American Physiological Society.

  13. Signaling hierarchy regulating human endothelial cell development

    Science.gov (United States)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  14. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  15. Endothelial RIG-I activation impairs endothelial function

    International Nuclear Information System (INIS)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-01-01

    Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  16. Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

    Science.gov (United States)

    Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

    2015-01-01

    During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

  17. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

    Science.gov (United States)

    Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

    2015-01-01

    Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

  18. CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

    Science.gov (United States)

    Chrifi, Ihsan; Louzao-Martinez, Laura; Brandt, Maarten; van Dijk, Christian G M; Burgisser, Petra; Zhu, Changbin; Kros, Johan M; Duncker, Dirk J; Cheng, Caroline

    2017-06-01

    Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development. Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM

  19. Hedgehog Signalling in the Embryonic Mouse Thymus

    Directory of Open Access Journals (Sweden)

    Alessandro Barbarulo

    2016-07-01

    Full Text Available T cells develop in the thymus, which provides an essential environment for T cell fate specification, and for the differentiation of multipotent progenitor cells into major histocompatibility complex (MHC-restricted, non-autoreactive T cells. Here we review the role of the Hedgehog signalling pathway in T cell development, thymic epithelial cell (TEC development, and thymocyte–TEC cross-talk in the embryonic mouse thymus during the last week of gestation.

  20. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    NARCIS (Netherlands)

    Guadix, Juan Antonio; Orlova, Valeria V.; Giacomelli, Elisa; Bellin, Milena; Ribeiro, Marcelo C.; Mummery, Christine L.; Pérez-Pomares, José M.; Passier, Robert

    2017-01-01

    Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA)

  1. Signaling hierarchy regulating human endothelial cell development.

    Science.gov (United States)

    Kelly, Melissa A; Hirschi, Karen K

    2009-05-01

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

  2. Epidermal growth factor-like domain 7 is a marker of the endothelial lineage and active angiogenesis.

    Science.gov (United States)

    Bambino, Kathryn; Lacko, Lauretta A; Hajjar, Katherine A; Stuhlmann, Heidi

    2014-07-01

    Epidermal growth factor-like domain 7 (Egfl7) expression in the developing embryo is largely restricted to sites of mesodermal progenitors of angioblasts/hemangioblasts and the vascular endothelium. We hypothesize that Egfl7 marks the endothelial lineage during embryonic development, and can be used to define the emergence of endothelial progenitor cells, as well as to visualize newly-forming vasculature in the embryo and during the processes of physiologic and pathologic angiogenesis in the adult. We have generated a transgenic mouse strain that expresses enhanced green fluorescent protein (eGFP) under the control of a minimal Egfl7 regulatory sequence (Egfl7:eGFP). Expression of the transgene recapitulated that of endogenous Egfl7 at sites of vasculogenesis and angiogenesis in the allantois, yolk sac, and in the embryo proper. The transgene was not expressed in the quiescent endothelium of most adult organs. However, the uterus and ovary, which undergo vascular growth and remodeling throughout the estrus cycle, expressed high levels of Egfl7:eGFP. Importantly, expression of the Egfl7:eGFP transgene was induced in adult neovasculature. We also found that increased Egfl7 expression contributed to pathologic revascularization in the mouse retina. To our knowledge, this is the first mouse model that enables monitoring of endothelial cells at sites of active vasculogenesis and angiogenesis. This model also facilitated the isolation and characterization of EGFL7(+) endothelial cell populations by fluorescence activated cell sorting (FACS). Together, our results demonstrate that the Egfl7:eGFP reporter mouse is a valuable tool that can be used to elucidate the mechanisms by which blood vessels form during development and under pathologic circumstances. © 2014 Wiley Periodicals, Inc.

  3. Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Shunsuke Tanigawa

    2016-04-01

    Full Text Available Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine.

  4. Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

    Science.gov (United States)

    Tanigawa, Shunsuke; Taguchi, Atsuhiro; Sharma, Nirmala; Perantoni, Alan O; Nishinakamura, Ryuichi

    2016-04-26

    Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Progenitor outgrowth from the niche in Drosophila trachea is guided by FGF from decaying branches.

    Science.gov (United States)

    Chen, Feng; Krasnow, Mark A

    2014-01-10

    Although there has been progress identifying adult stem and progenitor cells and the signals that control their proliferation and differentiation, little is known about the substrates and signals that guide them out of their niche. By examining Drosophila tracheal outgrowth during metamorphosis, we show that progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. The embryonic tracheal inducer branchless FGF (fibroblast growth factor) is expressed dynamically just ahead of progenitor outgrowth in decaying branches. Knockdown of branchless abrogates progenitor outgrowth, whereas misexpression redirects it. Thus, reactivation of an embryonic tracheal inducer in decaying branches directs outgrowth of progenitors that replace them. This explains how the structure of a newly generated tissue is coordinated with that of the old.

  6. Luminal progenitors restrict their lineage potential during mammary gland development.

    Science.gov (United States)

    Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

    2015-02-01

    The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

  7. Reporter-Based Isolation of Developmental Myogenic Progenitors

    Directory of Open Access Journals (Sweden)

    Eyemen Kheir

    2018-04-01

    Full Text Available The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS. The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors.

  8. NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

    Science.gov (United States)

    Heng, Yee Hsieh Evelyn; McLeay, Robert C.; Harvey, Tracey J.; Smith, Aaron G.; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M.; Bailey, Timothy L.; Richards, Linda J.; Piper, Michael

    2014-01-01

    Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix−/− mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix−/− mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix−/− mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure. PMID:23042739

  9. PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

    Science.gov (United States)

    Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

    2016-12-15

    During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

  10. Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

    NARCIS (Netherlands)

    Krenning, Guido; Moonen, Jan-Renier A. J.; van Luyn, Marja J. A.; Harmsen, Martin C.

    The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet,

  11. Effect of β-nerve growth factor on differentiation of endothelial ...

    African Journals Online (AJOL)

    (Ad-EGFP-hβ-NGF) on the differentiation of endothelial progenitor cells (EPCs) in rats. Methods: The successfully ... may contribute to angiopoiesis or vascular repair. Keywords: β-Nerve ... angiogenesis in damaged tissues [6]. In this study ...

  12. Insights into the molecular mechanisms of diabetes-induced endothelial dysfunction

    DEFF Research Database (Denmark)

    Saad, Mohamed I.; Abdelkhalek, Taha M.; Saleh, Moustafa M.

    2015-01-01

    -associated metabolic disturbances (IR, subclinical inflammation, dyslipidemia, hyperglycemia, dysregulated production of adipokines, defective incretin and gut hormones production/action, and oxidative stress) and ED, focusing on oxidative stress and endothelial progenitor cells (EPCs). In addition, we re...

  13. Endothelial cells and hematopoiesis: a light microscopic study of fetal, normal, and pathologic human bone marrow in plastic-embedded sections.

    Science.gov (United States)

    Islam, A; Glomski, C; Henderson, E S

    1992-07-01

    The origin and morphological identity of hematopoietic progenitor cells, as well as their precursor, the pleuripotential hematopoietic stem cell (HSC), has not been established. Our studies of 2 microns sectioned undecalcified plastic-embedded bone marrow (BM) from healthy human fetuses; normal adults; patients with acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic granulocytic leukemia (CGL) in various stages (chronic, accelerated, acute blastic phase, and after autografting); and patients recovering from therapy-induced marrow hypoplasia suggest that proliferative hematopoietic zones exist near the endosteum (endosteal marrow) and the vascular endothelium (capillary and sinus-lining endothelium) and a maturational zone distal to these regions. In some of these areas, morphologically recognizable hematopoietic cells were seen and interpreted as emerging and maturing in a sequential progression, suggesting an origin from the endosteal or endothelial progenitors. In other loci, early hematopoietic cells were seen in close contact with the endosteal or vascular endothelial (VE) cells. This latter relationship suggested that these areas of cellular contact were important and represented sites of cell to cell interaction that may be associated with the liberation of growth factors by endosteal and endothelial cells and their action on hematopoietic progenitor cells. Following treatment-induced hypoplasia, the endosteal and VE cells were seen to modulate, transform, and migrate into the surrounding empty and edematous marrow space as fibroblasts. Later, as hemopoietic regeneration began, clusters of regenerating hematopoietic cells were seen adjacent to bone trabecule (BT) and near the vascular endothelium. We postulate that endosteal and VE cells are the equivalent of embryonal-stage, undifferentiated mesenchyme and, under the appropriate regulatory influence, are capable of modulation and transformation (differentiation) into stromal

  14. DNMT1 Maintains Progenitor Function in Self-Renewing Somatic Tissue

    OpenAIRE

    Sen, George L.; Reuter, Jason A.; Webster, Daniel E.; Zhu, Lilly; Khavari, Paul A.

    2010-01-01

    Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation1,2. DNA methylation3,4,5 provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1)6,7 maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintena...

  15. Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

    Science.gov (United States)

    Miao, Z G; Zhang, L P; Fu, X; Yang, Q Y; Zhu, M J; Dodson, M V; Du, M

    2016-01-01

    The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

  16. In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133

    DEFF Research Database (Denmark)

    Barraud, Perrine; Stott, Simon; Møllgård, Kjeld

    2007-01-01

    The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression....... Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain....... decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133(+)), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we...

  17. Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

    Science.gov (United States)

    Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

    2015-01-01

    ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

  18. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Juan Antonio Guadix

    2017-12-01

    Full Text Available Summary: Human pluripotent stem cells (hPSCs are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA and bone morphogenetic protein 4 (BMP4 promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (proepicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (proepicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (proepicardial-like cells displaying functional properties (adhesion and spreading over the myocardium of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (proepicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. : The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair. Keywords: human pluripotent stem cells, proepicardium, progenitor cells, cardiovascular, differentiation

  19. Engineering bone tissue from human embryonic stem cells

    OpenAIRE

    Marolt, Darja; Campos, Iván Marcos; Bhumiratana, Sarindr; Koren, Ana; Petridis, Petros; Zhang, Geping; Spitalnik, Patrice F.; Grayson, Warren L.; Vunjak-Novakovic, Gordana

    2012-01-01

    In extensive bone defects, tissue damage and hypoxia lead to cell death, resulting in slow and incomplete healing. Human embryonic stem cells (hESC) can give rise to all specialized lineages found in healthy bone and are therefore uniquely suited to aid regeneration of damaged bone. We show that the cultivation of hESC-derived mesenchymal progenitors on 3D osteoconductive scaffolds in bioreactors with medium perfusion leads to the formation of large and compact bone constructs. Notably, the i...

  20. Renal blood flow and oxygenation drive nephron progenitor differentiation.

    Science.gov (United States)

    Rymer, Christopher; Paredes, Jose; Halt, Kimmo; Schaefer, Caitlin; Wiersch, John; Zhang, Guangfeng; Potoka, Douglas; Vainio, Seppo; Gittes, George K; Bates, Carlton M; Sims-Lucas, Sunder

    2014-08-01

    During kidney development, the vasculature develops via both angiogenesis (branching from major vessels) and vasculogenesis (de novo vessel formation). The formation and perfusion of renal blood vessels are vastly understudied. In the present study, we investigated the regulatory role of renal blood flow and O2 concentration on nephron progenitor differentiation during ontogeny. To elucidate the presence of blood flow, ultrasound-guided intracardiac microinjection was performed, and FITC-tagged tomato lectin was perfused through the embryo. Kidneys were costained for the vasculature, ureteric epithelium, nephron progenitors, and nephron structures. We also analyzed nephron differentiation in normoxia compared with hypoxia. At embryonic day 13.5 (E13.5), the major vascular branches were perfused; however, smaller-caliber peripheral vessels remained unperfused. By E15.5, peripheral vessels started to be perfused as well as glomeruli. While the interior kidney vessels were perfused, the peripheral vessels (nephrogenic zone) remained unperfused. Directly adjacent and internal to the nephrogenic zone, we found differentiated nephron structures surrounded and infiltrated by perfused vessels. Furthermore, we determined that at low O2 concentration, little nephron progenitor differentiation was observed; at higher O2 concentrations, more differentiation of the nephron progenitors was induced. The formation of the developing renal vessels occurs before the onset of blood flow. Furthermore, renal blood flow and oxygenation are critical for nephron progenitor differentiation. Copyright © 2014 the American Physiological Society.

  1. DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    EMOKE PALL

    2008-05-01

    Full Text Available Embryonic stem (ES cells, the undifferentiated cells of early embryos are established as permanent lines and are characterised by their self-renewal capacity and the ability to retain their developmental capacity in vivo and in vitro. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains with inactivated genes by homologous recombination. There are several methods to induce the formation of EBs. One of them the formation by aggregating ES cells in hanging drops, using gravity as an aggregation force. This method presents the advantage of obtaining well-calibrated EBs almost identical in size. We used at our experiment the mouse ES cell line KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical examination was aimed to identify tissue-restricted proteins for the two differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A (TROMA for the presence of mesenchymal progenitor cells, Oct-4 for the presence of the undifferentiated ES cells. The beating cardiac muscle clumps showed more synchronous rhythm than those seen in EBs obtained from suspension culture method, where the beating cardiac muscle clumps appeared later, had a lower frequency and were uneven. The synaptic networks of neuronal cells were best developed in EBs from suspension, compared to those observed in EBs from hanging-drop method.

  2. A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance

    DEFF Research Database (Denmark)

    Livigni, Alessandra; Peradziryi, Hanna; Sharov, Alexei A

    2013-01-01

    BACKGROUND: The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differ...

  3. Biomechanical forces promote embryonic haematopoiesis

    Science.gov (United States)

    Adamo, Luigi; Naveiras, Olaia; Wenzel, Pamela L.; McKinney-Freeman, Shannon; Mack, Peter J.; Gracia-Sancho, Jorge; Suchy-Dicey, Astrid; Yoshimoto, Momoko; Lensch, M. William; Yoder, Mervin C.; García-Cardeña, Guillermo; Daley, George Q.

    2009-01-01

    Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system1,2. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells4. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential6. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells7,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling8, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development. PMID:19440194

  4. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  5. Extraembryonic origin of circulating endothelial cells.

    Directory of Open Access Journals (Sweden)

    Luc Pardanaud

    Full Text Available Circulating endothelial cells (CEC are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

  6. Extraembryonic origin of circulating endothelial cells.

    Science.gov (United States)

    Pardanaud, Luc; Eichmann, Anne

    2011-01-01

    Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

  7. Efficiency of rat bone marrow-derived endothelial progenitor cells on VEGF and SDF-1 expression with the aid of Tanshinone ⅡA%丹参酮ⅡA增效大鼠骨髓源性内皮祖细胞VEGF、SDF-1表达

    Institute of Scientific and Technical Information of China (English)

    杨彦; 陈庆伟; 曹广煜; 李桂琼

    2012-01-01

    Objective:To investigate the effect of rat bone marrow-derived endothelial progenitor cells on VEGF expression with the aid of Tanshinone ⅡA.Methods:The mononuclear cells were isolated from rat femoral and tibial bone marrow using percoll density gradient centrifugation,then induced with vascular endothelial growth factor(VEGF),basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF)for two weeks.The expression of cell markers was assessed by immunocytochemistry,and the attached cells were stained with Dil-acLDL and FITC-UEA-1.Then this study included two groups: EPCs and EPCs+ Tanshinone ⅡA groups.The expression levels of VEGF and SDF-1 was detected by fluorescent qRT-PCR and western blot.Results:Compared with the EPCs group,EPCs+ Tanshinone IIA group had a higher VEGF and SDF-1 expression(P0.05).Conclusions:EPCs can increase VEGF and SDF-1 expression with combination of Tanshinone IIA.Homing rate and repairing efficacy of EPCs can improve also with the aid of Tanshinone IIA.EPCs can increase VEGF and SDF-1 expression in injured myocardium after homing,thus improve angiogenesis efficiently.%目的:研究在丹参酮ⅡA的辅助下,骨髓源性内皮祖细胞(EPCs)VEGF与SDF-1表达是否能增强,从而促进细胞归巢。方法:Percoll密度梯度离心法分离SD大鼠骨髓单个核细胞,血管内皮生长因子(VEGF)、表皮生长因子((EGF)、碱性成纤维细胞生长因子(bFGF)诱导培养,并进行形态学、免疫学(免疫细胞化学染色)、功能学(Dil-acLDL与FITC-UEA-1双荧光染色)鉴定。将鉴定为EPCs的细胞分为单纯EPCs组(对照组)、EPCs+丹参酮ⅡA组(加药组)。细胞培养第9d,Western blot与实时荧光定量PCR检测各组VEGF、SDF-1基因与蛋白表达。结果:EPCs+丹参酮IIA组VEGF、SDF-1基因与蛋白表达均高于EPCs组(p均〈0.05)。结论:丹参酮ⅡA可上调大鼠骨

  8. Progenitors of white dwarfs

    International Nuclear Information System (INIS)

    Drilling, J.S.; Schoenberner, D.

    1985-01-01

    Direct observational evidence is presented which indicates that the immediate progenitors of white dwarfs are the central stars of planetary nebulae (approximately 70%), other post-AGB objects (approximately 30%), and post-HB objects not massive enough to climb the AGB (approximately 0.3%). The combined birth rate for these objects is in satisfactory agreement with the death rate of main-sequence stars and the birth rate of white dwarfs

  9. Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

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    Abir A Rahman

    2017-01-01

    Full Text Available The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain's basal ganglia circuitry. This is evidenced by the loss of these neurons, resulting in the cardinal motor deficits associated with Parkinson's disease. In order to fully understand the physiology of these key neurons and develop potential therapies for their loss, it is essential to determine if and how dopaminergic neurons are replenished in the adult brain. Recent work has presented evidence for adult neurogenesis of these neurons by Nestin+/Sox2– neural progenitor cells. We sought to further validate this finding and explore a potential atypical origin for these progenitor cells. Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin+, we hypothesized that dopaminergic neural progenitors might share a common cell lineage. Therefore, we employed a VE-cadherin promoter-driven CREERT2:THlox/THlox transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals. After 26 weeks, but not 13 weeks, following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin+ cells, we observed a significant reduction in tyrosine hydroxylase+ neurons in the substantia nigra. The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin+ progenitor cell population potentially arising from an endothelial lineage.

  10. On the genesis of articular cartilage. Embryonic joint development and gene expression - implications for tissue engineering

    NARCIS (Netherlands)

    Jenner, F

    2013-01-01

    Articular chondrocytes descend from a distinct cohort of progenitor cells located in the embryonic joint anlagen, termed interzones. Their unique lineage might explain some of the problems encountered using chondrocytes of different lineages for articular cartilage tissue engineering. While it is

  11. Oxidative stress tolerance of early stage diabetic endothelial progenitor cell

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    Dewi Sukmawati

    2015-06-01

    Conclusions: Primitive BM-EPCs showed vasculogenic dysfunction in early diabetes. However the oxidative stress is not denoted as the major initiating factor of its cause. Our results suggest that primitive BM-KSL cell has the ability to compensate oxidative stress levels in early diabetes by increasing the expression of anti-oxidative enzymes.

  12. HOXB4 Promotes Hemogenic Endothelium Formation without Perturbing Endothelial Cell Development

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    Nadine Teichweyde

    2018-03-01

    Full Text Available Summary: Generation of hematopoietic stem cells (HSCs from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs. Through the use of Runx1(−/− ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression. : In this article, Klump and colleagues demonstrate that the human homeotic selector protein HOXB4 promotes ESC-derived hematopoiesis by inducing hemogenic endothelium formation, in vitro. It propels hematopoietic specification by upregulating the transcription of genes essential for hematopoietic development, such as those encoding members of the so-called heptad transcription factors. Keywords: HOXB4, hematopoietic stem cells, hemangioblast, hemogenic endothelium, hematopoietic specification, EHT, RUNX1, pluripotent stem cells

  13. PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

    Science.gov (United States)

    Gaber, Zachary B; Butler, Samantha J; Novitch, Bennett G

    2013-10-01

    Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

  14. Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls.

    Science.gov (United States)

    Tian, Xinghui; Kaufman, Dan S

    2008-07-01

    Hematopoietic development from embryonic stem cells has been one of the most productive areas of stem cell biology. Recent studies have progressed from work with mouse to human embryonic stem cells. Strategies to produce defined blood cell populations can be used to better understand normal and abnormal hematopoiesis, as well as potentially improve the generation of hematopoietic cells with therapeutic potential. Molecular profiling, phenotypic and functional analyses have all been utilized to demonstrate that hematopoietic cells derived from embryonic stem cells most closely represent a stage of hematopoiesis that occurs at embryonic/fetal developmental stages. Generation of hematopoietic stem/progenitor cells comparable to hematopoietic stem cells found in the adult sources, such as bone marrow and cord blood, still remains challenging. However, genetic manipulation of intrinsic factors during hematopoietic differentiation has proven a suitable approach to induce adult definitive hematopoiesis from embryonic stem cells. Concrete evidence has shown that embryonic stem cells provide a powerful approach to study the early stage of hematopoiesis. Multiple hematopoietic lineages can be generated from embryonic stem cells, although most of the evidence suggests that hematopoietic development from embryonic stem cells mimics an embryonic/fetal stage of hematopoiesis.

  15. Pdgfrb-Cre targets lymphatic endothelial cells of both venous and non-venous origins.

    Science.gov (United States)

    Ulvmar, Maria H; Martinez-Corral, Ines; Stanczuk, Lukas; Mäkinen, Taija

    2016-06-01

    The Pdgfrb-Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb-Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb-Cre does not, however, target YS HemEC or YS-derived erythro-myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous-derived LECs were targeted. Assessment of temporal Cre activity using the R26-mTmG double reporter suggested recent occurrence of Pdgfrb-Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb-Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb-Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb-Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb-Cre also warrants consideration for its use in studies of mural cells. genesis 54:350-358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

  16. Masses of supernova progenitors

    International Nuclear Information System (INIS)

    Tinsley, B.M.

    1977-01-01

    The possible nature and masses of supernovae progenitors, and the bearing of empirical results on some unsolved theoretical problems concerning the origin of supernovae, are discussed. The author concentrates on two main questions: what is the lower mass limit for stars to die explosively and what stars initiate type I supernovae. The evidence considered includes local supernova rates, empirical estimates of msub(w) (the upper mass limit for death as a white dwarf), the distributions of supernovae among stellar populations in galaxies and the colors of supernova producing galaxies. (B.D.)

  17. Hmga2 regulates self-renewal of retinal progenitors.

    Science.gov (United States)

    Parameswaran, Sowmya; Xia, Xiaohuan; Hegde, Ganapati; Ahmad, Iqbal

    2014-11-01

    In vertebrate retina, histogenesis occurs over an extended period. To sustain the temporal generation of diverse cell types, retinal progenitor cells (RPCs) must self-renew. However, self-renewal and regulation of RPCs remain poorly understood. Here, we demonstrate that cell-extrinsic factors coordinate with the epigenetic regulator high-mobility group AT-hook 2 (Hmga2) to regulate self-renewal of late retinal progenitor cells (RPCs). We observed that a small subset of RPCs was capable of clonal propagation and retained multipotentiality of parents in the presence of endothelial cells (ECs), known self-renewal regulators in various stem cell niches. The self-renewing effects, also observed in vivo, involve multiple intercellular signaling pathways, engaging Hmga2. As progenitors exhaust during retinal development, expression of Hmga2 progressively decreases. Analyses of Hmga2-expression perturbation, in vitro and in vivo, revealed that Hmga2 functionally helps to mediate cell-extrinsic influences on late-retinal progenitor self-renewal. Our results provide a framework for integrating the diverse intercellular influences elicited by epigenetic regulators for self-renewal in a dynamic stem cell niche: the developing vertebrate retina. © 2014. Published by The Company of Biologists Ltd.

  18. Development and molecular composition of the hepatic progenitor cell niche.

    Science.gov (United States)

    Vestentoft, Peter Siig

    2013-05-01

    , we examined several genes upregulated in a global gene expression array conducted on one of these models, in which progenitor cells are activated. The protein expression patterns were evaluated in our collections of human embryonic and fetal livers, human liver diseases, and rodent hepatic injury models. When analyzing standard histological liver sections underlying connections and tissue architecture are not immediately evident. We therefore developed models for digitally reconstructing not only protein expression in serially cut tissue sections, but also vessels of the portal area. Article I constituted our earliest attempts to create 3D reconstructions of biological material. Human embryonic stem cell cultures were previously thought to consist of homogenously undifferentiated cells. The protocols for 3D reconstructions developed in this study demonstrated micro heterogeneity in expression of differentiation markers and provided the basis for later reconstructions of hepatic tissues. In article II we examined the expression patterns of chosen proteins seen upregulated in the gene array as well as classical hepatocytic and cholangiocytic markers in human liver disease and during prenatal development. Previous studies had indicated direct connections between activated progenitor cells apparently isolated in the parenchyma and the intrahepatic biliary tree. Our developed protocols for 3D reconstructions visually demonstrated direct connections between these entities. Analysis of protein expression in prenatal liver revealed the formation of the intrahepatic tree to occur through a special form of asymmetric tubulogenesis, only recently described in mice. In order to describe the composition of the hepatic progenitor cell niche and the localization of cell surface proteins in article III, the expression patterns of certain genes upregulated in the gene array analysis were analyzed in different models of rodent liver regeneration. We observed that the extracellular

  19. Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

    Science.gov (United States)

    Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

    2009-07-01

    Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

  20. Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

    Directory of Open Access Journals (Sweden)

    Baumann Gert

    2010-05-01

    Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

  1. Tbx1 coordinates addition of posterior second heart field progenitor cells to the arterial and venous poles of the heart

    NARCIS (Netherlands)

    Rana, M. Sameer; Théveniau-Ruissy, Magali; de Bono, Christopher; Mesbah, Karim; Francou, Alexandre; Rammah, Mayyasa; Domínguez, Jorge N.; Roux, Marine; Laforest, Brigitte; Anderson, Robert H.; Mohun, Timothy; Zaffran, Stephane; Christoffels, Vincent M.; Kelly, Robert G.

    2014-01-01

    Cardiac progenitor cells from the second heart field (SHF) contribute to rapid growth of the embryonic heart, giving rise to right ventricular and outflow tract (OFT) myocardium at the arterial pole of the heart, and atrial myocardium at the venous pole. Recent clonal analysis and cell-tracing

  2. Porcine embryonic stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane

    2008-01-01

    The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

  3. Live imaging of mitosis in the developing mouse embryonic cortex.

    Science.gov (United States)

    Pilaz, Louis-Jan; Silver, Debra L

    2014-06-04

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

  4. [Circulating endothelial cells: biomarkers for monitoring activity of antiangiogenic therapy].

    Science.gov (United States)

    Farace, Françoise; Bidart, Jean-Michel

    2007-07-01

    Tumor vessel formation is largely dependent on the recruitment of endothelial cells. Rare in healthy individuals, circulating endothelial cells (CEC) are shed from vessel walls and enter the circulation reflecting endothelial damage or dysfunction. Increased numbers of CEC have been documented in different types of cancer. Recent studies have suggested the role for CEC in tumor angiogenesis, but whose presence could also reflect normal endothelium perturbation in cancer. Originating from the bone marrow rather than from vessel walls, endothelial progenitor cells (EPC) are mobilized following tissue ischemia and may be recruited to complement local angiogenesis supplied by existing endothelium. Recently, studies in mouse models suggest that the circulating fraction of endothelial progenitors (CEP) is involved in tumor angiogenesis but their contribution is less clear in humans. The detection of CEC and CEP is difficult and impeded by the rarity of these cells. They may have important clinical implication as novel biomarkers susceptible to predict more efficiently and rapidly the therapeutic response to anti-angiogenic treatments. However, a methodological consensus would be necessary in order to correctly evaluate the clinical interest of CEC and CEP in patients.

  5. 8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Lifeng [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Zhou, Yong [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Yu, Shanhe [Shanghai Institute of Hematology, RuiJin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025 (China); Ji, Guixiang [Nanjing Institute of Environmental Sciences/Key Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection, Nanjing 210042 (China); Wang, Lei [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Liu, Wei [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Gu, Aihua, E-mail: aihuagu@njmu.edu.cn [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China)

    2013-11-15

    Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.

  6. Absence of Rybp Compromises Neural Differentiation of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Gergo Kovacs

    2016-01-01

    Full Text Available Rybp (Ring1 and Yy1 Binding Protein is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs, exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp+/+ and rybp null mutant (rybp-/- embryonic stem cells (ESCs and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found that rybp null mutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack of rybp coincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes.

  7. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    Science.gov (United States)

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Canonical Wnt signaling transiently stimulates proliferation and enhances neurogenesis in neonatal neural progenitor cultures

    International Nuclear Information System (INIS)

    Hirsch, Cordula; Campano, Louise M.; Woehrle, Simon; Hecht, Andreas

    2007-01-01

    Canonical Wnt signaling triggers the formation of heterodimeric transcription factor complexes consisting of β-catenin and T cell factors, and thereby controls the execution of specific genetic programs. During the expansion and neurogenic phases of embryonic neural development canonical Wnt signaling initially controls proliferation of neural progenitor cells, and later neuronal differentiation. Whether Wnt growth factors affect neural progenitor cells postnatally is not known. Therefore, we have analyzed the impact of Wnt signaling on neural progenitors isolated from cerebral cortices of newborn mice. Expression profiling of pathway components revealed that these cells are fully equipped to respond to Wnt signals. However, Wnt pathway activation affected only a subset of neonatal progenitors and elicited a limited increase in proliferation and neuronal differentiation in distinct subsets of cells. Moreover, Wnt pathway activation only transiently stimulated S-phase entry but did not support long-term proliferation of progenitor cultures. The dampened nature of the Wnt response correlates with the predominant expression of inhibitory pathway components and the rapid actuation of negative feedback mechanisms. Interestingly, in differentiating cell cultures activation of canonical Wnt signaling reduced Hes1 and Hes5 expression suggesting that during postnatal neural development, Wnt/β-catenin signaling enhances neurogenesis from progenitor cells by interfering with Notch pathway activity

  9. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

    Science.gov (United States)

    Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

    2016-01-06

    Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. DNMT1 maintains progenitor function in self-renewing somatic tissue.

    Science.gov (United States)

    Sen, George L; Reuter, Jason A; Webster, Daniel E; Zhu, Lilly; Khavari, Paul A

    2010-01-28

    Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

  11. Requirement of mouse BCCIP for neural development and progenitor proliferation.

    Directory of Open Access Journals (Sweden)

    Yi-Yuan Huang

    Full Text Available Multiple DNA repair pathways are involved in the orderly development of neural systems at distinct stages. The homologous recombination (HR pathway is required to resolve stalled replication forks and critical for the proliferation of progenitor cells during neural development. BCCIP is a BRCA2 and CDKN1A interacting protein implicated in HR and inhibition of DNA replication stress. In this study, we determined the role of BCCIP in neural development using a conditional BCCIP knock-down mouse model. BCCIP deficiency impaired embryonic and postnatal neural development, causing severe ataxia, cerebral and cerebellar defects, and microcephaly. These development defects are associated with spontaneous DNA damage and subsequent cell death in the proliferative cell populations of the neural system during embryogenesis. With in vitro neural spheroid cultures, BCCIP deficiency impaired neural progenitor's self-renewal capability, and spontaneously activated p53. These data suggest that BCCIP and its anti-replication stress functions are essential for normal neural development by maintaining an orderly proliferation of neural progenitors.

  12. Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

    2017-01-01

    Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

  13. Secondhand smoke exposure and endothelial stress in children and adolescents.

    Science.gov (United States)

    Groner, Judith A; Huang, Hong; Nagaraja, Haikady; Kuck, Jennifer; Bauer, John Anthony

    2015-01-01

    Links between secondhand smoke exposure and cardiovascular disease in adults are well established. Little is known about the impact of this exposure on cardiovascular status during childhood. The purpose of this study was to investigate relationships between secondhand smoke exposure in children and adolescents and cardiovascular disease risk--systemic inflammation, endothelial stress, and endothelial repair. A total of 145 subjects, aged 9 to 18 years, were studied. Tobacco smoke exposure was determined by hair nicotine level. Cardiovascular risk was assessed by markers of systemic inflammation (C-reactive protein [CRP] and adiponectin); by soluble intercellular adhesion molecule 1 (s-ICAM1), which measures endothelial activation after surface vascular injury; and by endothelial repair. This was measured by prevalence of endothelial progenitor cells (EPCs), which are bone marrow-derived cells that home preferentially to sites of vascular damage. Hair nicotine was directly correlated with s-ICAM1 (r = 0.4090, P Secondhand smoke exposure during childhood and adolescence is detrimental to vascular health because s-ICAM1 is a marker for endothelial activation and stress after vascular surface injury, and EPCs contribute to vascular repair. The fact that body mass index is also a factor in the model predicting s-ICAM1 is concerning, in that 2 risk factors may both contribute to endothelial stress. Copyright © 2015 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

  14. Role of liver progenitors in liver regeneration.

    Science.gov (United States)

    Best, Jan; Manka, Paul; Syn, Wing-Kin; Dollé, Laurent; van Grunsven, Leo A; Canbay, Ali

    2015-02-01

    During massive liver injury and hepatocyte loss, the intrinsic regenerative capacity of the liver by replication of resident hepatocytes is overwhelmed. Treatment of this condition depends on the cause of liver injury, though in many cases liver transplantation (LT) remains the only curative option. LT for end stage chronic and acute liver diseases is hampered by shortage of donor organs and requires immunosuppression. Hepatocyte transplantation is limited by yet unresolved technical difficulties. Since currently no treatment is available to facilitate liver regeneration directly, therapies involving the use of resident liver stem or progenitor cells (LPCs) or non-liver stem cells are coming to fore. LPCs are quiescent in the healthy liver, but may be activated under conditions where the regenerative capacity of mature hepatocytes is severely impaired. Non-liver stem cells include embryonic stem cells (ES cells) and mesenchymal stem cells (MSCs). In the first section, we aim to provide an overview of the role of putative cytokines, growth factors, mitogens and hormones in regulating LPC response and briefly discuss the prognostic value of the LPC response in clinical practice. In the latter section, we will highlight the role of other (non-liver) stem cells in transplantation and discuss advantages and disadvantages of ES cells, induced pluripotent stem cells (iPS), as well as MSCs.

  15. Identification of derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization

    International Nuclear Information System (INIS)

    Ran Yuliang; Jiang Yangfu; Zhong Xing; Zhou Zhuan; Liu Haiyan; Hu Hai; Lou Jinning; Yang Zhihua

    2006-01-01

    Endothelial cells play an important regulatory role in embryonic development, reproductive functions, tumor growth and progression. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify differentially expressed genes between non-stimulated endothelial cells and activated endothelial cells. Following mRNA isolation of non-stimulated and hepatocellular carcinoma homogenate-stimulated cells, cDNAs of both populations were prepared and subtracted by suppressive PCR. Sequencing of the enriched cDNAs identified a couple of genes differentially expressed, including derlin-1. Derlin-1 was significantly up-regulated by tumor homogenates, VEGF, and endothelial growth supplements in a dose-dependent manner. Knock-down of derlin-1 triggered endothelial cell apoptosis, inhibited endothelial cell proliferation, and blocked the formation of a network of tubular-like structures. Our data reveal that derlin-1 is a novel growth factor-responsive endothelial antigen that promotes endothelial cell survival and growth

  16. Progenitor cells for regenerative medicine and consequences of ART and cloning-associated epimutations.

    Science.gov (United States)

    Laprise, Shari L

    2010-06-01

    The "holy grail" of regenerative medicine is the identification of an undifferentiated progenitor cell that is pluripotent, patient specific, and ethically unambiguous. Such a progenitor cell must also be able to differentiate into functional, transplantable tissue, while avoiding the risks of immune rejection. With reports detailing aberrant genomic imprinting associated with assisted reproductive technologies (ART) and reproductive cloning, the idea that human embryonic stem cells (hESCs) derived from surplus in vitro fertilized embryos or nuclear transfer ESCs (ntESCs) harvested from cloned embryos may harbor dangerous epigenetic errors has gained attention. Various progenitor cell sources have been proposed for human therapy, from hESCs to ntESCs, and from adult stem cells to induced pluripotent stem cells (iPS and piPS cells). This review highlights the advantages and disadvantages of each of these technologies, with particular emphasis on epigenetic stability.

  17. The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions

    NARCIS (Netherlands)

    Gremmels, Hendrik; De Jong, Olivier G.; Hazenbrink, Diënty H.; Fledderus, Joost O.; Verhaar, Marianne C.

    2017-01-01

    Background. Endothelial colony forming cells (ECFCs) have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS). The transcription factor Nrf2

  18. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...... germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth...

  19. Mesenchymal progenitor cells for the osteogenic lineage.

    Science.gov (United States)

    Ono, Noriaki; Kronenberg, Henry M

    2015-09-01

    Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

  20. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

    Directory of Open Access Journals (Sweden)

    Akira Niwa

    Full Text Available Elucidating the in vitro differentiation of human embryonic stem (ES and induced pluripotent stem (iPS cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

  1. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

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    Michele Bertacchi

    2015-10-01

    Full Text Available Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

  2. Towards the therapeutic use of vascular smooth muscle progenitor cells.

    Science.gov (United States)

    Merkulova-Rainon, Tatyana; Broquères-You, Dong; Kubis, Nathalie; Silvestre, Jean-Sébastien; Lévy, Bernard I

    2012-07-15

    Recent advances in the development of alternative proangiogenic and revascularization processes, including recombinant protein delivery, gene therapy, and cell therapy, hold the promise of greater efficacy in the management of cardiovascular disease in the coming years. In particular, vascular progenitor cell-based strategies have emerged as an efficient treatment approach to promote vessel formation and repair and to improve tissue perfusion. During the past decade, considerable progress has been achieved in understanding therapeutic properties of endothelial progenitor cells, while the therapeutic potential of vascular smooth muscle progenitor cells (SMPC) has only recently been explored; the number of the circulating SMPC being correlated with cardiovascular health. Several endogenous SMPC populations with varying phenotypes have been identified and characterized in the peripheral blood, bone marrow, and vascular wall. While the phenotypic entity of vascular SMPC is not fully defined and remains an evolving area of research, SMPC are increasingly recognized to play a special role in cardiovascular biology. In this review, we describe the current approaches used to define vascular SMPC. We further summarize the data on phenotype and functional properties of SMPC from various sources in adults. Finally, we discuss the role of SMPC in cardiovascular disease, including the contribution of SMPC to intimal proliferation, angiogenesis, and atherosclerotic plaque instability as well as the benefits resulting from the therapeutic use of SMPC.

  3. Carcino-Embryonic Antigen

    International Nuclear Information System (INIS)

    Akute, O.

    1999-02-01

    Tumour marker analysis has increased our understanding of the presence of tumours in the body. Carcino-embryonic antigen, CEA, is one of the best studied tumour markers and has proved an ideal diagnostic adjuvant. It has helped in quantifying the amount of disease present in a patient and thence to make accurate prognosis on the various diagnosed ailments. At UCH, it is observed that there is an increase in cancer related ailments and therefore the need for early diagnosis is more compelling in our environment to mitigate future cost of managing advanced manifestation

  4. The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

    International Nuclear Information System (INIS)

    Clayton, Elizabeth; Forbes, Stuart J.

    2009-01-01

    The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1 + CD45 - cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1 + cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture or as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1 + cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.

  5. Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

    Directory of Open Access Journals (Sweden)

    Nicolas Christoforou

    Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

  6. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

    Science.gov (United States)

    El Harane, Nadia; Kervadec, Anaïs; Bellamy, Valérie; Pidial, Laetitia; Neametalla, Hany J; Perier, Marie-Cécile; Lima Correa, Bruna; Thiébault, Léa; Cagnard, Nicolas; Duché, Angéline; Brunaud, Camille; Lemitre, Mathilde; Gauthier, Jeanne; Bourdillon, Alexandra T; Renault, Marc P; Hovhannisyan, Yeranuhi; Paiva, Solenne; Colas, Alexandre R; Agbulut, Onnik; Hagège, Albert; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

    2018-05-21

    We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left

  7. The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages.

    Science.gov (United States)

    Xie, Jingwei; Willerth, Stephanie M; Li, Xiaoran; Macewan, Matthew R; Rader, Allison; Sakiyama-Elbert, Shelly E; Xia, Younan

    2009-01-01

    Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.

  8. Embryonic Blood-Cerebrospinal Fluid Barrier Formation and Function

    Directory of Open Access Journals (Sweden)

    David eBueno

    2014-10-01

    Full Text Available During embryonic development and adult life, brain cavities and ventricles are filled with cerebrospinal fluid (CSF. CSF has attracted interest as an active signaling medium that regulates brain development, homeostasis and disease. CSF is a complex protein-rich fluid containing growth factors and signaling molecules that regulate multiple cell functions in the central nervous system (CNS. The composition and substance concentrations of CSF are tightly controlled. In recent years, it has been demonstrated that embryonic CSF (eCSF has a key function as a fluid pathway for delivering diffusible signals to the developing brain, thus contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the expansion and patterning of the brain. From fetal stages through to adult life, CSF is primarily produced by the choroid plexus. The development and functional activities of the choroid plexus and other blood–brain barrier (BBB systems in adults and fetuses have been extensively analyzed. However, eCSF production and control of its homeostasis in embryos, from the closure of the anterior neuropore when the brain cavities become physiologically sealed, to the formation of the functional fetal choroid plexus, has not been studied in as much depth and remains open to debate. This review brings together the existing literature, some of which is based on experiments conducted by our research group, concerning the formation and function of a temporary embryonic blood–CSF barrier in the context of the crucial roles played by the molecules in eCSF.

  9. Sphingosine-1-Phosphate (S1P) Signaling in Neural Progenitors.

    Science.gov (United States)

    Callihan, Phillip; Alqinyah, Mohammed; Hooks, Shelley B

    2018-01-01

    Sphingosine-1-phosphate (S1P) and its receptors are important in nervous system development. Reliable in vitro human model systems are needed to further define specific roles for S1P signaling in neural development. We have described S1P-regulated signaling, survival, and differentiation in a human embryonic stem cell-derived neuroepithelial progenitor cell line (hNP1) that expresses functional S1P receptors. These cells can be further differentiated to a neuronal cell type and therefore represent a good model system to study the role of S1P signaling in human neural development. The following sections describe in detail the culture and differentiation of hNP1 cells and two assays to measure S1P signaling in these cells.

  10. [Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

    Science.gov (United States)

    Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

    2008-10-14

    To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

  11. Ablation of cdk4 and cdk6 affects proliferation of basal progenitor cells in the developing dorsal and ventral forebrain.

    Science.gov (United States)

    Grison, Alice; Gaiser, Carine; Bieder, Andrea; Baranek, Constanze; Atanasoski, Suzana

    2018-03-23

    Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin-dependent kinase (cdk) family using cdk-deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018. © 2018 Wiley Periodicals, Inc.

  12. Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Hongyan Tao

    2018-01-01

    Full Text Available With their properties of self-renewal and differentiation, embryonic stem (ES cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc and red fluorescent protein (RFP to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc, was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

  13. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

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    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  14. Gene expression response to EWS–FLI1 in mouse embryonic cartilage

    Directory of Open Access Journals (Sweden)

    Miwa Tanaka

    2014-12-01

    Full Text Available Ewing's sarcoma is a rare bone tumor that affects children and adolescents. We have recently succeeded to induce Ewing's sarcoma-like small round cell tumor in mice by expression of EWS–ETS fusion genes in murine embryonic osteochondrogenic progenitors. The Ewing's sarcoma precursors are enriched in embryonic superficial zone (eSZ cells of long bone. To get insights into the mechanisms of Ewing's sarcoma development, gene expression profiles between EWS–FLI1-sensitive eSZ cells and EWS–FLI1-resistant embryonic growth plate (eGP cells were compared using DNA microarrays. Gene expression of eSZ and eGP cells (total, 30 samples was evaluated with or without EWS–FLI1 expression 0, 8 or 48 h after gene transduction. Our data provide useful information for gene expression responses to fusion oncogenes in human sarcoma.

  15. Nephron progenitor cell death elicits a limited compensatory response associated with interstitial expansion in the neonatal kidney

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    Sree Deepthi Muthukrishnan

    2018-01-01

    Full Text Available The final nephron number in an adult kidney is regulated by nephron progenitor cell availability and collecting duct branching in the fetal period. Fetal environmental perturbations that cause reductions in cell numbers in these two compartments result in low nephron endowment. Previous work has shown that maternal dietary factors influence nephron progenitor cell availability, with both caloric restriction and protein deprivation leading to reduced cell numbers through apoptosis. In this study, we evaluate the consequences of inducing nephron progenitor cell death on progenitor niche dynamics and on nephron endowment. Depletion of approximately 40% of nephron progenitor cells by expression of diphtheria toxin A at embryonic day 15 in the mouse results in 10-20% nephron reduction in the neonatal period. Analysis of cell numbers within the progenitor cell pool following induction of apoptosis reveals a compensatory response in which surviving progenitor cells increase their proliferation and replenish the niche. The proliferative response is temporally associated with infiltration of macrophages into the nephrogenic zone. Colony stimulating factor 1 (CSF1 has a mitogenic effect on nephron progenitor cells, providing a potential explanation for the compensatory proliferation. However, CSF1 also promotes interstitial cell proliferation, and the compensatory response is associated with interstitial expansion in recovering kidneys which can be pharmacologically inhibited by treatment with clodronate liposomes. Our findings suggest that the fetal kidney employs a macrophage-dependent compensatory regenerative mechanism to respond to acute injury caused by death of nephron progenitor cells, but that this regenerative response is associated with neonatal interstitial expansion.

  16. Nitric oxide synthase-3 promotes embryonic development of atrioventricular valves.

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    Yin Liu

    Full Text Available Nitric oxide synthase-3 (NOS3 has recently been shown to promote endothelial-to-mesenchymal transition (EndMT in the developing atrioventricular (AV canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT and NOS3(-/- mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/- compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/- mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+ cells in the AV cushion were decreased in NOS3(-/- compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ, bone morphogenetic protein (BMP2 and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/- compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.

  17. Lin28 sustains early renal progenitors and induces Wilms tumor

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    Urbach, Achia; Yermalovich, Alena; Zhang, Jin; Spina, Catherine S.; Zhu, Hao; Perez-Atayde, Antonio R.; Shukrun, Rachel; Charlton, Jocelyn; Sebire, Neil; Mifsud, William; Dekel, Benjamin; Pritchard-Jones, Kathy; Daley, George Q.

    2014-01-01

    Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. Here we show that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, we observed Wilms tumor only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. We show that withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures and that tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, we demonstrate overexpression of the LIN28B paralog in a significant percentage of human Wilms tumor. Our data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis. PMID:24732380

  18. Haematopoietic stem and progenitor cells from human pluripotent stem cells

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    Sugimura, Ryohichi; Jha, Deepak Kumar; Han, Areum; Soria-Valles, Clara; da Rocha, Edroaldo Lummertz; Lu, Yi-Fen; Goettel, Jeremy A.; Serrao, Erik; Rowe, R. Grant; Malleshaiah, Mohan; Wong, Irene; Sousa, Patricia; Zhu, Ted N.; Ditadi, Andrea; Keller, Gordon; Engelman, Alan N.; Snapper, Scott B.; Doulatov, Sergei; Daley, George Q.

    2018-01-01

    A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. PMID:28514439

  19. Physalis minima Leaves Extract Induces Re-Endothelialization in Deoxycorticosterone Acetate-Salt-Induced Endothelial Dysfunction in Rats

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    Dian Nugrahenny

    2018-02-01

    Full Text Available The administration of deoxy-corticosterone acetate (DOCA-salt can induce oxidative stress leading to decrease the bioavailability of nitric oxide (NO, increase senescence of circulating endothelial progenitor cells (EPCs, thus contributing to endothelial dysfunction. This study was aimed to investigate the effects of Physalis minima L. leaves extract on serum NO levels, circulating EPCs number, and histopathology of tail artery endothelial cells in DOCA-salt-induced endothelial dysfunction in rats. Twenty-five male Wistar rats were randomly divided into five groups: rats without any treatment (normal, rats treated with DOCA (10 mg/kgBW s.c. twice weekly and given 0.9% NaCl to drink ad libitum for 6 weeks, and DOCA-salt-induced rats orally supplemented with P. minima leaves extract at doses of 500, 1500, or 2500 mg/kgBW for 4 weeks. Serum NO levels were measured by colorimetry. The number of circulating EPCs (CD34+/CD133+ cells was determined by flow cytometry. The tail artery sections were histologically processed with hematoxylin-eosin staining. DOCA-salt-induced rats showed significantly (p<0.05 decrease in serum NO levels and circulating EPCs number compared to the normal. There was also more detached tail artery endothelial cells in DOCA-salt-induced rats. P. minima leaves extract at a dose of 500 mg/kgBW significantly (p<0.05 increased serum NO level and circulating EPCs number, and also induced an optimal re-endothelialization in DOCA-salt-induced rats. P. minima leave extract dose-dependently increases NO bioavailability contributing to enhanced EPCs mobilization, thereby promoting re-endothelialization in DOCA-salt-induced endothelial dysfunction in rats.

  20. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

    International Nuclear Information System (INIS)

    Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko; Teramoto, Kenichi; Nishida, Tomohiro; Shimizu-Saito, Keiko; Ota, Masato; Eto, Kazuhiro; Teraoka, Hirobumi

    2009-01-01

    Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

  1. Synergistic actions of hematopoietic and mesenchymal stem/progenitor cells in vascularizing bioengineered tissues.

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    Eduardo K Moioli

    Full Text Available Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs and mesenchymal stem/progenitor cells (MSCs were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP scaffolds, followed by infusion of gel-suspended CD34(+ hematopoietic cells. Co-transplantation of CD34(+ HSCs and CD34(- MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromised mice yielded vascularized tissue. The average vascular number of co-transplanted CD34(+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34(+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34(+ cells. Based on additional in vitro results of endothelial differentiation of CD34(+ cells by vascular endothelial growth factor (VEGF, we adsorbed VEGF with co-transplanted CD34(+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34(+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone

  2. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

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    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  3. Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

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    Forrester Jeff

    2009-09-01

    Full Text Available Abstract Background Neural differentiation of embryonic stem (ES cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming. Results Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of Oct4 and NANOG and increased expression of nestin. ES cells containing a GFP gene under the control of the Sox1 regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents. Conclusion Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.

  4. The poster as modernist progenitor

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    Katherine Hauser

    2015-12-01

    Full Text Available Ruth E. Iskin’s The Poster: Art, Advertising. Design, and Collecting, 1860s-1900s positions the late-nineteenth-century advertising poster as the progenitor of valued modernist practices typically attached solely to photography and film. Modernist biases separating high art from mass culture account for scholars ignoring posters, however the poster ushered in an innovative reductive graphic style as well as pioneered the notion of multiple originals.

  5. Embryonic duplications in sheep.

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    Dennis, S M

    1975-02-01

    Twenty-seven embryonic duplications were examined during a 3-year investigation into the causes of perinatal lamb mortality. Twenty of the 27 were anomalous twins with 19 being conjoined (diplopagus 9 and heteropagus 10). The various duplications were: haloacardius acephalus 1, diprosopus 2, dicephalus 2, dipypus 3, diprosopus dipygus 1, syncephalus dipygus 1, pygopagus parasiticus 1, heteropagus dipygus 3, melodidymus 6, polyury 4, penile duplication 2, and bilateral otognathia 1. Four lambs were living and the time of death of the others was: parturient 8, and post-parturient 15. Average dry weight of the lambs was 3.35 kg (range 1.59 to 5.45 kg). Breed distribution was: Merino 77.8%, Crossbred 14.8%, Dorset Horn 3.7%, and Corriedale 3.7%. The caudal region was involved in 10 of the conjoined twins (52.6%), anterior region in 7 (36.9%), and both anterior and caudal regions in 2 (10.5%). Associated defects were present in 70.4% of the 27 lambs, the most common being atresia ani.

  6. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

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    Moriya, Takashi J., E-mail: takashi.moriya@ipmu.jp [Kavli Institute for the Physics and Mathematics of the Universe, Todai Institutes for Advanced Study, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8583 (Japan)

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  7. Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors.

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    Takeshi Takarada

    Full Text Available BACKGROUND: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. METHODOLOGY/PRINCIPAL FINDINGS: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl-N-methyl-(3E-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice

  8. Autoantigens targeted in scleroderma patients with vascular disease are enriched in endothelial lineage cells

    Science.gov (United States)

    McMahan, Zsuzsanna H.; Cottrell, Tricia R.; Wigley, Fredrick M.; Antiochos, Brendan; Zambidis, Elias T.; Park, Tea Soon; Halushka, Marc K.; Gutierrez-Alamillo, Laura; Cimbro, Raffaello; Rosen, Antony; Casciola-Rosen, Livia

    2016-01-01

    Objective Scleroderma patients with autoantibodies to centromere proteins (CENPs) and/or interferon-inducible protein 16 (IFI16) are at increased risk of severe vascular complications. We set out to define whether these autoantigens are enriched in cells of the vasculature. Methods Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI16 and CD31 expression were defined in skin paraffin sections from scleroderma patients and from healthy controls. IFI16 expression was determined by flow cytometry in circulating endothelial cells (CECs) and circulating progenitor cells (CPCs). Results Expression of CENP-A, IFI16 and CD31 was enriched in EBs at days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI16, CD31, CENPs A and-B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of skin paraffin sections showed enrichment of IFI16 in CD31-positive vascular endothelial cells in biopsies from scleroderma patients and normal controls. Flow cytometry analysis revealed IFI16 expression in CPCs, but minimal expression in CECs. Conclusion Expression of scleroderma autoantigens IFI16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens. PMID:27159521