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Sample records for vessels endothelial cells

  1. Dynamic Endothelial Cell Rearrangements Drive Developmental Vessel Regression

    Science.gov (United States)

    Franco, Claudio A.; Jones, Martin L.; Bernabeu, Miguel O.; Geudens, Ilse; Mathivet, Thomas; Rosa, Andre; Lopes, Felicia M.; Lima, Aida P.; Ragab, Anan; Collins, Russell T.; Phng, Li-Kun; Coveney, Peter V.; Gerhardt, Holger

    2015-01-01

    Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments. PMID:25884288

  2. Role of endothelial cell metabolism in vessel sprouting.

    Science.gov (United States)

    De Bock, Katrien; Georgiadou, Maria; Carmeliet, Peter

    2013-11-05

    Endothelial cells (ECs) are quiescent for years but can plastically switch to angiogenesis. Vascular sprouting relies on the coordinated activity of migrating tip cells at the forefront and proliferating stalk cells that elongate the sprout. Past studies have identified genetic signals that control vascular branching. Prominent are VEGF, activating tip cells, and Notch, which stimulates stalk cells. After the branch is formed and perfused, ECs become quiescent phalanx cells. Now, emerging evidence has accumulated indicating that ECs not only adapt their metabolism when switching from quiescence to sprouting but also that metabolism regulates vascular sprouting in parallel to the control by genetic signals. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Smooth muscle–endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

    Science.gov (United States)

    Lutter, Sophie; Xie, Sherry; Tatin, Florence

    2012-01-01

    Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels—smooth muscle and endothelial cells—and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function. PMID:22665518

  4. Netrin-1 Promotes Inflammation Resolution to Achieve Endothelialization of Small-Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ.

    Science.gov (United States)

    Li, Yanzhao; Wan, Simin; Liu, Ge; Cai, Wang; Huo, Da; Li, Gang; Yang, Mingcan; Wang, Yuxin; Guan, Ge; Ding, Ning; Liu, Feila; Zeng, Wen; Zhu, Chuhong

    2017-12-01

    The transplant of small-diameter tissue engineering blood vessels (small-diameter TEBVs) (vascular replacement therapy often fails because of early onset thrombosis and long-standing chronic inflammation. The specific inflammation state involved in small-diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small-diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin-1) is found present in endothelial cells of natural blood vessels and has anti-inflammatory effects. This work generates netrin-1-modified small-diameter TEBVs by using layer-by-layer self-assembly to resolve the inflammation. The results show that netrin-1 reprograms macrophages (MΦ) to assume an anti-inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small-diameter TEBVs modified by netrin-1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small-diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ-derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

  5. Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

    Science.gov (United States)

    Toh, Huishi; Cao, Mingju; Daniels, Eugene; Bateman, Andrew

    2013-01-01

    Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5) and later (E15.5-17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in

  6. Responses of endothelial cells from different vessels to inflammatory cytokines and shear stress: evidence for the pliability of endothelial phenotype.

    Science.gov (United States)

    Luu, N Thin; Rahman, Mahbub; Stone, Phil C; Rainger, G Ed; Nash, Gerard B

    2010-01-01

    Local haemodynamic and stromal microenvironments may determine the phenotype of endothelial cells (EC) and regulate their inflammatory responses. We compared neutrophil recruitment by EC from human umbilical veins (HUVEC) or arteries (HUAEC) or from human coronary arteries (HCAEC) after 'static' culture or exposure to shear stress (2 Pa for 24 h) and treatment with tumour necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). Static cultures of each type of EC recruited flowing neutrophils efficiently after treatment with TNF-alpha or IL-1beta; differences in culture media caused minor variations. After shear conditioning, the response of HUVEC to TNF-alpha (but not IL-1beta) was much reduced, while the responses of HUAEC and HCAEC to both cytokines were reduced. However, swapping the culture media suggested that the differences in the shear response arose largely from medium constituents, particularly basic fibroblast growth factor. When gene expression profiles for HUVEC were examined immediately after isolation, after 5 days in static culture and after re-exposure to shear, variations in gene expression were only partially attributable to the effects of changes in shear stress. The behaviour of cultured EC may depend as much on the physico-chemical culture conditions as on their origins. The EC phenotype appears to be highly pliable, with environmental factors, such as shear stress and growth factors, modifying responses in an inter-linked manner.

  7. High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

    Directory of Open Access Journals (Sweden)

    Nistri Silvia

    2002-01-01

    Full Text Available We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i good reproducibility, (ii accurate sterility that can be maintained throughout the isolation procedure and (iii high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.

  8. 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.

  9. Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep

    Science.gov (United States)

    Seedorf, Gregory J.; Brown, Alicia; Roe, Gates; O'Meara, Meghan C.; Gien, Jason; Tang, Jen-Ruey; Abman, Steven H.

    2011-01-01

    Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% (P IUGR fetuses. Pulmonary vessel density was decreased 44% (P IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% (P IUGR PAECs by 29% at baseline (P IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR. PMID:21873446

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice.

    Science.gov (United States)

    Liang, Qianqian; Ju, Yawen; Chen, Yan; Wang, Wensheng; Li, Jinlong; Zhang, Li; Xu, Hao; Wood, Ronald W; Schwarz, Edward M; Boyce, Brendan F; Wang, Yongjun; Xing, Lianping

    2016-03-12

    In this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis. We used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice. LECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N(6)-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage. iNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory

  12. Endothelial cells, fibroblasts and vasculitis.

    Science.gov (United States)

    Buckley, Christopher D; Rainger, G Ed; Nash, Gerard B; Raza, Karim

    2005-07-01

    One of the most important questions in vasculitis research is not why inflammation of blood vessels occurs but why it persists, often in a site-specific manner. In this review we illustrate how stromal cells, such as fibroblasts and pericytes, might play an important role in regulating the site at which vasculitis occurs. Smooth muscle cells and fibroblasts directly influence the behaviour of overlying vascular cells, amplifying the response of the endothelium to proinflammatory agents such as TNF-alpha and allowing enhanced and inappropriate leucocyte recruitment. An abnormal local vascular stromal environment can therefore influence local endothelial function and drive the persistence of local vascular inflammation. However, such local vascular inflammation can have distant effects on the systemic vascular system, leading to widespread endothelial cell dysfunction. Vascular endothelial dysfunction is common in a range of immune-mediated inflammatory diseases, is seen in multiple vascular beds, and is reversible following the induction of disease remission. The mechanisms that drive such systemic vascular endothelial dysfunction are unclear but factors such as TNF-alpha and CRP may play a role. Persistence of such widespread endothelial dysfunction in systemic vasculitis appears to have long-term consequences, leading to the acceleration of atherosclerosis and premature ischaemic heart disease. It may also underlie the accelerated atherosclerosis seen in other immune-mediated rheumatic diseases, such as rheumatoid arthritis.

  13. Down-regulation of miR-101 in endothelial cells promotes blood vessel formation through reduced repression of EZH2.

    Directory of Open Access Journals (Sweden)

    Michiel Smits

    2011-01-01

    Full Text Available Angiogenesis is a balanced process controlled by pro- and anti-angiogenic molecules of which the regulation is not fully understood. Besides classical gene regulation, miRNAs have emerged as post-transcriptional regulators of angiogenesis. Furthermore, epigenetic changes caused by histone-modifying enzymes were shown to modulate angiogenesis as well. However, a possible interplay between miRNAs and histone-modulating enzymes during angiogenesis has not been described. Here we show that VEGF-mediated down-regulation of miR-101 caused pro-angiogenic effects. We found that the pro-angiogenic effects are partly mediated through reduced repression by miR-101 of the histone-methyltransferase EZH2, a member of the Polycomb group family, thereby increasing methylation of histone H3 at lysine 27 and transcriptome alterations. In vitro, the sprouting and migratory properties of primary endothelial cell cultures were reduced by inhibiting EZH2 through up-regulation of miR-101, siRNA-mediated knockdown of EZH2, or treatment with 3-Deazaneplanocin-A (DZNep, a small molecule inhibitor of EZH2 methyltransferase activity. In addition, we found that systemic DZNep administration reduced the number of blood vessels in a subcutaneous glioblastoma mouse model, without showing adverse toxicities. Altogether, by identifying a pro-angiogenic VEGF/miR-101/EZH2 axis in endothelial cells we provide evidence for a functional link between growth factor-mediated signaling, post-transcriptional silencing, and histone-methylation in the angiogenesis process. Inhibition of EZH2 may prove therapeutic in diseases in which aberrant vascularization plays a role.

  14. Synchronization of endothelial Dll4-Notch dynamics switch blood vessels from branching to expansion.

    Science.gov (United States)

    Ubezio, Benedetta; Blanco, Raquel Agudo; Geudens, Ilse; Stanchi, Fabio; Mathivet, Thomas; Jones, Martin L; Ragab, Anan; Bentley, Katie; Gerhardt, Holger

    2016-04-13

    Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.

  15. Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization.

    Science.gov (United States)

    Phng, Li-Kun; Gebala, Véronique; Bentley, Katie; Philippides, Andrew; Wacker, Andrin; Mathivet, Thomas; Sauteur, Loïc; Stanchi, Fabio; Belting, Heinz-Georg; Affolter, Markus; Gerhardt, Holger

    2015-01-12

    During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  17. Radiation-induced apoptosis in microvascular endothelial cells.

    OpenAIRE

    Langley, R. E.; Bump, E A; Quartuccio, S. G.; Medeiros, D; Braunhut, S. J.

    1997-01-01

    The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary en...

  18. Receptor-Mediated Transport of Insulin across Endothelial Cells

    Science.gov (United States)

    King, George L.; Johnson, Sandra M.

    1985-03-01

    Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.

  19. Mechanical cell-substrate feedback explains pairwise and collective endothelial cell behavior in vitro

    NARCIS (Netherlands)

    R.F.M. van Oers (Rene); R.M.H. Merks (Roeland)

    2013-01-01

    htmlabstractIn vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in a extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and

  20. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells.

  1. Vascular Mechanobiology Endothelial Cell Responses to Fluid Shear Stress

    National Research Council Canada - National Science Library

    Ando, Joji; Yamamoto, Kimiko

    2009-01-01

    Endothelial cells (ECs) lining blood vessel walls respond to shear stress, a fluid mechanical force generated by flowing blood, and the EC responses play an important role in the homeostasis of the circulatory system...

  2. Cell-cell junctional mechanotransduction in endothelial remodeling.

    Science.gov (United States)

    Dorland, Yvonne L; Huveneers, Stephan

    2017-01-01

    The vasculature is one of the most dynamic tissues that encounter numerous mechanical cues derived from pulsatile blood flow, blood pressure, activity of smooth muscle cells in the vessel wall, and transmigration of immune cells. The inner layer of blood and lymphatic vessels is covered by the endothelium, a monolayer of cells which separates blood from tissue, an important function that it fulfills even under the dynamic circumstances of the vascular microenvironment. In addition, remodeling of the endothelial barrier during angiogenesis and trafficking of immune cells is achieved by specific modulation of cell-cell adhesion structures between the endothelial cells. In recent years, there have been many new discoveries in the field of cellular mechanotransduction which controls the formation and destabilization of the vascular barrier. Force-induced adaptation at endothelial cell-cell adhesion structures is a crucial node in these processes that challenge the vascular barrier. One of the key examples of a force-induced molecular event is the recruitment of vinculin to the VE-cadherin complex upon pulling forces at cell-cell junctions. Here, we highlight recent advances in the current understanding of mechanotransduction responses at, and derived from, endothelial cell-cell junctions. We further discuss their importance for vascular barrier function and remodeling in development, inflammation, and vascular disease.

  3. Epigenetic regulation of tumor endothelial cell anergy : Silencing of intercellular adhesion molecule-1 by histone modifications

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Castermans, Karolien; Vire, Emmanuelle; Dings, Ruud P. M.; Hoebers, Nicole T. H.; Mayos, Kevin H.; Egbrink, Mirjam G. A. Oude; Molema, Grietje; Fuks, Francois; Griffloen, Arjan W.

    2006-01-01

    Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel

  4. Endothelial Cell Implantation and Survival within Experimental Gliomas

    Science.gov (United States)

    Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

    1994-10-01

    The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

  5. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    Science.gov (United States)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  6. Optical Investigations of Endothelial Cell Motility

    DEFF Research Database (Denmark)

    Rossen, Ninna Struck

    A monolayer of endothelial cells lines the entire circulatory system and create a barrier between the circulatory system and the tissues. To create and maintain an intact barrier, the individual cells have to connect tightly with their neighbors, which causes a highly correlated motion between...... the cells within the monolayer. The cells have to maintain this barrier while apoptotic cells are being replaced and even while new blood vessels are being created. Meanwhile they are constantly exposed to a shear stress from the ow of blood through the vessels. These extreme micro-environmental conditions....... Below is a summary of the thesis' general outline to give an overview of the thesis and provide the structure of how the di erent projects described in individual chapters relate to each other. Each chapter describes a separate project and will contain a small introduction that address the issues...

  7. An Important Method in the Investigation of Vascular Pathologies: Endothelial Cell Culture

    Directory of Open Access Journals (Sweden)

    Yusufhan Yazır

    2012-12-01

    Full Text Available Endothelial cells line the interior surface of blood vessels and form an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells are involved in many aspects of vascular biology, including barrier function, vasoconstriction, coagulation and inflamation. The endothelial cells in different organs have different functions and surface phenotype. These cells express prostoglandin-I2, platelet activating factor, collagen, endothelin-1, laminin, fibronectin and growth factors including platelet derived growth factor, fibroblast growth factor. İn the cell culture, cells can be isolated, maintened and proliferate in the laboratory conditions. The techniques of the cell culture have allowed scientists to use the cells in vitro for experimental studies, such as the production of vaccine, antibody and enzime, drug research, cell-cell interactions. Human umbilical vein endothelial cell is a good source for endothelial cell, because it is cheaper, easy to find and has the basic features of the normal endothelial cells.

  8. Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature.

    Directory of Open Access Journals (Sweden)

    Andrew Scott

    2010-07-01

    Full Text Available Vascular endothelial growth factor (VEGF plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.

  9. Regulation of tissue morphogenesis by endothelial cell-derived signals

    Science.gov (United States)

    Ramasamy, Saravana K.; Kusumbe, Anjali P.; Adams, Ralf H.

    2016-01-01

    Summary Endothelial cells form an extensive network of blood vessels that has numerous essential functions in the vertebrate body. In addition to their well-established role as a versatile transport network, blood vessels can induce organ formation or direct growth and differentiation processes by providing signals in a paracrine (angiocrine) fashion. Tissue repair also requires the local restoration of vasculature. Endothelial cells are emerging as important signaling centers that coordinate regeneration and help to prevent deregulated, disease-promoting processes. Vascular cells are also part of stem cell niches and play key roles in hematopoiesis, bone formation and neurogenesis. Here, we will review these newly identified roles of endothelial cells in the regulation of organ morphogenesis, maintenance and regeneration. PMID:25529933

  10. The Bipartite Rac1 Guanine Nucleotide Exchange Factor Engulfment and Cell Motility 1/Dedicator of Cytokinesis 180 (Elmo1/Dock180) Protects Endothelial Cells from Apoptosis in Blood Vessel Development

    NARCIS (Netherlands)

    Schaeker, Kathrin; Bartsch, Susanne; Patry, Christian; Stoll, Sandra J.; Hillebrands, Jan-Luuk; Wieland, Thomas; Kroll, Jens

    2015-01-01

    Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmol/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Racl). Elmol/Dock180 regulates Racl activity in a specific spatiotemporal manner in endothelial cells

  11. Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells.

    Science.gov (United States)

    Coso, Sanja; Zeng, Yiping; Sooraj, Dhanya; Williams, Elizabeth D

    2011-10-15

    Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  12. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  13. Endothelial cells are intrinsically defective in xenophagy of Streptococcus pyogenes.

    Directory of Open Access Journals (Sweden)

    Shiou-Ling Lu

    2017-07-01

    Full Text Available Group A Streptococcus (GAS is deleterious pathogenic bacteria whose interaction with blood vessels leads to life-threatening bacteremia. Although xenophagy, a special form of autophagy, eliminates invading GAS in epithelial cells, we found that GAS could survive and multiply in endothelial cells. Endothelial cells were competent in starvation-induced autophagy, but failed to form double-membrane structures surrounding GAS, an essential step in xenophagy. This deficiency stemmed from reduced recruitment of ubiquitin and several core autophagy proteins in endothelial cells, as demonstrated by the fact that it could be rescued by exogenous coating of GAS with ubiquitin. The defect was associated with reduced NO-mediated ubiquitin signaling. Therefore, we propose that the lack of efficient clearance of GAS in endothelial cells is caused by their intrinsic inability to target GAS with ubiquitin to promote autophagosome biogenesis for xenophagy.

  14. Selective expression of erg isoforms in human endothelial cells.

    Science.gov (United States)

    Hewett, P W; Nishi, K; Daft, E L; Clifford Murray, J

    2001-04-01

    Erg and Fli-1 are closely related members of the ets family of transcription factors. There are at least five human Erg isoforms (Erg-1, Erg-2, Erg-3/p55(Erg), p49(Erg) and p38(Erg)) produced through differential mRNA splicing and alternative use of translational start codons. However, relatively little is known about the expression or function of these isoforms in vitro or their distribution in vivo. We used RT-PCR to screen a panel of primary and established human cell lines for erg and fli-1 consensus sequences. Whilst fli-1 was expressed in several human cell types, erg was detected mainly in endothelial cells. To identify which erg isoforms are expressed in endothelial cells we used RT-PCR, Northern blotting and 5'-RACE. Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like transcripts were detected in both microvascular and large vessel endothelial cells affinity-purified from different vascular beds. Moreover, these erg isoforms were present in both freshly isolated, and confluent endothelial cells following several passages in culture, indicating that endothelial erg expression in vitro may be broadly representative of that in vivo. The selective expression of the Erg-3/p55(Erg) and p38(Erg)/p38(Erg)-like isoforms in endothelial cells indicates their involvement in the regulation of endothelial-restricted genes.

  15. Effects of hypergravity on the angiogenic potential of endothelial cells

    NARCIS (Netherlands)

    Costa-Almeida, R. (Raquel); Carvalho, D.T.O. (Daniel T.O.); Ferreira, M.J.S. (Miguel J.S.); Aresta, G. (Guilherme); Gomes, M.E. (Manuela E.); Van Loon, J.J.W.A. (Jack J.W.A.); K. van der Heiden (Kim); Granja, P.L. (Pedro L.)

    2016-01-01

    textabstractAngiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying

  16. High endothelial venule-like vessels and lymphocyte recruitment in diffuse sclerosing variant of papillary thyroid carcinoma.

    Science.gov (United States)

    Low, Shulin; Sakai, Yasuhiro; Hoshino, Hitomi; Hirokawa, Mitsuyoshi; Kawashima, Hiroto; Higuchi, Kayoko; Imamura, Yoshiaki; Kobayashi, Motohiro

    2016-12-01

    Diffuse sclerosing variant of papillary thyroid carcinoma (DSPTC) is a rare subtype of papillary thyroid carcinoma with a high incidence of lymph node metastasis. One of its characteristic histological features is the presence of dense lymphocyte infiltrates; however, how these lymphocytes are recruited in this pathological setting remains unclear. Here, we analysed 17 DSPTC cases immunohistologically for cell adhesion molecules expressed on endothelial cells. We found that venules morphologically similar to high endothelial venules (HEVs) in secondary lymphoid organs were induced in lymphoid aggregates in DSPTC, and such HEV-like vessels expressed 6-sulfo sialyl Lewis X (sLeX) glycans as well as intercellular adhesion molecule 1 (ICAM-1). Triple immunohistochemistry revealed that CD8+ cytotoxic T cells were the major lymphocyte subset attached to the luminal surface of HEV-like vessels. sLeX-type glycans were also expressed on DSPTC carcinoma cells, which in binding assays were decorated with E-selectin•IgM chimaeras calcium-dependently. These findings collectively suggest that 6-sulfo sLeX glycans, together with ICAM-1, on HEV-like vessels may function to recruit CD8+ cytotoxic T cells in DSPTC. Additionally, sLeX-type glycans on carcinoma cells might partly contribute to highly metastatic properties of DSPTC through interaction with E-selectin expressed on endothelial cells. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

  17. Protective effects of vascular endothelial growth factor in cultured brain endothelial cells against hypoglycemia.

    Science.gov (United States)

    Zhao, Fei; Deng, Jiangshan; Yu, Xiaoyan; Li, Dawei; Shi, Hong; Zhao, Yuwu

    2015-08-01

    Hypoglycemia is a common and serious problem among patients with type 1 diabetes receiving treatment with insulin. Clinical studies have demonstrated that hypoglycemic edema is involved in the initiation of hypoglycemic brain damage. However, the mechanisms of this edema are poorly understood. Vascular endothelial growth factor (VEGF), a potent regulator of blood vessel function, has been observed an important candidate hormone induced by hypoglycemia to protect neurons by restoring plasma glucose. Whether VEGF has a protective effect against hypoglycemia-induced damage in brain endothelial cells is still unknown. To investigate the effects of hypoglycemia on cerebral microvascular endothelial cells and assess the protective effect of exogenous VEGF on endothelial cells during hypoglycemia, confluent monolayers of the brain endothelial cell line bEnd.3 were treated with normal (5.5 mM glucose), hypoglycemic (0, 0.5, 1 mM glucose) medium or hypoglycemic medium in the presence of VEGF. The results clearly showed that hypoglycemia significantly downregulated the expression of claudin-5 in bEnd.3 cells, without affecting ZO-1 and occludin expression and distribution. Besides, transendothelial permeability significantly increased under hypoglycemic conditions compared to that under control conditions. Moreover, the hypoglycemic medium in presence of VEGF decreased endothelial permeability via the inhibition of claudin-5 degradation and improved hypoglycemia-induced cell toxicity. Furthermore, Glucose transporter-1 (Glut-1) and apoptosis regulator Bcl-2 expression were significantly upregulated. Taken together, hypoglycemia can significantly increase paraendocellular permeability by downregulating claudin-5 expression. We further showed that VEGF protected brain endothelial cells against hypoglycemia by enhancing glucose passage, reducing endothelial cell death, and ameliorating paraendocellular permeability.

  18. Smooth muscle cell recruitment to lymphatic vessels requires PDGFB and impacts vessel size but not identity.

    Science.gov (United States)

    Wang, Yixin; Jin, Yi; Mäe, Maarja Andaloussi; Zhang, Yang; Ortsäter, Henrik; Betsholtz, Christer; Mäkinen, Taija; Jakobsson, Lars

    2017-10-01

    Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of Pdgfb prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly, Pdgfb overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin. © 2017. Published by The Company of Biologists Ltd.

  19. Smooth muscle cell recruitment to lymphatic vessels requires PDGFB and impacts vessel size but not identity

    Science.gov (United States)

    Wang, Yixin; Jin, Yi; Mäe, Maarja Andaloussi; Zhang, Yang; Ortsäter, Henrik; Betsholtz, Christer; Mäkinen, Taija

    2017-01-01

    Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of Pdgfb prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly, Pdgfb overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin. PMID:28851707

  20. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  1. Endothelial cells, fibroblasts and vasculitis

    OpenAIRE

    Buckley, Christopher D.; Rainger, G.Ed; Nash, Gerard B; Raza, Karim

    2005-01-01

    One of the most important questions in vasculitis research is not why inflammation of blood vessels occurs but why it persists, often in a site-specific manner. In this review we illustrate how stromal cells, such as fibroblasts and pericytes, might play an important role in regulating the site at which vasculitis occurs. Smooth muscle cells and fibroblasts directly influence the behaviour of overlying vascular cells, amplifying the response of the endothelium to proinflammatory agents such a...

  2. Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes

    OpenAIRE

    1994-01-01

    In contrast to the endothelial cells in large vessels where LDL receptors are downregulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced threefold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL recept...

  3. Lonidamine Causes Inhibition of Angiogenesis-Related Endothelial Cell Functions

    Directory of Open Access Journals (Sweden)

    Donatella Del Bufalo

    2004-09-01

    Full Text Available The aim of this study was to assess whether lonidamine (LND interferes with some steps in angiogenesis progression. We report here, for the first time, that LND inhibited angiogenic-related endothelial cell functions in a dose-dependent manner (1-50 μg/ml. In particular, LND decreased proliferation, migration, invasion, and morphogenesis on matrigel of different endothelial cell lines. Zymographic and Western blot analysis assays showed that LND treatment produced a reduction in the secretion of matrix metalloproteinase-2 and metalloproteinase-9 by endothelial cells. Vessel formation in a matrigel plug was also reduced by LND. The viability, migration, invasion, and matrix metalloproteinase production of different tumor cell lines were not affected by low doses of LND (1-10 μg/ml, whereas 50 μg/ml LND, which corresponds to the dose used in clinical management of tumors, triggered apoptosis both in endothelial and tumor cells. Together, these data demonstrate that LND is a compound that interferes with endothelial cell functions, both at low and high doses. Thus, the effect of LND on endothelial cell functions, previously undescribed, may be a significant contributor to the antitumor effect of LND observed for clinical management of solid tumors.

  4. GLP-1 inhibits VEGFA-mediated signaling in isolated human endothelial cells and VEGFA-induced dilation of rat mesenteric arteries

    DEFF Research Database (Denmark)

    Rotbøl, Cecilie Egholm; Khammy, Makhala Michell; Dalsgaard, Thomas

    2016-01-01

    to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries...

  5. Endothelial cell loss after autologous rotational keratoplasty.

    Science.gov (United States)

    Birnbaum, Florian; Reinhard, Thomas; Böhringer, Daniel; Sundmacher, Rainer

    2005-01-01

    To investigate whether it may be possible to ascertain the influence of immunological factors on chronic endothelial cell loss by comparing chronic endothelial cell loss after autologous rotational penetrating keratoplasty and after homologous penetrating keratoplasty. For six patients who had undergone autologous rotational penetrating keratoplasty the relative annual loss of endothelial cells was calculated by means of an exponential regression analysis. The findings were compared with those in a homogeneous historical control group (53 patients undergoing homologous penetrating keratoplasty for keratoconus). After autologous rotational keratoplasty relative annual loss of endothelial cells was 1.1%+/-2.6% (mean +/- standard deviation). Relative annual loss of endothelial cells in the control-group was 16.7%+/-20.8%. The results of the study lead to the assumption that immunological influences might be the main cause for chronic endothelial cell loss after homologous penetrating keratoplasty.

  6. Morph or Move? How Distinct Endothelial Cell Responses to Blood Flow Shape Vascular Networks.

    Science.gov (United States)

    Franco, Claudio A; Gerhardt, Holger

    2017-06-19

    Blood vessel shape is malleable and dynamically regulated. In two recent papers in Nature Cell Biology, Jin et al. (2017) and Sugden et al. (2017) show that endoglin alters endothelial cell shape or behavior in response to blood flow, thus regulating vessel structure. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. [Transplantation of corneal endothelial cells].

    Science.gov (United States)

    Amano, Shiro

    2002-12-01

    Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells. In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs). We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum. We performed the following analysis utilizing these cultured HCECs. The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs. The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting. HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases. Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages. These results indicated that shortening of telomere length is not related to senescence of HCECs. We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs. The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs. HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions. Primary and passage

  8. High Endothelial Venules and Other Blood Vessels: Critical Regulators of Lymphoid Organ Development and Function.

    Science.gov (United States)

    Ager, Ann

    2017-01-01

    The blood vasculature regulates both the development and function of secondary lymphoid organs by providing a portal for entry of hemopoietic cells. During the development of lymphoid organs in the embryo, blood vessels deliver lymphoid tissue inducer cells that initiate and sustain the development of lymphoid tissues. In adults, the blood vessels are structurally distinct from those in other organs due to the requirement for high levels of lymphocyte recruitment under non-inflammatory conditions. In lymph nodes (LNs) and Peyer's patches, high endothelial venules (HEVs) especially adapted for lymphocyte trafficking form a spatially organized network of blood vessels, which controls both the type of lymphocyte and the site of entry into lymphoid tissues. Uniquely, HEVs express vascular addressins that regulate lymphocyte entry into lymphoid organs and are, therefore, critical to the function of lymphoid organs. Recent studies have demonstrated important roles for CD11c+ dendritic cells in the induction, as well as the maintenance, of vascular addressin expression and, therefore, the function of HEVs. Tertiary lymphoid organs (TLOs) are HEV containing LN-like structures that develop inside organized tissues undergoing chronic immune-mediated inflammation. In autoimmune lesions, the development of TLOs is thought to exacerbate disease. In cancerous tissues, the development of HEVs and TLOs is associated with improved patient outcomes in several cancers. Therefore, it is important to understand what drives the development of HEVs and TLOs and how these structures contribute to pathology. In several human diseases and experimental animal models of chronic inflammation, there are some similarities between the development and function of HEVs within LN and TLOs. This review will summarize current knowledge of how hemopoietic cells with lymphoid tissue-inducing, HEV-inducing, and HEV-maintaining properties are recruited from the bloodstream to induce the development and

  9. Circulating Endothelial Cells and Endothelial Progenitor Cells in Pediatric Sepsis.

    Science.gov (United States)

    Zahran, Asmaa Mohamad; Elsayh, Khalid Ibrahim; Mohamad, Ismail Lotfy; Hassan, Gamal Mohamad; Abdou, Madleen Adel A

    2016-03-01

    The aim of the study was to measure the number of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) in pediatric patients with sepsis and correlating it with the severity of the disease and its outcome. The study included 19 children with sepsis, 26 with complicated sepsis, and 30 healthy controls. The patients were investigated within 48 hours of pediatric intensive care unit admission together with flow cytometric detection of CECs and CEPs. The levels of both CECs and CEPs were significantly higher in patient with sepsis and complicated sepsis than the controls. The levels of CECs were higher in patients with complicated sepsis, whereas the levels of CEPs were lower in patients with complicated sepsis. Comparing the survival and nonsurvival septic patients, the levels of CEPs were significantly higher in the survival than in nonsurvival patients, whereas the levels of CECs were significantly lower in the survival than in nonsurvival patients. Serum albumin was higher in survival than in nonsurvival patients. Estimation of CECs and CEPs and their correlation with other parameters such as serum albumen could add important information regarding prognosis in septic pediatric patients.

  10. Biophysical Cueing and Vascular Endothelial Cell Behavior

    Directory of Open Access Journals (Sweden)

    Joshua A. Wood

    2010-03-01

    Full Text Available Human vascular endothelial cells (VEC line the vessels of the body and are critical for the maintenance of vessel integrity and trafficking of biochemical cues. They are fundamental structural elements and are central to the signaling environment. Alterations in the normal functioning of the VEC population are associated with a number of vascular disorders among which are some of the leading causes of death in both the United States and abroad. VECs attach to their underlying stromal elements through a specialization of the extracellular matrix, the basement membrane. The basement membrane provides signaling cues to the VEC through its chemical constituents, by serving as a reservoir for cytoactive factors and through its intrinsic biophysical properties. This specialized matrix is composed of a topographically rich 3D felt-like network of fibers and pores on the nano (1–100 nm and submicron (100–1,000 nm size scale. The basement membrane provides biophysical cues to the overlying VECs through its intrinsic topography as well as through its local compliance (relative stiffness. These biophysical cues modulate VEC adhesion, migration, proliferation, differentiation, and the cytoskeletal signaling network of the individual cells. This review focuses on the impact of biophysical cues on VEC behaviors and demonstrates the need for their consideration in future vascular studies and the design of improved prosthetics.

  11. PPAR Gamma and Angiogenesis: Endothelial Cells Perspective

    Directory of Open Access Journals (Sweden)

    Jerzy Kotlinowski

    2016-01-01

    Full Text Available We summarize the current knowledge concerning PPARγ function in angiogenesis. We discuss the mechanisms of action for PPARγ and its role in vasculature development and homeostasis, focusing on endothelial cells, endothelial progenitor cells, and bone marrow-derived proangiogenic cells.

  12. Molecular expression in transfected corneal endothelial cells

    Science.gov (United States)

    Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

    2017-10-01

    To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

  13. Chemosensitizing AML cells by targeting bone marrow endothelial cells.

    Science.gov (United States)

    Bosse, Raphael C; Wasserstrom, Briana; Meacham, Amy; Wise, Elizabeth; Drusbosky, Leylah; Walter, Glenn A; Chaplin, David J; Siemann, Dietmar W; Purich, Daniel L; Cogle, Christopher R

    2016-05-01

    Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  14. Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway

    Science.gov (United States)

    Uemura, Mami; Nagasawa, Ayumi; Terai, Kenta

    2016-01-01

    Osteogenesis is categorized into two groups based on developmental histology, intramembranous and endochondral ossification. The role of blood vessels during endochondral ossification is well known, while their role in intramembranous ossification, especially the intertissue pathway, is poorly understood. Here, we demonstrate endothelial Yap/Taz is a novel regulator of intramembranous ossification in zebrafish. Appropriate blood flow is required for Yap/Taz transcriptional activation in endothelial cells and intramembranous ossification. Additionally, Yap/Taz transcriptional activity in endothelial cells specifically promotes intramembranous ossification. BMP expression by Yap/Taz transactivation in endothelial cells is also identified as a bridging factor between blood vessels and intramembranous ossification. Furthermore, the expression of Runx2 in pre-osteoblast cells is a downstream target of Yap/Taz transcriptional activity in endothelial cells. Our results provide novel insight into the relationship between blood flow and ossification by demonstrating intertissue regulation. PMID:27273480

  15. Estetrol modulates endothelial nitric oxide synthesis in human endothelial cells

    Directory of Open Access Journals (Sweden)

    Maria Magdalena eMontt-Guevara

    2015-07-01

    Full Text Available Estetrol (E4 is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO is a key player for vascular function and disease during pregnancy and throughout ageing in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS in cultured human umbilical vein endothelial cells (HUVEC. E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2 and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use.

  16. An artificial blood vessel implanted three-dimensional microsystem for modeling transvascular migration of tumor cells.

    Science.gov (United States)

    Wang, Xue-Ying; Pei, Ying; Xie, Min; Jin, Zi-He; Xiao, Ya-Shi; Wang, Yang; Zhang, Li-Na; Li, Yan; Huang, Wei-Hua

    2015-02-21

    Reproducing a tumor microenvironment consisting of blood vessels and tumor cells for modeling tumor invasion in vitro is particularly challenging. Here, we report an artificial blood vessel implanted 3D microfluidic system for reproducing transvascular migration of tumor cells. The transparent, porous and elastic artificial blood vessels are obtained by constructing polysaccharide cellulose-based microtubes using a chitosan sacrificial template, and possess excellent cytocompatibility, permeability, and mechanical characteristics. The artificial blood vessels are then fully implanted into the collagen matrix to reconstruct the 3D microsystem for modeling transvascular migration of tumor cells. Well-defined simulated vascular lumens were obtained by proliferation of the human umbilical vein endothelial cells (HUVECs) lining the artificial blood vessels, which enables us to reproduce structures and functions of blood vessels and replicate various hemodynamic parameters. Based on this model, the adhesion and transvascular migration of tumor cells across the artificial blood vessel have been well reproduced.

  17. Blood-Vessel Mimicking Structures by Stereolithographic Fabrication of Small Porous Tubes Using Cytocompatible Polyacrylate Elastomers, Biofunctionalization and Endothelialization

    Directory of Open Access Journals (Sweden)

    Birgit Huber

    2016-04-01

    Full Text Available Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM, biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.

  18. Blood-Vessel Mimicking Structures by Stereolithographic Fabrication of Small Porous Tubes Using Cytocompatible Polyacrylate Elastomers, Biofunctionalization and Endothelialization

    Science.gov (United States)

    Huber, Birgit; Engelhardt, Sascha; Meyer, Wolfdietrich; Krüger, Hartmut; Wenz, Annika; Schönhaar, Veronika; Tovar, Günter E. M.; Kluger, Petra J.; Borchers, Kirsten

    2016-01-01

    Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors. PMID:27104576

  19. The Influence of Hyaluronic Acid and Glioblastoma Cell Coculture on the Formation of Endothelial Cell Networks in Gelatin Hydrogels.

    Science.gov (United States)

    Ngo, Mai T; Harley, Brendan A

    2017-09-22

    Glioblastoma (GBM) is the most common and deadly form of brain cancer. Interactions between GBM cells and vasculature in vivo contribute to poor clinical outcomes, with GBM-induced vessel co-option, regression, and subsequent angiogenesis strongly influencing GBM invasion. Here, elements of the GBM perivascular niche are incorporated into a methacrylamide-functionalized gelatin hydrogel as a means to examine GBM-vessel interactions. The complexity of 3D endothelial cell networks formed from human umbilical vein endothelial cells and normal human lung fibroblasts as a function of hydrogel properties and vascular endothelial growth factor (VEGF) presentation is presented. While overall length and branching of the endothelial cell networks decrease with increasing hydrogel stiffness and incorporation of brain-mimetic hyaluronic acid, it can be separately altered by changing the vascular cell seeding density. It is shown that covalent incorporation of VEGF supports network formation as robustly as continuously available soluble VEGF. The impact of U87-MG GBM cells on the endothelial cell networks is subsequently investigated. GBM cells localize in proximity to the endothelial cell networks and hasten network regression in vitro. Together, this in vitro platform recapitulates the close association between GBM cells and vessel structures as well as elements of vessel co-option and regression preceding angiogenesis in vivo. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Apicobasal polarity of brain endothelial cells.

    Science.gov (United States)

    Worzfeld, Thomas; Schwaninger, Markus

    2016-02-01

    Normal brain homeostasis depends on the integrity of the blood-brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood-brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. © The Author(s) 2015.

  1. Endothelial progenitor cell dysfunction in diabetes mellitus

    NARCIS (Netherlands)

    Loomans, Cindy Johanna Maria

    2007-01-01

    Postnatally, Endothelial Progenitor Cells are needed to maintain the integrity of the endothelium (re-endothelialization) and to augment wound healing or vascularize hypoxic areas (neovascularization). Complex networks of different signals and regulators have been identified to be involved in these

  2. Mechanical cell-matrix feedback explains pairwise and collective endothelial cell behavior in vitro.

    Directory of Open Access Journals (Sweden)

    René F M van Oers

    2014-08-01

    Full Text Available In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a the contractile forces that endothelial cells exert on the ECM, (b the resulting strains in the extracellular matrix, and (c the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.

  3. Cataract phacoemulsification and corneal endothelial cell damage

    Directory of Open Access Journals (Sweden)

    Ni Zhu

    2013-07-01

    Full Text Available Phacoemulsification with small incision, reduced number of inflammation cells, and better postoperative recovery has been recognized as the world's most popular option for cataract surgery. Modern cataract surgery is developing gradually from sight rehabilitating to refractive surgery with better vision acuity. Being the most important part of the eye refractive system, maintenance of the cornea's transparency relies heavily upon the healthy endothelial cells. It is well known that there will be endothelial cell loss after phacoemulsification and the damage of the endothelial cells may lead to corneal swellings and opacity, or even the corneal descompensation, which often severely influenced the postoperative vision recovery. This is a review of phacoemulsification and the risk factors of corneal endothelial damage pre-and postoperation.

  4. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct

    NARCIS (Netherlands)

    Rouwkema, Jeroen; de Boer, Jan; van Blitterswijk, Clemens

    2006-01-01

    To engineer tissues with clinically relevant dimensions, one must overcome the challenge of rapidly creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products. We tested the hypothesis that endothelial cells, cocultured with osteoprogenitor cells, can

  5. Dysfunctional Endothelial Progenitor Cells in Metabolic Syndrome

    Science.gov (United States)

    Devaraj, Sridevi; Jialal, Ishwarlal

    2012-01-01

    The metabolic syndrome (MetS) is highly prevalent and confers an increased risk of diabetes and cardiovascular disease. A key early event in atherosclerosis is endothelial dysfunction. Numerous groups have reported endothelial dysfunction in MetS. However, the measurement of endothelial function is far from optimum. There has been much interest recently in a subtype of progenitor cells, termed endothelial progenitor cells (EPCs), that can circulate, proliferate, and dfferentiate into mature endothelial cells. EPCs can be characterized by the assessment of surface markers, CD34 and vascular endothelial growth factor receptor-2, VEGFR-2 (KDR). The CD34+KDR+ phenotype has been demonstrated to be an independent predictor of cardiovascular outcomes. MetS patients without diabetes or cardiovascular diseases have decreased EPC number and functionality as evidenced by decreased numbers of colony forming units, decreased adhesion and migration, and decreased tubule formation. Strategies that have been shown to upregulate and enhance EPC number and functionality include statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and peroxisome-proliferator-activating-receptor gamma agonists. Mechanisms by which they affect EPC number and functionality need to be studied. Thus, EPC number and/or functionality could emerge as novel cellular biomarkers of endothelial dysfunction and cardiovascular disease risk in MetS. PMID:21941528

  6. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

    2016-01-01

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters. PMID:26936382

  7. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

    2016-03-03

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  8. Heterologous corneal endothelial cell transplantation--human corneal endothelial cell transplantation in Lewis rats.

    OpenAIRE

    Tchah, H.

    1992-01-01

    A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous imm...

  9. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  10. The NR1 subunit of NMDA receptor regulates monocyte transmigration through the brain endothelial cell barrier

    NARCIS (Netherlands)

    Reijerkerk, A.; Kooij, G.; van der Pol, S.M.A.; Leyen, T.A.; Lakeman, K.; Van Het Hof, B; Vivien, D.; de Vries, H.E.

    2010-01-01

    Normal neuronal functioning is dependent on the blood-brain barrier. This barrier is confined to specialized brain endothelial cells lining the inner vessel wall, and tightly controlling transport of nutrients, efflux of potentially harmful molecules and entry of immune cells into the brain. Loss of

  11. Bacteria and endothelial cells: a toxic relationship.

    Science.gov (United States)

    Lubkin, Ashira; Torres, Victor J

    2017-02-01

    Pathogenic bacteria use the bloodstream as a highway for getting around the body, and thus have to find ways to enter and exit through the endothelium. Many bacteria approach this problem by producing toxins that can breach the endothelial barrier through diverse creative mechanisms, including directly killing endothelial cells (ECs), weakening the cytoskeleton within ECs, and breaking the junctions between ECs. Toxins can also modulate the immune response by influencing endothelial biology, and can modulate endothelial function by influencing the response of leukocytes. Understanding these interactions, in both the in vitro and in vivo contexts, is of critical importance for designing new therapies for sepsis and other severe bacterial diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells.

    Science.gov (United States)

    Dath, C; Dethy, A; Van Langendonckt, A; Van Eyck, A S; Amorim, C A; Luyckx, V; Donnez, J; Dolmans, M M

    2011-06-01

    Grafting of isolated follicles represents an approach to prevent the risk of reimplanting malignant cells with cryopreserved ovarian fragments. Optimal conditions and cell types required to sustain human follicular growth need to be identified. To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization. In human ovary, CD34 selectively labels endothelial cells of blood vessels. A CD34-replete ovarian stromal cell group, including stromal and endothelial cells, was obtained after enzymatic digestion of fresh human ovarian cortex. Magnetic-activated cell sorting was used to establish a CD34-depleted ovarian stromal cell group. Proportions of CD34-positive cells were evaluated by flow cytometry and immunocytochemistry. Cell suspensions were embedded in human plasma clots and grafted (n = 10 for each group, 7 days) to the ovarian bursa of nude mice. Angiogenesis was quantified after human/mouse CD34 immunostaining. CD34-replete grafts had a well-organized and vascularized stromal structure, containing tubular components staining for human CD34 and corresponding to functional vessels, as evidenced by intraluminal red blood cells. CD34-depleted grafts tended to be smaller than CD34-replete grafts and poorly vascularized with central necrosis. Global microvessel density was higher in the CD34-replete than depleted group (337.9 versus 187.3 vessels/mm(2), P ovarian endothelial and stromal cells to ensure the formation of a well-vascularized and structured ovarian-like stroma after short-term xenografting, for future application in the transplantation of isolated follicles.

  13. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  14. Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts.

    Science.gov (United States)

    Laurenzana, Anna; Cencetti, Francesca; Serratì, Simona; Bruno, Gennaro; Japtok, Lukasz; Bianchini, Francesca; Torre, Eugenio; Fibbi, Gabriella; Del Rosso, Mario; Bruni, Paola; Donati, Chiara

    2015-10-01

    The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions. • Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB. • H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB. • CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB.

  15. Tumor-Derived Factors and Reduced p53 Promote Endothelial Cell Centrosome Over-Duplication.

    Directory of Open Access Journals (Sweden)

    Zhixian Yu

    Full Text Available Approximately 30% of tumor endothelial cells have over-duplicated (>2 centrosomes, which may contribute to abnormal vessel function and drug resistance. Elevated levels of vascular endothelial growth factor A induce excess centrosomes in endothelial cells, but how other features of the tumor environment affect centrosome over-duplication is not known. To test this, we treated endothelial cells with tumor-derived factors, hypoxia, or reduced p53, and assessed centrosome numbers. We found that hypoxia and elevated levels of bone morphogenetic protein 2, 6 and 7 induced excess centrosomes in endothelial cells through BMPR1A and likely via SMAD signaling. In contrast, inflammatory mediators IL-8 and lipopolysaccharide did not induce excess centrosomes. Finally, down-regulation in endothelial cells of p53, a critical regulator of DNA damage and proliferation, caused centrosome over-duplication. Our findings suggest that some tumor-derived factors and genetic changes in endothelial cells contribute to excess centrosomes in tumor endothelial cells.

  16. CD13 is a novel mediator of monocytic/endothelial cell adhesion

    DEFF Research Database (Denmark)

    Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine

    2008-01-01

    During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown...... to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal...... rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept...

  17. Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo.

    Science.gov (United States)

    Moyon, D; Pardanaud, L; Yuan, L; Bréant, C; Eichmann, A

    2001-09-01

    Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Doyle, Margaret F; 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.

  20. Adenovirus vectors can induce activation of endothelial cells: CD40 ...

    African Journals Online (AJOL)

    Adenovirus vectors can induce activation of endothelial cells: CD40-CD40L interactions partly participate in the endothelial cells activation induced by adenovirus vectors in an NF-kappaB-dependent manner.

  1. Identification of epigenetically silenced genes in tumor endothelial cells

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Melotte, Veerle; Vire, Emmanuelle; Langenkamp, Elise; Molema, Grietje; Fuks, Francois; Herman, James G.; Van Criekinge, Wim; Griffioen, Arjan W.; van Engeland, Manon

    2007-01-01

    Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated

  2. Endothelial cells present antigens in vivo

    Directory of Open Access Journals (Sweden)

    Tellides George

    2004-03-01

    Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

  3. Spreading endothelial cell dysfunction in response to necrotic trophoblasts. Soluble factors released from endothelial cells that have phagocytosed necrotic shed trophoblasts reduce the proliferation of additional endothelial cells.

    Science.gov (United States)

    Chen, Q; Ding, J X; Liu, B; Stone, P; Feng, Y J; Chamley, L

    2010-11-01

    The pathogenesis of preeclampsia is not clear but the disease is characterised by systemic endothelial cell dysfunction that is considered to be triggered by a placental factor. Necrotic trophoblastic debris that is deported in the maternal blood is one possible placental trigger for preeclampsia. Syncytial knots were first associated with preeclampsia over 100 years ago. However, syncytial knots are very large and most are trapped in the pulmonary capillaries making it difficult to envisage how they could lead to widespread systemic endothelial cell dysfunction. This study was undertaken to examine whether conditioned medium from endothelial cells that have phagocytosed necrotic trophoblastic debris could adversely affect the proliferation or survival of fresh endothelial cells. Trophoblastic cellular debris, harvested from placental explants was added to endothelial cell monolayers directly or after induction of necrosis by freeze-thawing. Conditioned medium from the endothelial cell cultures was exposed to fresh endothelial cells and their proliferation measured by Alamar Blue, and CyQUANTNF cell proliferation assays. Endothelial cell death was examined by a fluorogenic caspase-3 activity assay and LDH release. Conditioned medium from endothelial cells that had phagocytosed necrotic but not apoptotic trophoblastic debris significantly inhibited the proliferation of fresh endothelial cells but did not induce their death. The conditioned medium also reduced cell-surface endoglin expression by fresh endothelial cells. These results confirm that phagocytosis of necrotic trophoblastic debris by endothelial cells results in the secretion of soluble factors that might explain how necrotic trophoblastic debris trapped in the pulmonary capillaries could induce systemic endothelial cell dysfunction. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Functional endothelial progenitor cells from cryopreserved umbilical cord blood

    Science.gov (United States)

    Lin, Ruei-Zeng; Dreyzin, Alexandra; Aamodt, Kristie; Dudley, Andrew C.; Melero-Martin, Juan M.

    2010-01-01

    Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies. PMID:20887663

  5. Blood-compatible poly(2-methoxyethyl acrylate) for the adhesion and proliferation of endothelial and smooth muscle cells.

    Science.gov (United States)

    Sato, Chikako; Aoki, Makiko; Tanaka, Masaru

    2016-09-01

    Thrombus formation presents a serious hindrance in the development of functional artificial blood vessels, especially those with a small diameter. Endothelialization can prevent thrombus formation; however, the adhesion of endothelial cells to existing polymer materials is generally weak. Therefore, polymers that have both anti-thrombotic and endothelialization properties do not currently exist. We previously reported that platelets do not adhere to poly(2-methoxyethyl acrylate) (PMEA) or poly(tetrahydrofurfuryl acrylate)(PTHFA). Here, we investigated whether endothelial cells and smooth muscle cells, both of which are blood vessel components, could adhere to these synthetic polymers. Polyethylene terephthalate films were coated with PMEA and PTHFA using a spin-coater. Human umbilical vein endothelial cells or aorta smooth muscle cells were seeded on the polymer surfaces, after which we analyzed the number of adherent cells, their morphologies and vinculin expression. We found that both endothelial and smooth muscle cells adhered to PMEA and PTHFA, while platelets did not. We propose that, by using PMEA and PTHFA with no modifications, it should be possible to develop artificial blood vessels with both anti-thrombotic and endothelialization properties. In addition, we discuss the mechanism of selective cell adhesion in PMEA and PTHFA. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction.

    Science.gov (United States)

    Ryu, Ji-Kan; Zhang, Lu Wei; Jin, Hai-Rong; Piao, Shuguang; Choi, Min Ji; Tuvshintur, Buyankhuu; Tumurbaatar, Munkhbayar; Shin, Sun Hwa; Han, Jee-Young; Kim, Woo Jean; Suh, Jun-Kyu

    2009-07-01

    Endothelial cell-to-cell junctions are crucial for vascular formation, networking, and remodeling of blood vessels as well as for inducing and integrating intracellular signals. We investigated the differential expression and distribution of endothelial cell-to-cell junction proteins in the penis of mice with hypercholesterolemia-induced erectile dysfunction. Two-month-old C57BL/6J mice were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. We performed dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) (Seegene, Seoul, Korea) to screen the differential gene expression of 21 endothelial cell-to-cell junctions. At 5 months, erectile function was measured by electrical stimulation of the cavernous nerve, and the penis was harvested and stained with antibody to claudin-5, vascular endothelial (VE)-cadherin, and platelet/endothelial cell adhesion molecule (PECAM)-1 (N = 8 per group). Cavernous specimens from a separate group of animals were used for claudin-5, VE-cadherin, and PECAM-1 reverse transcriptase-PCR and Western blot analysis. Erectile function was significantly lower in hypercholesterolemic mice than in controls. DPO-based multiplex PCR revealed a profound decrease in the gene expression of endothelium-specific cell-to-cell junction proteins, including claudin-5, VE-cadherin, and PECAM-1, in hypercholesterolemic mice compared with that in controls. The expression of claudin-5, VE-cadherin, and PECAM-1 protein evaluated by Western blot or immunohistochemistry was significantly lower in hypercholesterolemic mice than in controls. These endothelial cell-to-cell junction proteins were more sparsely distributed in the endothelium of cavernous sinusoids than in the endothelium of cavernous artery and dorsal blood vessels. Down-regulation of the endothelial cell-to-cell junctions and decreased endothelial content in the corpus cavernosum might play a major

  7. Endothelial junction regulation: a prerequisite for leukocytes crossing the vessel wall

    NARCIS (Netherlands)

    Daniel, Anna E.; van Buul, Jaap D.

    2013-01-01

    The leukocytes of the innate immune system, especially neutrophils and monocytes, exit the circulation early in the response to local inflammation and infection. This is necessary to control and prevent the spread of infections before an adaptive immune response can be raised. The endothelial cells

  8. Endothelial cell marker PAL-E reactivity in brain tumor, developing brain, and brain disease

    NARCIS (Netherlands)

    Leenstra, S.; Troost, D.; Das, P. K.; Claessen, N.; Becker, A. E.; Bosch, D. A.

    1993-01-01

    The endothelial cell marker PAL-E is not reactive to vessels in the normal brain. The present study concerns the PAL-E reactivity in brain tumors in contrast to normal brain and nonneoplastic brain disease. A total of 122 specimens were examined: brain tumors (n = 94), nonneoplastic brain disease (n

  9. Human very Small Embryonic-like Cells Support Vascular Maturation and Therapeutic Revascularization Induced by Endothelial Progenitor Cells.

    Science.gov (United States)

    Guerin, Coralie L; Rossi, Elisa; Saubamea, Bruno; Cras, Audrey; Mignon, Virginie; Silvestre, Jean-Sébastien; Smadja, David M

    2017-08-01

    Very small embryonic-like stem cells (VSELs) are major pluripotent stem cells defined as cells of small size being Lineage- negative, CD133-positive, and CD45-negative. We previously described that human bone marrow VSELs were able to differentiate into endothelial cells and promoted post-ischemic revascularization in mice with surgically induced critical limb ischemia. In the present work, we isolated bone marrow VSELs from patients with critical limb ischemia and studied their ability to support endothelial progenitor cells therapeutic capacity and revascularization potential. Sorted bone marrow VSELs cultured in angiogenic media were co-injected with endothelial progenitor cells and have been show to trigger post-ischemic revascularization in immunodeficient mice, and support vessel formation in vivo in Matrigel implants better than human bone marrow mesenchymal stem cells. In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial and perivascular lineage in humans. VSELs are the first real vasculogenic stem cells able to differentiate in endothelial and perivascular lineage in human adult described from now. Thus, because VSELs presence have been proposed in adult tissues, we think that VSELs are CD45 negative stem cells able to give rise to vascular regeneration in human tissues and vessels.

  10. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

    Science.gov (United States)

    Greene, Jennifer A.; Portillo, Jose-Andres C.; Lopez Corcino, Yalitza; Subauste, Carlos S.

    2015-01-01

    CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand) upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF) are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina. PMID:26710229

  11. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer A Greene

    Full Text Available CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

  12. Endothelial progenitor cell biology in ankylosing spondylitis.

    Science.gov (United States)

    Verma, Inderjeet; Syngle, Ashit; Krishan, Pawan

    2015-03-01

    Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). EPCs were depleted in AS patients as compared to healthy controls (CD34(+) /CD133(+) : 0.027 ± 0.010% vs. 0.044 ± 0.011%, P < 0.001). EPC depletions were significantly associated with disease duration (r = -0.52, P = 0.01), BASDAI (r = -0.45, P = 0.04) and C-reactive protein (r = -0.5, P = 0.01). This is the first study to demonstrate endothelial progenitor cell depletion in AS patients. EPC depletions inversely correlate with disease duration, disease activity and inflammation, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  13. cKit Lineage Hemogenic Endothelium-Derived Cells Contribute to Mesenteric Lymphatic Vessels

    Directory of Open Access Journals (Sweden)

    Lukas Stanczuk

    2015-03-01

    Full Text Available Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.

  14. Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice.

    Science.gov (United States)

    Guerreiro, Susana G; Brochhausen, Christoph; Negrão, Rita; Barbosa, Mário A; Unger, Ronald E; Kirkpatrick, C James; Soares, Raquel; Granja, Pedro L

    2012-01-01

    The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The vessels exhibited higher levels of hemoglobin, compared with the control matrix, implanted without fibroblasts, in which no vessel formation could be observed. No significant differences were detected in systemic inflammation. The presence of vessels originated from the host vasculature suggested that host vascular response was involved, which constitutes a fundamental aspect in the process of neovascularization. Fibroblasts implanted within matrigel increased the presence of endothelial cells with positive staining for CD31 and for CD34 and the production of collagen influencing the angiogenic process and promoting the formation of microvessels. New strategies in tissue engineering could be delineated with improved angiogenesis using neonatal fibroblasts.

  15. Endothelial cell oxidative stress and signal transduction

    Directory of Open Access Journals (Sweden)

    ROCIO FONCEA

    2000-01-01

    Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

  16. The Effects of kisspeptin-10 on Migration and Proliferation of Endothelial Cell

    Directory of Open Access Journals (Sweden)

    Fatemeh Golzar

    2015-01-01

    Full Text Available Background: Migration, expansion and survival of endothelial cells that are an important cellular component of blood vessels plays an important role in the induction of tumor growth. Kisspeptins (kp, peptides that bind to coupled-G protein receptor (GPR54, inhibit each step of metastatic cascade include invasion, migration and homing, angiogenesis, survival and proliferation. In this study we investigated effects of kisspeptin-10, the most potent member of kisspeptin family, on Migration and proliferation of endothelial cells that are necessary for angiogenesis and tumor metastasis. Materials and Methods: We compared migration of Human Umbilical Vein Endothelial Cells (HUVECs were treated with 10-100 or 500 nM kp-10 for 24 hours and no treated cells using an in vitro trans membrane migration assay and HUVEC proliferation of treated endothelial cells with 10-100 or 500 nM kp-10 for 48 hours and no treated cells was measured by MTT Cell Proliferation Assay Kit. Analysis of data was performed using the Kruskal-Wallis test followed by the Mann-Whitney test. Results: Migration and proliferation of endothelial cells were increased at lower concentration of kp-10 specially at 100 nM while higher concentration reduced both migration and proliferation. Conclusion: Our data showed that different concentrations of kp-10 have distinct effects on migration and proliferation of endothelial cells.

  17. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, B; Holck, Susanne; Christensen, Ib Jarle

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  18. Oxidative stress induced pulmonary endothelial cell proliferation is ...

    African Journals Online (AJOL)

    Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

  19. Distribution of blood-group-related carbohydrate antigens on oral endothelial cells.

    Science.gov (United States)

    Bryne, M; Lilleholt, S; Thrane, P S; Koppang, H S; Dabelsteen, E

    1993-05-01

    Recent studies indicate that carbohydrate structures are involved in various endothelial functions such as inflammatory processes, adhesion of metastatic cells to endothelium and endothelial differentiation. In this paper we report the endothelial expression of various blood-group-related carbohydrate structures in normal oral mucosa using 15 different monoclonal antibodies reacting with type 1, 2 or 3 carbohydrate chains. Twenty biopsies, including normal oral mucosa, from secretor individuals comprised nine blood group O, nine A, one B and one AB. Endothelial staining was compared with epithelial staining in the same biopsies. Five blood-group-related carbohydrate antigens were detected on endothelial cells. The H type 2 antigen, which is the precursor of A, B and AB antigens, has previously been believed to be a universal marker for endothelial cells. All blood group O individuals (n = 9) showed strong H antigen staining in the endothelium of most vessels. However, of blood group A, B and AB individuals (n = 11), four showed heterogenous H antigen staining. In addition, we found that six out of ten blood group A or AB individuals, who expressed A or A and B antigens on spinous squamous cells and glandular epithelium, showed either heterogenous or no staining for these structures on their endothelial cells. It is concluded that there are differences between the biosynthesis of blood-group-related carbohydrate antigens in oral endothelium and epithelium.

  20. Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/bioglass nanofibrous scaffolds.

    Science.gov (United States)

    Shamosi, Atefeh; Mehrabani, Davood; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Siavashi, Vahid; Ghanbari, Hossein; Sharifi, Esmaeel; Roozafzoon, Reza; Ai, Jafar

    2017-02-01

    The capacity of gelatin/chitosan/bioactive glass nanopowders (GEL/CS/BGNPs) scaffolds was investigated for increasing human endometrial stem cells (hEnSCs) differentiation into the endothelial cells in the presence of angiogenic factors. GEL/CS nanofibrous scaffold with different contents of BGNPs were fabricated and assessed. Expression of endothelial markers (CD31, vascular endothelial cadherin (VE-cadherin), and KDR) in differentiated cells was evaluated. Results showed the diameter of nanofiber increases with decreasing the BG content in GEL/CS scaffolds. Moreover, in vitro study indicated that the GEL/CS/BGNPs scaffold with 1.5% BGNPs content provided a suitable three-dimensional structure for endothelial cells differentiation. Thus, the GEL/CS/BGNPs scaffold can be recommended for blood vessels repair.

  1. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  2. Effects of hypergravity on the angiogenic potential of endothelial cells.

    Science.gov (United States)

    Costa-Almeida, Raquel; Carvalho, Daniel T O; Ferreira, Miguel J S; Aresta, Guilherme; Gomes, Manuela E; van Loon, Jack J W A; Van der Heiden, Kim; Granja, Pedro L

    2016-11-01

    Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modulation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1g), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3g and 10g for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cytoskeleton organization was observed for cells exposed to high g's, a slight loss of the perinuclear localization of β-tubulin was observed for cells exposed to 3g with less pronounced peripheral bodies of actin when compared with 1g control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10g level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and g-level-dependent manner. © 2016 The Author(s).

  3. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Silicon microgrooves for contact guidance of human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Sara Fernández-Castillejo

    2017-03-01

    Full Text Available Background: Micro- and nanoscale substrates have been fabricated in order to study the influence of the topography on the cellular response. The aim of this work was to prepare different collagen-coated silicon substrates displaying grooves and ridges to mimic the aligned and elongated endothelium found in linear vessels, and to use them as substrates to study cell growth and behaviour.Results: The influence of groove-shaped substrates on cell adhesion, morphology and proliferation were assessed, by comparing them to flat silicon substrates, used as control condition. Using human aortic endothelial cells, microscopy images demonstrate that the cellular response is different depending on the silicon surface, when it comes to cell adhesion, morphology (alignment, circularity and filopodia presence and proliferation. Moreover, these structures exerted no cytotoxic effect.Conclusion: The results suggest that topographical patterning influences cell response. Silicon groove substrates can be used in developing medical devices with microscale features to mimic the endothelium in lineal vessels.

  5. Transition of mesenchymal stem/stromal cells to endothelial cells

    NARCIS (Netherlands)

    M. Crisan (Mihaela)

    2013-01-01

    textabstractMesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial

  6. Soluble endothelial cell molecules and circulating endothelial cells in patients with venous thromboembolism.

    Science.gov (United States)

    Torres, Cláudia; Matos, Rui; Morais, Sara; Campos, Manuel; Lima, Margarida

    2017-12-01

    : To evaluate the plasma levels of soluble endothelial cell molecules in patients with venous thromboembolism (VTE) out of the acute phase as compared with healthy individuals. We also investigated the possible associations of the soluble endothelial cell molecules among them, as well as with other clinical and laboratory data, including the numbers of circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and CEC expressing activation-related [cluster of differentiation (CD)54 and CD62E] and procoagulant (CD142) markers. In total, 15 patients with VTE and 20 normal individuals were studied. The CEC and CEP were quantified and characterized by flow cytometry. The soluble molecules studied included P-selectin, E-selectin, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1 and tissue factor (ELISA), and von Willebrand factor antigen (immunoturbidimetry). VTE patients had significantly higher levels of vascular cell adhesion molecule 1 and von Willebrand factor antigen and lower levels of soluble E-selectin than controls. They also showed significantly higher numbers of CEC, as of activated/procoagulant CEC and lower numbers of CEP, compared with controls. We did not find any correlation between the levels of soluble molecules and the numbers of endothelial cell in circulation, but there was with several clinical and laboratory data in VTE patients. Our results would suggest that in VTE patients, the endothelium remains activated and in some hypercoagulable state. The levels of soluble endothelial cell molecules did not seem to be directly related to the numbers of CEC and CEP neither reflected the number of activated CEC, which may be because of the different function that surface and soluble molecules may have.

  7. Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction.

    Directory of Open Access Journals (Sweden)

    Nicola N Lynskey

    2015-09-01

    Full Text Available The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology.

  8. Reciprocal interactions between endothelial cells and macrophages in angiogenic vascular niches

    Energy Technology Data Exchange (ETDEWEB)

    Baer, Caroline; Squadrito, Mario Leonardo [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland); Iruela-Arispe, M. Luisa, E-mail: arispe@mcdb.ucla.edu [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland); Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California, Los Angeles 90095, CA (United States); De Palma, Michele, E-mail: michele.depalma@epfl.ch [The Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne (Switzerland)

    2013-07-01

    The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sprouting blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis. -- Highlights: • Macrophages promote angiogenesis by secreting proangiogenic factors. • Macrophages modulate angiogenesis via cell-to-cell contacts with endothelial cells. • Endothelial cells promote the differentiation of proangiogenic macrophages. • Macrophages and endothelial cells may cooperate to form angiogenic vascular niches.

  9. Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels

    Science.gov (United States)

    Berkowitz, Dan E.; White, Ron; Li, Dechun; Minhas, Khalid M.; Cernetich, Amy; Kim, Soonyul; Burke, Sean; Shoukas, Artin A.; Nyhan, Daniel; Champion, Hunter C.; hide

    2003-01-01

    BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

  10. Fibroblast growth factor signaling pathway in endothelial cells is activated by BMPER to promote angiogenesis.

    Science.gov (United States)

    Esser, Jennifer S; Rahner, Susanne; Deckler, Meike; Bode, Christoph; Patterson, Cam; Moser, Martin

    2015-02-01

    Previously, we have identified bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project, we now investigate how BMPER acts in concert with key molecules of angiogenesis to promote blood vessel formation. To assess the effect of BMPER on angiogenesis-related signaling pathways, we performed an angiogenesis antibody array with BMPER-stimulated endothelial cells. We detected increased basic fibroblast growth factor (bFGF/FGF-2) expression after BMPER stimulation and decreased expression of thrombospondin-1. Additionally, FGF receptor-1 expression, phosphorylation, FGF signaling pathway activity, and cell survival were increased. Consistently, silencing of BMPER by small interfering RNA decreased bFGF and FGF receptor-1 expression and increased thrombospondin-1 expression and cell apoptosis. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased endothelial cell angiogenic activity in migration, Matrigel, and spheroid assays. To block FGF signaling, an anti-bFGF antibody was used, which effectively inhibited the proangiogenic BMPER effect. Accordingly, BMPER-silenced endothelial cells under bFGF stimulation showed decreased angiogenic activity compared with bFGF control. We confirmed these findings in vivo by subcutaneous Matrigel injections with and without bFGF in C57BL/6_Bmper(+/-) mice. Aortic ring assays of C57BL/6_Bmper(+/-) mice confirmed a specific effect for bFGF but not for vascular endothelial growth factor. Taken together, the proangiogenic BMPER effect in endothelial cells is mediated by inhibition of antiangiogenic thrombospondin-1 and enhanced expression and activation of the FGF signaling pathway that is crucial in the promotion of angiogenesis. © 2014 American Heart Association, Inc.

  11. SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells.

    Science.gov (United States)

    Zhang, Lianghui; Jambusaria, Ankit; Hong, Zhigang; Marsboom, Glenn; Toth, Peter T; Herbert, Brittney-Shea; Malik, Asrar B; Rehman, Jalees

    2017-06-20

    The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and

  12. CXCL12 enhances angiogenesis through CXCR7 activation in human umbilical vein endothelial cells

    OpenAIRE

    ZHANG Min; Qiu, Lisha; Zhang, Yanyan; Xu, Dongsheng; Zheng, Jialin C.; Jiang, Li

    2017-01-01

    Angiogenesis is the process by which new vessels form from existing vascular networks. Human umbilical vein endothelial cells (HUVECs) may contribute to the study of vascular repair and angiogenesis. The chemokine CXCL12 regulates multiple cell functions, including angiogenesis, mainly through its receptor CXCR4. In contrast to CXCL12/CXCR4, few studies have described roles for CXCR7 in vascular biology, and the downstream mechanism of CXCR7 in angiogenesis remains unclear. The results of the...

  13. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  14. First trimester trophoblasts forming endothelial-like tubes in vitro emulate a 'blood vessel development' gene expression profile.

    Science.gov (United States)

    Highet, Amanda R; Buckberry, Sam; Mayne, Benjamin T; Khoda, Sultana M; Bianco-Miotto, Tina; Roberts, Claire T

    2016-07-01

    Extravillous cytotrophoblasts isolated from first trimester placenta, and immortalised cell lines derived from them, have the intrinsic ability to form endothelial-like tubes when cultured on Matrigel™ extracellular matrix. This in vitro tube formation may model placental angiogenesis and/or endovascular differentiation by trophoblasts. To interpret the relevance of this phenomenon to placental development, we used a gene expression microarray approach to identify which genes and pathways are associated with the tube-forming phenotype of HTR8/SVneo first trimester trophoblasts (HTR8-M), compared with HTR8/SVneo not forming tubes on plastic culture surface (HTR8-P). Furthermore, we used weighted gene co-expression network analysis (WGCNA) of microarray data to identify modules of co-expressed genes underlying the biological processes. There were 481 genes differentially expressed between HTR8-M and HTR8-P and these were significantly enriched for blood vessel development and related gene ontologies. WGCNA clustered the genes into 9 co-expression modules. One module was significantly associated with HTR8-M (p = 1.15E-05) and contained genes involved in actin cytoskeleton organization, cell migration and blood vessel development, consistent with tube formation on Matrigel. Another module was significantly associated with HTR8-P (p = 1.94E-05) and was enriched for genes involved in mitosis, consistent with proliferation by cells on plastic which do not differentiate. Up-regulation of angiogenesis and vascular development pathways in endovascular trophoblasts in vivo could underpin spiral artery remodelling processes, which are defective in preeclamptic pregnancies. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. The role of caveolin1 and sprouty1 in genistein's regulation of vascular smooth muscle cell and endothelial cell proliferation.

    Science.gov (United States)

    Xiang, QiuLing; Lin, GuiPing; Xu, JinWen; Zheng, ShuHui; Chen, SiJuan; Zhou, KeWen; Wang, TingHuai

    2010-12-01

    Genistein prevents atherosclerosis by exerting protective effects on blood vessels. The aim of this study is to investigate the role of caveolin1 and sprouty1 in the regulation of proliferation of vascular smooth muscle cell (VSMC) and endothelial cell by genistein. Using thiazolyl blue tetrazolium bromide(MTT) and [3H]-TdR assay, we found genistein inhibited angiotensin II-induced proliferation in primary cultured VSMC while it stimulated proliferation of quiescent endothelial cells. The effects were attenuated by caveolin1 or sprouty1 siRNA. Western blot analysis indicated that genistein attenuated the phosphorylation of extracellular regulated kinase1/2(ERK1/2) in angiotensin II-induced proliferated VSMC but stimulated the phosphorylation of ERK1/2 in quiescent endothelial cell. Double staining immunofluorescence identified caveolin1 and sprouty1 coexpressed in the cytoplasm of both VSMC and endothelial cell. Genistein increased the expression of caveolin1, p-caveolin1 and sprouty1 in VSMC, while it had opposite effects in quiescent endothelial cell. Co-immunoprecipitation suggested that genistein exerted its effects through interaction of caveolin1 and sprouty1. Our results demonstrate that the inhibition of angiotensin II-induced proliferation of VSMC and stimulation of quiescent endothelial cell by genistein are regulated by caveolin1 and sprouty1, which are implemented through Ras/MAPK pathway. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties.

    Science.gov (United States)

    Wakabayashi, Taku; Naito, Hisamichi; Suehiro, Jun-Ichi; Lin, Yang; Kawaji, Hideya; Iba, Tomohiro; Kouno, Tsukasa; Ishikawa-Kato, Sachi; Furuno, Masaaki; Takara, Kazuhiro; Muramatsu, Fumitaka; Weizhen, Jia; Kidoya, Hiroyasu; Ishihara, Katsuhiko; Hayashizaki, Yoshihide; Nishida, Kohji; Yoder, Mervin C; Takakura, Nobuyuki

    2018-02-01

    The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157 + VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Distinct tissue formation by heterogeneous printing of osteo- and endothelial progenitor cells.

    Science.gov (United States)

    Fedorovich, Natalja E; Wijnberg, Hans M; Dhert, Wouter J A; Alblas, Jacqueline

    2011-08-01

    The organ- or tissue-printing approach, based on layered deposition of cell-laden hydrogels, is a new technique in regenerative medicine suitable to investigate whether mimicking the anatomical organization of cells, matrix, and bioactive molecules is necessary for obtaining or improving functional engineered tissues. Currently, data on performance of multicellular printed constructs in vivo are limited. In this study we illustrate the ability of the system to print intricate porous constructs containing two different cell types--endothelial progenitors and multipotent stromal cells--and show that these grafts retain heterogeneous cell organization after subcutaneous implantation in immunodeficient mice. We demonstrate that cell differentiation leading to the expected tissue formation occurs at the site of the deposited progenitor cell type. While perfused blood vessels are formed in the endothelial progenitor cell-laden part of the constructs, bone formation is taking place in the multipotent stromal cell-laden part of the printed grafts.

  18. Culture and detection of primary cilia in endothelial cell models.

    Science.gov (United States)

    Lim, Yi Chung; McGlashan, Sue R; Cooling, Michael T; Long, David S

    2015-01-01

    The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length. HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003). We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

  19. Endothelial Progenitor Cells Enter the Aging Arena.

    Directory of Open Access Journals (Sweden)

    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  20. Immunohistochemical endothelial localisation--a novel method of vessel delineation in tendon tissue.

    Science.gov (United States)

    Jones, Martin; Clayton, Elizabeth; Noel, Carole; Ladhani, Kaetan; Grobbelaar, Addie

    2003-09-01

    The mechanism of tendon healing is still not fully understood. A dual source of nutrition for the tendon is important at times of injury either from synovial-type fluid bathing the tendon or its own blood supply. In addition, neovascularisation occurs at the site of injury from the time of the insult. The aim of this study was to develop a method of precise endothelial localisation in archived paraffin tendon sections, therefore facilitating the study of the healing of tendons within different species. The sections had to retain a high degree of cytoarchitecture and the stain be of enough contrast to allow quantitative assessment of tendon vascularity in different sites and different species. Endothelial staining was produced using an antibody to the endothelial cell surface marker CD-31 (Dako, Cambridge, UK). The signal was intensified using the Catalytic Signal Amplification kit (Dako). It resulted in a dark brown staining of the tendon endothelium, which was in sufficient contrast to allow automated image analysis.

  1. Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model

    Directory of Open Access Journals (Sweden)

    Bur Monica

    2006-02-01

    Full Text Available Abstract Background Cancer growth, invasion and metastasis are highly related to tumor-associated neovasculature. The presence and progression of endothelial cells in cancer is chaotic, unorganized, and angiogenic vessels are less functional. Therefore, not all markers appearing on the chaotic endothelial cells are accessible if a drug is given through the vascular route. Identifying endothelial cell markers from functional cancer angiogenic vessels will indicate the accessibility and potential efficacy of vascular targeted therapies. Results In order to quickly and effectively identify endothelial cell markers on the functional and accessible tumor vessels, we developed a novel technique by which tumor angiogenic vessels are labeled in vivo followed by Laser Capture Microdissection of microscopically isolated endothelial cells for genomic screening. Female C3H mice (N = 5 with established SCCVII tumors were treated with Rhodamine-RCA lectin by tail vein injection, and after fluorescence microscopy showed a successful vasculature staining, LCM was then performed on frozen section tissue using the PixCell II instrument with CapSure HS caps under the Rhodamine filter. By this approach, the fluorescent angiogenic endothelial cells were successfully picked up. As a result, the total RNA concentration increased from an average of 33.4 ng/ul +/- 24.3 (mean +/- S.D. to 1913.4 ng/ul +/- 164. Relatively pure RNA was retrieved from both endothelial and epithelial cells as indicated by the 260/280 ratios (range 2.22–2.47. RT-PCR and gene electrophoresis successfully detected CD31 and Beta-Actin molecules with minimal Keratin 19 expression, which served as the negative control. Conclusion Our present study demonstrates that in vivo Rhodamine RCA angiogenic vessel labeling provided a practical approach to effectively guide functional endothelial cell isolation by laser capture microdissection with fluorescent microscopy, resulting in high quality RNA and

  2. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    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: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  3. Endothelial progenitor cells, cardiovascular risk factors and lifestyle modifications.

    Science.gov (United States)

    Di Stefano, Rossella; Felice, Francesca; Feriani, Roberto; Balbarini, Alberto

    2013-04-01

    Endothelial progenitor cells (EPCs) contribute substantially to preservation of a structurally and functionally intact endothelium. EPCs home in to the sites of endothelial injury and ischemia, where they proliferate, differentiate and integrate into the endothelial layer or exert a paracrine function by producing vascular growth factors. This review will focus on successful lifestyle interventions that aim to maintain vascular health through beneficial actions on cell populations with vasculogenic potential. The results of the studies proving the role of healthy lifestyle are particularly emphasized.

  4. Dual role of lipoproteins in endothelial cell dysfunction in atherosclerosis.

    Science.gov (United States)

    Stancu, Camelia S; Toma, Laura; Sima, Anca V

    2012-08-01

    The endothelium is a key constituent of the vascular wall, being actively involved in maintaining the structural integrity and proper functioning of blood vessels. Hyperlipidemia, diabetes, hypertension, smoking and aging are important risk factors for the dysfunction of endothelial cells (EC). Circulating lipoproteins (Lp) synthesized and secreted from the intestine or liver have an important role in supplying peripheral tissues with fatty acids from triglyceride rich lipoproteins (TGRLp) for energy production or storage, and cholesterol from low density lipoproteins (LDL) or high density lipoproteins (HDL) for the synthesis of cellular membranes and steroid hormones. Under pathological conditions, Lp may suffer alterations in concentration and composition and become aggressors for EC. Modified LDL, remnant Lp, TGRLp lipolysis products, dysfunctional HDL are involved in the changes induced in EC morphology (reduced glycocalyx, overdeveloped endoplasmic reticulum, Golgi apparatus and basement membrane), loose intercellular junctions, increased oxidative and inflammatory stress, nitric oxide/redox imbalance, excess Lp transport and storage, as well as loss of anti-thrombotic properties, all of these being characteristics of endothelial dysfunction. Normal HDL are able to counteract the harmful effects of atherogenic Lp in EC but under persistent pathological conditions they lose the protective properties and become pro-atherogenic. This review summarises recent advances in understanding the role of Lp in the induction of endothelial dysfunction and the initiation and progression of atherosclerotic lesions. Its main focus is the antagonistic role of atherogenic Lp (LDL, VLDL, dysfunctional HDL) versus anti-atherogenic Lp (HDL), also pointing out the potential targets for arresting or reversing this process.

  5. Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

    Science.gov (United States)

    Shenoy, Anitha K; Lu, Jianrong

    2016-10-01

    Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells.

    Science.gov (United States)

    Suschek, C; Kolb, H; Kolb-Bachofen, V

    1997-12-01

    1. Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants. 2. In each of the different endothelial cells Mg-Dobesilate incubation (0.25-1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor N(G)-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects. 3. iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT-PCR.

  7. Msx genes define a population of mural cell precursors required for head blood vessel maturation.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Lallemand, Yvan; Cumano, Ana; Robert, Benoît

    2011-07-01

    Vessels are primarily formed from an inner endothelial layer that is secondarily covered by mural cells, namely vascular smooth muscle cells (VSMCs) in arteries and veins and pericytes in capillaries and veinules. We previously showed that, in the mouse embryo, Msx1(lacZ) and Msx2(lacZ) are expressed in mural cells and in a few endothelial cells. To unravel the role of Msx genes in vascular development, we have inactivated the two Msx genes specifically in mural cells by combining the Msx1(lacZ), Msx2(lox) and Sm22α-Cre alleles. Optical projection tomography demonstrated abnormal branching of the cephalic vessels in E11.5 mutant embryos. The carotid and vertebral arteries showed an increase in caliber that was related to reduced vascular smooth muscle coverage. Taking advantage of a newly constructed Msx1(CreERT2) allele, we demonstrated by lineage tracing that the primary defect lies in a population of VSMC precursors. The abnormal phenotype that ensues is a consequence of impaired BMP signaling in the VSMC precursors that leads to downregulation of the metalloprotease 2 (Mmp2) and Mmp9 genes, which are essential for cell migration and integration into the mural layer. Improper coverage by VSMCs secondarily leads to incomplete maturation of the endothelial layer. Our results demonstrate that both Msx1 and Msx2 are required for the recruitment of a population of neural crest-derived VSMCs.

  8. Radioprotection of mouse CNS endothelial cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimova, N.; Coultas, P.; Martin, R. [Peter McCallum Cancer Institute, Melbourne, VIC (Australia)

    1996-12-31

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy {gamma}-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  9. 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.

  10. Recent Insights in the Paracrine Modulation of Cardiomyocyte Contractility by Cardiac Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Jacques Noireaud

    2014-01-01

    Full Text Available The cardiac endothelium is formed by a continuous monolayer of cells that line the cavity of the heart (endocardial endothelial cells (EECs and the luminal surface of the myocardial blood vessels (intramyocardial capillary endothelial cells (IMCEs. EECs and IMCEs can exercise substantial control over the contractility of cardiomyocytes by releasing various factors such as nitric oxide (NO via a constitutive endothelial NO-synthase (eNOS, endothelin-1, prostaglandins, angiotensin II, peptide growth factors, and neuregulin-1. The purpose of the present paper is actually to shortly review recent new information concerning cardiomyocytes as effectors of endothelium paracrine signaling, focusing particularly on contractile function. The modes of action and the regulatory paracrine role of the main mediators delivered by cardiac endothelial cells upon cardiac contractility identified in cardiomyocytes are complex and not fully described. Thus, careful evaluation of new therapeutic approaches is required targeting important physiological signaling pathways, some of which have been until recently considered as deleterious, like reactive oxygen species. Future works in the field of cardiac endothelial cells and cardiac function will help to better understand the implication of these mediators in cardiac physiopathology.

  11. Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro.

    Science.gov (United States)

    Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

    2014-12-06

    Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-L-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  12. Vessel involvement in giant cell arteritis : an imaging approach

    NARCIS (Netherlands)

    Holm, Pieter W.; Sandovici, Maria; Slart, Riemer H. J. A.; Glaudemans, Andor W. J. M.; Rutgers, Abraham; Brouwer, Elisabeth

    Vasculitis is classified based on the size of the involved vessels. The two major forms are small vessel vasculitis and large vessel vasculitis (LVV). Main forms of LVV are Takayasu arteritis, giant cell arteritis (GCA), isolated aortitis and chronic periaortitis. This manuscript will focus on GCA,

  13. Comparison of Endothelial Cell Loss by Specular Microscopy ...

    African Journals Online (AJOL)

    Aim: To compare the endothelial cell loss between phacoemulsification and manual small‑incision cataract surgery (SICS). Endothelial cell loss was also compared in phacoemulsification group by temporal clear corneal incision (CCI) and by superior scleral incision (SI) technique. Materials and Methods: A total of 200 ...

  14. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  15. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  16. Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues.

    Science.gov (United States)

    Laitinen, L

    1987-04-01

    The binding of Griffonia simplicifolia agglutinin-I (GSA-I) and the isolectins GSA-I-AB3 and GSA-I-B4, having affinity for some alpha-D-galactosyl and N-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver and kidney cortex the GSA-I-conjugates selectively reacted with endothelial cells of the sinusoids and with intertubular and glomerular capillaries, respectively. However, a strong reactivity with the GSA-I-conjugates was additionally seen in the acinar cells of the pancreas, in the stratified squamous epithelia of skin and tongue, and in transitional epithelium. SDS-PAGE electrophoresis combined with the lectin-blotting technique indicated that a similar set of glycoproteins are responsible for the GSA-I binding, even in different tissues. Another lectin with specificity for alpha-D-galactose, the Maclura pomifera agglutinin, displayed a distinctly different distribution of binding sites, mainly in the basement membranes, of all mouse tissues studied. The results suggest that some alpha-D-galactosyl residues, recognized by the binding of GSA-I lectins, are preferentially expressed in endothelial cells of mouse tissues, and also provide further evidence that endothelial cells can present a highly specific surface glycosylation pattern.

  17. Obstructive sleep apnea and endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    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

  18. Role of Rutin on Nitric Oxide Synthesis in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Azizah Ugusman

    2014-01-01

    Full Text Available Nitric oxide (NO, produced by endothelial nitric oxide synthase (eNOS, is a major antiatherogenic factor in the blood vessel. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Decreased availability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis. Rutin is a flavonoid with multiple cardiovascular protective effects. This study aimed to investigate the effects of rutin on eNOS and NO production in cultured human umbilical vein endothelial cells (HUVEC. HUVEC were divided into four groups: control; oxidative stress induction with 180 μM H2O2; treatment with 300 μM rutin; and concomitant induction with rutin and H2O2 for 24 hours. HUVEC treated with rutin produced higher amount of NO compared to control (P<0.01. In the oxidative stress-induced HUVEC, rutin successfully induced cells’ NO production (P<0.01. Rutin promoted NO production in HUVEC by inducing eNOS gene expression (P<0.05, eNOS protein synthesis (P<0.01, and eNOS activity (P<0.05. Treatment with rutin also led to increased gene and protein expression of basic fibroblast growth factor (bFGF in HUVEC. Therefore, upregulation of eNOS expression by rutin may be mediated by bFGF. The results showed that rutin may improve endothelial function by augmenting NO production in human endothelial cells.

  19. Human brain microvascular endothelial cells resist elongation due to shear stress.

    Science.gov (United States)

    Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

    2015-05-01

    Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Transition of mesenchymal stem/stromal cells to endothelial cells.

    Science.gov (United States)

    Crisan, Mihaela

    2013-08-14

    Mesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial cells remains controversial. Isolation and in vitro manipulation of MSCs before clinical application are important steps. High numbers of MSCs are needed, requiring the in vitro expansion of these clinically important cells. To this end, a well-controlled procedure for MSC isolation and maintenance in culture is necessary.

  1. Cell Structure Controls Endothelial Cell Migration under Fluid Shear Stress.

    Science.gov (United States)

    Lin, Xiefan; Helmke, Brian P

    2009-06-01

    Cobblestone-shaped endothelial cells in confluent monolayers undergo triphasic mechanotaxis in response to steady unidirectional shear stress, but cells that are elongated and aligned on micropatterned substrates do not change their migration behavior in response to either perpendicular or parallel flow. Whether mechanotaxis of micropatterned endothelial cell layers is suppressed by elongated cytoskeletal structure or limited availability of adhesion area remains unknown. In this study, cells were examined on wide (100-200 μm) micropatterned lines after onset of shear stress. Cells in center regions of the lines exhibited cobblestone morphology and triphasic mechanotaxis behavior similar to that in unpatterned monolayers, whereas cells along the edges migrated parallel to the line axis regardless of the flow direction. When scratch wounds were created perpendicular to the micropatterned lines, the cells became less elongated before migrating into the denuded area. In sparsely populated lines oriented perpendicular to the flow direction, elongated cells along the upstream edge migrated parallel to the edge for 7 h before migrating parallel to the shear stress direction, even though adhesion area existed in the downstream direction. Thus, cytoskeletal structure and not available adhesion area serves as the dominant factor in determining whether endothelial mechanotaxis occurs in response to shear stress.

  2. Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane.

    Science.gov (United States)

    Scheppke, Lea; Murphy, Eric A; Zarpellon, Alessandro; Hofmann, Jennifer J; Merkulova, Alona; Shields, David J; Weis, Sara M; Byzova, Tatiana V; Ruggeri, Zaverio M; Iruela-Arispe, M Luisa; Cheresh, David A

    2012-03-01

    Vascular development and angiogenesis initially depend on endothelial tip cell invasion, which is followed by a series of maturation steps, including lumen formation and recruitment of perivascular cells. Notch ligands expressed on the endothelium and their cognate receptors expressed on perivascular cells are involved in blood vessel maturation, though little is known regarding the Notch-dependent effectors that facilitate perivascular coverage of nascent vessels. Here, we report that vascular smooth muscle cell (VSMC) recognition of the Notch ligand Jagged1 on endothelial cells leads to expression of integrin αvβ3 on VSMCs. Once expressed, integrin αvβ3 facilitates VSMC adhesion to VWF in the endothelial basement membrane of developing retinal arteries, leading to vessel maturation. Genetic or pharmacologic disruption of Jagged1, Notch, αvβ3, or VWF suppresses VSMC coverage of nascent vessels and arterial maturation during vascular development. Therefore, we define a Notch-mediated interaction between the developing endothelium and VSMCs leading to adhesion of VSMCs to the endothelial basement membrane and arterial maturation.

  3. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  4. DC8 and DC13 var genes associated with severe malaria bind avidly to diverse endothelial cells.

    Directory of Open Access Journals (Sweden)

    Marion Avril

    Full Text Available During blood stage infection, Plasmodium falciparum infected erythrocytes (IE bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1, a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow. To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

  5. Human Endothelial Cell Models in Biomaterial Research.

    Science.gov (United States)

    Hauser, Sandra; Jung, Friedrich; Pietzsch, Jens

    2017-03-01

    Endothelial cell (EC) models have evolved as important tools in biomaterial research due to ubiquitously occurring interactions between implanted materials and the endothelium. However, screening the available literature has revealed a gap between material scientists and physiologists in terms of their understanding of these biomaterial-endothelium interactions and their relative importance. Consequently, EC models are often applied in nonphysiological experimental setups, or too extensive conclusions are drawn from their results. The question arises whether this might be one reason why, among the many potential biomaterials, only a few have found their way into the clinic. In this review, we provide an overview of established EC models and possible selection criteria to enable researchers to determine the most reliable and relevant EC model to use. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Tumor-Endothelium Cross Talk Blocks Recruitment of Neutrophils to Endothelial Cells: A Novel Mechanism of Endothelial Cell Anergy1

    OpenAIRE

    Blaheta, Roman A; Powerski, Maciej; Hudak, Lukasz; Juengel, Eva; Jonas, Dietger; von Knethen, Andreas; Doerr, Hans Willhelm; Cinatl, Jindrich

    2009-01-01

    Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours wit...

  7. Tumor-Endothelium Cross Talk Blocks Recruitment of Neutrophils to Endothelial Cells: A Novel Mechanism of Endothelial Cell Anergy

    OpenAIRE

    Blaheta, Roman A.; Powerski, Maciej; Hudak, Lukasz; Juengel, Eva; Jonas, Dietger; von Knethen, Andreas; Doerr, Hans Willhelm; Cinatl Jr, Jindrich

    2009-01-01

    Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours wit...

  8. Coordinated Molecular Cross-Talk between Staphylococcus aureus, Endothelial Cells and Platelets in Bloodstream Infection

    Directory of Open Access Journals (Sweden)

    Carolina D. Garciarena

    2015-12-01

    Full Text Available Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the human body. Upon entry to the otherwise sterile environment of the cardiovascular system, S. aureus can lead to serious complications resulting in organ failure and death. The success of S. aureus as a pathogen in the bloodstream is due to its ability to express a wide array of cell wall proteins on its surface that recognise host receptors, extracellular matrix proteins and plasma proteins. Endothelial cells and platelets are important cells in the cardiovascular system and are a major target of bloodstream infection. Endothelial cells form the inner lining of a blood vessel and provide an antithrombotic barrier between the vessel wall and blood. Platelets on the other hand travel throughout the cardiovascular system and respond by aggregating around the site of injury and initiating clot formation. Activation of either of these cells leads to functional dysregulation in the cardiovascular system. In this review, we will illustrate how S. aureus establish intimate interactions with both endothelial cells and platelets leading to cardiovascular dysregulation.

  9. Endothelial progenitor cells regenerate infracted myocardium with neovascularisation development

    Directory of Open Access Journals (Sweden)

    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.

  10. Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles

    Science.gov (United States)

    Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C.; Haverich, Axel; Hilfiker, Andres

    2017-08-01

    In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

  11. The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

    2010-07-16

    Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

  12. Elongation, proliferation & migration differentiate endothelial cell phenotypes and determine capillary sprouting

    Directory of Open Access Journals (Sweden)

    Popel Aleksander S

    2009-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of capillaries from preexisting blood vessels, has been extensively studied experimentally over the past thirty years. Molecular insights from these studies have lead to therapies for cancer, macular degeneration and ischemia. In parallel, mathematical models of angiogenesis have helped characterize a broader view of capillary network formation and have suggested new directions for experimental pursuit. We developed a computational model that bridges the gap between these two perspectives, and addresses a remaining question in angiogenic sprouting: how do the processes of endothelial cell elongation, migration and proliferation contribute to vessel formation? Results We present a multiscale systems model that closely simulates the mechanisms underlying sprouting at the onset of angiogenesis. Designed by agent-based programming, the model uses logical rules to guide the behavior of individual endothelial cells and segments of cells. The activation, proliferation, and movement of these cells lead to capillary growth in three dimensions. By this means, a novel capillary network emerges out of combinatorially complex interactions of single cells. Rules and parameter ranges are based on literature data on endothelial cell behavior in vitro. The model is designed generally, and will subsequently be applied to represent species-specific, tissue-specific in vitro and in vivo conditions. Initial results predict tip cell activation, stalk cell development and sprout formation as a function of local vascular endothelial growth factor concentrations and the Delta-like 4 Notch ligand, as it might occur in a three-dimensional in vitro setting. Results demonstrate the differential effects of ligand concentrations, cell movement and proliferation on sprouting and directional persistence. Conclusion This systems biology model offers a paradigm closely related to biological phenomena and highlights previously

  13. Transcriptional Remodeling of Ion Channel Subunits by Flow Adaptation in Human Coronary Artery Endothelial Cells

    Science.gov (United States)

    Kefaloyianni, Eirini; Coetzee, William A.

    2011-01-01

    Endothelial cells (ECs) are constantly exposed to blood flow-induced shear forces in the vessels and this is a major determinant of endothelial function. Ion channels have a major role in endothelial function and in the control of vascular tone. We hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. We examined this hypothesis using large-scale quantitative real-time RT-PCR. Human coronary artery ECs were exposed to two levels of flow-induced shear stress for 24 h, while control cells were grown under static conditions. The expression of ion channel subunits was compared between control and flow-adapted cells. We used primers against 55 ion channel and exchanger subunits and were able to detect 54 subunits. Five dyn/cm2 of shear induced downregulation of 1 (NCX1) and upregulation of 18 subunits, including KCa2.2, KCa2.3, CX37, Kv1.5 and HCN2. Fifteen dyn/cm2 of shear stress induced the expression of 30 ion channel subunits, including KCa2.3, KCa2.2, CX37, Kir2.3 and KCa3.1. Our data demonstrate that substantial remodeling of endothelial ion channel subunit expression occurs with flow adaptation and suggest that altered ion channel expression may significantly contribute to vascular pathology associated with flow-induced alterations. PMID:21389733

  14. Radioprotection of mouse CNS endothelial cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimova, N.; Coultas, P.; Martin, R. [Peter MacCallum Cancer institute, Melbourne, Victoria (Australia)

    1997-03-01

    After treatments with monoamine oxidase inhibitors and L-DOPA, the blood brain barrier causes a build-up of dopamine in brain capillary endothelial cells. Conversion of the dopamine to a fluorophore provides a marker which can be used to measure endothelial cell density by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed loss within 24 hours of a sub-population of about 15 % of the endothelial cells. As a first step, rather than use the later endpoints of radionecrosis it was decided to examine directly whether Hoechst 33342 could protect against this rapid initial endothelial cell loss. Ten minutes after intravenous injection of Joechst 33342, in mouse brain the ligand was confined to endothelial cells and, for irradiation at this time, there was protection against endothelial cell loss over the first 24 hours after after exposure. Ablation of the sensitive subpopulation in unprotected mice took place over a dose range of 1 to 3 Gy {gamma}-rays but doses between 12 to 20 Gy were required in the presence of the ligand. This protection equated to a high dose modification factor of approximately 7 and may reflect suppression of apoptosis in this sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole, and how the observed protection affects late CNS necrosis development, has yet to be determined. However these results suggest a potential use of DNA-binding radioprotectors with limited penetration in investigations of the relative significance of endothelial and parenchymal damage in normal tissue responses to ionising radiation. (authors)

  15. Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

    Science.gov (United States)

    Rakocevic, Jelena; Kojic, Snezana; Orlic, Dejan; Stankovic, Goran; Ostojic, Miodrag; Petrovic, Olga; Zaletel, Ivan; Puskas, Nela; Todorovic, Vera; Labudovic-Borovic, Milica

    2016-02-01

    Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (5days old). Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. SOCS1 prevents graft arteriosclerosis by preserving endothelial cell function.

    Science.gov (United States)

    Qin, Lingfeng; Huang, Qunhua; Zhang, Haifeng; Liu, Renjing; Tellides, George; Min, Wang; Yu, Luyang

    The aim of this study was to determine the role of suppressor of cytokine signaling 1 (SOCS1) in graft arteriosclerosis (GA). GA, the major cause of late cardiac allograft failure, is initiated by immune-mediated endothelial activation resulting in vascular inflammation and consequent neointima formation. SOCS1, a negative regulator of cytokine signaling, is highly expressed in endothelial cells (ECs) and may prevent endothelial inflammatory responses and phenotypic activation. Clinical specimens of coronary arteries with GA, with atherosclerosis, or without disease were collected for histological analysis. SOCS1 knockout or vascular endothelial SOCS1 (VESOCS1) transgenic mice were used in an aorta transplant model of GA. Mouse aortic ECs were isolated for in vitro assays. Dramatic but specific reduction of endothelial SOCS1 was observed in human GA and atherosclerosis specimens, which suggested the importance of SOCS1 in maintaining normal endothelial function. SOCS1 deletion in mice resulted in basal EC dysfunction. After transplantation, SOCS1-deficient aortic grafts augmented leukocyte recruitment and neointima formation, whereas endothelial overexpression of SOCS1 diminished arterial rejection. Induction of endothelial adhesion molecules in early stages of GA was suppressed by the VESOCS1 transgene, and this effect was confirmed in cultured aortic ECs. Moreover, VESOCS1 maintained better vascular function during GA progression. Mechanistically, endothelial SOCS1, by modulating both basal and cytokine-induced expression of the adhesion molecules platelet/endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, restrained leukocyte adhesion and transendothelial migration during inflammatory cell infiltration. SOCS1 prevents GA progression by preserving endothelial function and attenuating cytokine-induced adhesion molecule expression in vascular endothelium. Copyright © 2014 American College of Cardiology

  17. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities

    OpenAIRE

    Aman, Jurjan; Weijers, Ester M.; Geerten P van Nieuw Amerongen; Malik, Asrar B.; van Hinsbergh, Victor W.M.

    2016-01-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the micr...

  18. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

    Directory of Open Access Journals (Sweden)

    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  19. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    Science.gov (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  20. Identification of Cell Surface Markers to Differentiate Rat Endothelial and Fibroblast Cells Using Lectin Arrays and LC–ESI-MS/MS

    Science.gov (United States)

    Lee, Ji Eun; Mirza, Shama P.; Didier, Daniela N.; Scalf, Mark; Olivier, Michael; Greene, Andrew S.; Smith, Lloyd M.

    2010-01-01

    Vascular endothelial cells located at the inner surface of blood vessels are a key component in angiogenesis and are employed as a primary cell type in the study of angiogenesis. These endothelial cells are, however, easily contaminated with fibroblast cells, which are located in proximity to the endothelial cells, during their isolation from tissue. It is thus important to find markers to distinguish the two cell types. In the present work, lectin arrays were prepared using aldehyde-terminated self-assembled monolayers (SAMs) and utilized to explore cell surface carbohydrate expression patterns on endothelial and fibroblast cells. It was found that the lectins Griffonia simplicifolia II (GS II) and Ulex europaeus agglutinin I (UEA I) selectively bind to rat fibroblast cells and not to rat endothelial cells. GS II-binding glycoproteins on fibroblast cells, which are potential cell surface markers to differentiate endothelial and fibroblast cells, were captured on a GS II lectin column and analyzed by LC–ESI-MS/MS. Six candidate cell surface glycoproteins were identified. Differential expression was confirmed by Western blot analysis for two of these proteins, lysosome-associated membrane glycoprotein-1 and transmembrane glycoprotein NMB. PMID:18821777

  1. Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

    Science.gov (United States)

    Howland, Shanshan W; Poh, Chek Meng; Rénia, Laurent

    2015-06-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

  2. Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

    Directory of Open Access Journals (Sweden)

    Shanshan W Howland

    2015-06-01

    Full Text Available In the murine model of cerebral malaria caused by P. berghei ANKA (PbA, parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

  3. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    Science.gov (United States)

    Howland, Shanshan W.; Poh, Chek Meng; Rénia, Laurent

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

  4. Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

    Science.gov (United States)

    Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

    2017-11-01

    The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

  5. Variety of corneal endothelial cell in glaucoma by confocal microscope

    Directory of Open Access Journals (Sweden)

    Hong-Liang Gao

    2014-10-01

    Full Text Available ATM: To define the causes of corneal endothelial cell damage, to investigate the preventive methods, and to observe the variety of corneal endothelial cell in glaucoma using confocal microscope.METHODS: Totally, 143 eyes of 97 patients with different types of glaucoma, and matched normal people were 20 cases, all 40 eyes. The cell density, cell area and cell variable coefficient were measured used confocal microscope. These indicatives of every kind of glaucoma were compared.RESULTS: The corneal endothelial cell density of normal group was 2 893.88±255.026/mm2, the group of acute angle-closure glaucoma(AACGwas 1 674.11±683.95/mm2, and the group of open angle glaucoma(OAGwas 2 687.22±391.87/mm2, the group of chronic angle-closure glaucoma(CACGwas 2 706.97±351.27/mm2. In all index the average cell density of corneal endothelial and the average area have statistical significance(F=62.950, 8.795; P=0.000, especially the group of AACG. CONCLUSION: The index of corneal endothelial cell in AACG is lower than that of normal. All index in OAG and CACG is difference with that of normal, but the difference has no statistical significance. And the dominant factor of damaged corneal endothelial is the time of intraocular hypertension.

  6. Tumor-associated endothelial cells display GSTP1 and RARβ2 promoter methylation in human prostate cancer

    Directory of Open Access Journals (Sweden)

    Pohida Thomas J

    2006-03-01

    Full Text Available Abstract Background A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. Methods Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARβ2 promoters via the QMS-PCR method. Results Comparing GSTP1 and RARβ2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. Conclusion We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.

  7. Mesenchymal status of lymphatic endothelial cell: enlightening treatment of lymphatic malformation.

    Science.gov (United States)

    Cai, Xu; Zhang, Wei; Chen, Gang; Li, Rui-Fang; Sun, Yan-Fang; Zhao, Yi-Fang

    2015-01-01

    In contrast to blood capillaries, lymphatic capillaries in peripheral tissues are composed of a single-cell layer of lymphatic endothelial cells (LECs) without a covering of mural cells. However, in lymphatic malformations, the enlarged lymphatic vessels were covered with mural cells. This study aimed to understand the molecular mechanism of differences between human dermal lymphatic endothelial cells (HDLECs) and human umbilical vein endothelial cells (HUVECs) and to determine the changes of LECs in the pathological condition of lymphatic malformation. Results showed that HDLECs exhibited lower expression of endothelial proteins, including VE-cadherin and CD31, than HUVECs; HDLECs also showed higher expression of mesenchymal proteins, including α-SMA, SM22α, calponin, and epithelial mesenchymal transition-related transcription factor Slug, than HUVECs. Likewise, HDLECs displayed higher permeability and weaker recruitment of SMCs than HUVECs; HDLECs also exhibited low PDGF-BB expression. TGF-β2 treatment and FGF2 depletion enhanced mesenchymal marker expression with increased permeability and reduced SMC recruitment. By contrast, Slug depletion in HDLECs enhanced VE-cadherin expression, inhibited α-SMA expression, decreased permeability, and enhanced PDGF-BB expression. These results suggested that HDLECs were in a mesenchymal status, which contributed to their functions and might determine their identities. Our data also revealed that miR143/145 was implicated in the mesenchymal status of HDLECs. In lymphatic malformations (LMs) treated with OK-432 sclerotherapy, immunohistochemistry results showed that Prox1 expression was reduced and mural cell investment was increased; these results indicated that LECs lost their mesenchymal status after OK-432 treatment was administered. The decreased mesenchymal status of LECs in LMs may induce dilated vessel constriction, which could be the mechanism of OK-432 sclerotherapy.

  8. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway.

    Science.gov (United States)

    Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

    2017-05-11

    During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned 'ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials.

  9. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  10. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    Science.gov (United States)

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  11. Magnetizable stent-grafts enable endothelial cell capture

    Energy Technology Data Exchange (ETDEWEB)

    Tefft, Brandon J. [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Uthamaraj, Susheil [Division of Engineering, Mayo Clinic, Rochester, MN (United States); Harburn, J. Jonathan [School of Medicine, Pharmacy and Health, Durham University, Stockton-on-Tees (United Kingdom); Hlinomaz, Ota [Department of Cardioangiology, St. Anne' s University Hospital, Brno (Czech Republic); Lerman, Amir [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Dragomir-Daescu, Dan [Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN (United States); Sandhu, Gurpreet S., E-mail: sandhu.gurpreet@mayo.edu [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States)

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance. - Highlights: • Magnetic stent-grafts were made from 2205 steel stents and polyurethane nanofibers. • Stent-grafts remained patent and formed a thin and uniform neointima when implanted. • Stent-grafts captured endothelial cells labeled with magnetic nanoparticles.

  12. Uterine Vasculature Remodeling in Human Pregnancy Involves Functional Macrochimerism by Endothelial Colony Forming Cells of Fetal Origin

    Science.gov (United States)

    Sipos, Peter I; Rens, Willem; Schlecht, HÉlène; Fan, Xiaohu; Wareing, Mark; Hayward, Christina; Hubel, Carl A; Bourque, Stephane; Baker, Philip N; Davidge, Sandra T; Sibley, Colin P; Crocker, Ian P

    2013-01-01

    The potency of adult-derived circulating progenitor endothelial colony forming cells (ECFCs) is drastically surpassed by their fetal counterparts. Human pregnancy is associated with robust intensification of blood flow and vascular expansion in the uterus, crucial for placental perfusion and fetal supply. Here, we investigate whether fetal ECFCs transmigrate to maternal bloodstream and home to locations of maternal vasculogenesis, primarily the pregnant uterus. In the first instance, endothelial-like cells, originating from mouse fetuses expressing paternal eGFP, were identified within uterine endothelia. Subsequently, LacZ or enhanced green fluorescent protein (eGFP)-labeled human fetal ECFCs, transplanted into immunodeficient (NOD/SCID) fetuses on D15.5 pregnancy, showed similar integration into the mouse uterus by term. Mature endothelial controls (human umbilical vein endothelial cells), similarly introduced, were unequivocally absent. In humans, SRY was detected in 6 of 12 myometrial microvessels obtained from women delivering male babies. The copy number was calculated at 175 [IQR 149–471] fetal cells per millimeter square endothelium, constituting 12.5% of maternal vessel lumina. Cross-sections of similar human vessels, hybridized for Y-chromosome, positively identified endothelial-associated fetal cells. It appears that through ECFC donation, fetuses assist maternal uterine vascular expansion in pregnancy, potentiating placental perfusion and consequently their own fetal supply. In addition to fetal growth, this cellular mechanism holds implications for materno-fetal immune interactions and long-term maternal vascular health. PMID:23554274

  13. Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

    Directory of Open Access Journals (Sweden)

    Yumiko Mitome-Mishima

    Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

  14. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  15. Effects of NM-3 on lymphatic vessel density and vascular endothelial growth factor of colon cancer in orthotopic implantation model of a severe combined immune deficiency mice

    Directory of Open Access Journals (Sweden)

    Jin-Shui Zhu

    2010-12-01

    Full Text Available The molecular mechanisms involved colon cancer tumorigenesis and development of colon cancer remain unclear. The aim of this study is to explore the inhibitive effects of NM-3 on lymphatic vessel density and vascular endothelial growth factor of micrometastatic lesion of orthotopic implantated colon cancer in the severe combined immune deficiency (SCID nude mice. Human colon cancer SW1116 cells were orthotopically implantated into the colon of the nude mice. Twenty-eight SCID nude mice were randomly divided into four groups (7 mice for each group after one week feeding and then the nude mice were treated with carboplatin and NM-3 via intraperitoneal injection twice a week for 8 weeks. The mice were sacrificed after 8 weeks and the vascular endothelial growth factor-C (VEGF-C, VEGF-D, VEGF-R-3 and lymphatic vessel density (LVD were analyzed using immunohistochemistry staining assay. LVD in NM-3 treated mice was significantly lower than that of control (normal saline treated mice. The expression of VEGF-C, VEGF-D, and VEGF-R-3 and the expression of mRNA of VEGF-C, VEGF-D, and VEGF-R-3 in NM-3 treated mice were significantly lower than that of control mice. The NM-3 inhibited the growth of colon cancer in the SCID mice of orthotopic implantatation model, and this effect may be related to the inhibitive effects of NM-3 on the lymphangiogenesis and vascular endothelial growth factor in colon cancer. NM-3 and carboplatin played a synergistic role in inhibiting lymphangiogenesis of human colon cancer in SCID nude mice and the further investigation of molecular mechanisms involved in colon cancer metastasis will provide an important evidence for understanding of lymphangiogenesis of human colon cancer.

  16. Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells

    Science.gov (United States)

    Fujie, Tomoya; Murakami, Masaki; Yoshida, Eiko; Yasuike, Shuji; Kimura, Tomoki; Fujiwara, Yasuyuki; Yamamoto, Chika; Kaji, Toshiyuki

    2016-01-01

    Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1–MRE and Nrf2–ARE pathways, whereas MT-2A expression requires only activation of the MTF-1–MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism. PMID:27563876

  17. Paradoxic effects of metformin on endothelial cells and angiogenesis

    Science.gov (United States)

    Bruno, Antonino; Cantelmo, Anna R.; Albini, Adriana

    2014-01-01

    The biguanide metformin is used in type 2 diabetes management and has gained significant attention as a potential cancer preventive agent. Angioprevention represents a mechanism of chemoprevention, yet conflicting data concerning the antiangiogenic action of metformin have emerged. Here, we clarify some of the contradictory effects of metformin on endothelial cells and angiogenesis, using in vitro and in vivo assays combined with transcriptomic and protein array approaches. Metformin inhibits formation of capillary-like networks by endothelial cells; this effect is partially dependent on the energy sensor adenosine-monophosphate-activated protein kinase (AMPK) as shown by small interfering RNA knockdown. Gene expression profiling of human umbilical vein endothelial cells revealed a paradoxical modulation of several angiogenesis-associated genes and proteins by metformin, with short-term induction of vascular endothelial growth factor (VEGF), cyclooxygenase 2 and CXC chemokine receptor 4 at the messenger RNA level and downregulation of ADAMTS1. Antibody array analysis shows an essentially opposite regulation of numerous angiogenesis-associated proteins in endothelial and breast cancer cells including interleukin-8, angiogenin and TIMP-1, as well as selective regulation of angiopioetin-1, -2, endoglin and others. Endothelial cell production of the cytochrome P450 member CYP1B1 is upregulated by tumor cell supernatants in an AMPK-dependent manner, metformin blocks this effect. Metformin inhibits VEGF-dependent activation of extracellular signal-regulated kinase 1/2, and the inhibition of AMPK activity abrogates this event. Metformin hinders angiogenesis in matrigel pellets in vivo, prevents the microvessel density increase observed in obese mice on a high-fat diet, downregulating the number of white adipose tissue endothelial precursor cells. Our data show that metformin has an antiangiogenic activity in vitro and in vivo associated with a contradictory short

  18. Paradoxic effects of metformin on endothelial cells and angiogenesis.

    Science.gov (United States)

    Dallaglio, Katiuscia; Bruno, Antonino; Cantelmo, Anna R; Esposito, Alessia I; Ruggiero, Luca; Orecchioni, Stefania; Calleri, Angelica; Bertolini, Francesco; Pfeffer, Ulrich; Noonan, Douglas M; Albini, Adriana

    2014-05-01

    The biguanide metformin is used in type 2 diabetes management and has gained significant attention as a potential cancer preventive agent. Angioprevention represents a mechanism of chemoprevention, yet conflicting data concerning the antiangiogenic action of metformin have emerged. Here, we clarify some of the contradictory effects of metformin on endothelial cells and angiogenesis, using in vitro and in vivo assays combined with transcriptomic and protein array approaches. Metformin inhibits formation of capillary-like networks by endothelial cells; this effect is partially dependent on the energy sensor adenosine-monophosphate-activated protein kinase (AMPK) as shown by small interfering RNA knockdown. Gene expression profiling of human umbilical vein endothelial cells revealed a paradoxical modulation of several angiogenesis-associated genes and proteins by metformin, with short-term induction of vascular endothelial growth factor (VEGF), cyclooxygenase 2 and CXC chemokine receptor 4 at the messenger RNA level and downregulation of ADAMTS1. Antibody array analysis shows an essentially opposite regulation of numerous angiogenesis-associated proteins in endothelial and breast cancer cells including interleukin-8, angiogenin and TIMP-1, as well as selective regulation of angiopioetin-1, -2, endoglin and others. Endothelial cell production of the cytochrome P450 member CYP1B1 is upregulated by tumor cell supernatants in an AMPK-dependent manner, metformin blocks this effect. Metformin inhibits VEGF-dependent activation of extracellular signal-regulated kinase 1/2, and the inhibition of AMPK activity abrogates this event. Metformin hinders angiogenesis in matrigel pellets in vivo, prevents the microvessel density increase observed in obese mice on a high-fat diet, downregulating the number of white adipose tissue endothelial precursor cells. Our data show that metformin has an antiangiogenic activity in vitro and in vivo associated with a contradictory short

  19. Gap-junctional coupling between neutrophils and endothelial cells

    OpenAIRE

    Zahler, Stefan; Hoffmann, Anke; Gloe, Torsten; Pohl, Ulrich

    2003-01-01

    Communication between leukocytes and endothelial cells is crucial for inflammatory reactions. Paracrine cross-talk and outside-in signaling (via adhesion molecules) have been characterized as communication pathways to date. As leukocytes and endothelial cells express connexins, we considered intercellular communication via gap junctions an intriguing additional concept. We found that gap-junctional coupling between neutrophils and endothelium occurred in a time-dependent, bidirectional manner...

  20. Cellular and molecular biology of aging endothelial cells.

    Science.gov (United States)

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  1. Expansion and cryopreservation of porcine and human corneal endothelial cells.

    Science.gov (United States)

    Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

    2017-08-01

    Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Intracellular cytoskeleton and junction proteins of endothelial cells in the porcine iris microvasculature.

    Science.gov (United States)

    Yang, Hongfang; Yu, Paula K; Cringle, Stephen J; Sun, Xinghuai; Yu, Dao-Yi

    2015-11-01

    Recently we reported studies of the iris microvasculature and its endothelial cells using intra-luminal micro-perfusion, fixation, and silver staining, suggesting that the iris vascular endothelium may be crucial for maintaining homeostasis in the ocular anterior segment. Here we present information regarding the intracellular structure and cell junctions of the iris endothelium. Thirty-seven porcine eyes were used for this study. The temporal long posterior ciliary artery was cannulated to assess the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining with phalloidin for intracellular cytoskeleton f-actin, and with antibodies against claudin-5 and VE-cadherin for junction proteins. Nuclei were counterstained with Hoechst. The iris was flat-mounted for confocal imaging. The iris microvasculature was studied for its distribution, branch orders and endothelial morphometrics with endothelial cell length measured for each vessel order. Our results showed that morphometrics of the iris microvasculature was comparable with our previous silver staining. Abundant stress fibres and peripheral border staining were seen within the endothelial cells in larger arteries. An obvious decrease in cytoplasmic stress fibres was evident further downstream in the smaller arterioles, and they tended to be absent from capillaries and veins. Endothelial intercellular junctions throughout the iris vasculature were VE-cadherin and claudin-5 immuno-positive, indicating the presence of both adherent junctions and tight junctions between vascular endothelial cells throughout the iris microvasculature. Unevenness of claudin-5 staining was noted along the endothelial cell borders in almost every order of vessels, especially in veins and small arterioles. Our results suggest that significant heterogeneity of intracellular structure and junction proteins is present in different orders of the iris vasculature in addition to vascular diameter

  3. A unique pulmonary microvascular endothelial cell niche revealed by Weibel-Palade bodies and Griffonia simplicifolia.

    Science.gov (United States)

    Wu, Songwei; Zhou, Chun; King, Judy A C; Stevens, Troy

    2014-03-01

    Pulmonary endothelium displays considerable heterogeneity along the vascular axis, from arteries to capillaries to veins. Griffonia simplicifolia is a lectin that recognizes pulmonary microvascular endothelium with preference over extra-alveolar endothelium in both arteries and veins, yet the precise vascular location where this phenotypic shift occurs is poorly resolved. We gelatin-filled the circulation and agarose-loaded the airways and then labeled the lung with Griffonia lectin to enable visualization of the endothelial transition zone. Endothelium in vessels with internal diameters less than 38 μm were uniformly Griffonia positive, whereas vessels with internal diameters greater than 60 μm were always Griffonia negative. Two populations of endothelium were identified in vessels ranging from 38 to 60 μm in diameter, including some that were positive and others that were negative for binding to G. simplicifolia. To better resolve this endothelial transition zone, we performed morphology studies to measure the distribution of Weibel-Palade bodies (WPbs), since WPbs are present in conduit vessel endothelium and absent in capillary endothelium. WPbs were found in endothelium with vascular dimensions as small as 18 μm in diameter but were not found in capillaries. Thus, we identify with precision that the endothelial phenotype transition from a cell that does not interact with Griffonia lectin to one that does occurs in blood vessels with internal diameters of approximately 38 μm, and we reveal an unappreciated vascular zone, between 18 and 38 μm in diameter, where endothelium both is Griffonia positive and possesses WPbs.

  4. Aged Lymphatic Vessels and Mast Cells in Perilymphatic Tissues.

    Science.gov (United States)

    Pal, Sarit; Meininger, Cynthia J; Gashev, Anatoliy A

    2017-05-03

    This review provides a comprehensive summary of research on aging-associated alterations in lymphatic vessels and mast cells in perilymphatic tissues. Aging alters structure (by increasing the size of zones with low muscle cell investiture), ultrastructure (through loss of the glycocalyx), and proteome composition with a concomitant increase in permeability of aged lymphatic vessels. The contractile function of aged lymphatic vessels is depleted with the abolished role of nitric oxide and an increased role of lymphatic-born histamine in flow-dependent regulation of lymphatic phasic contractions and tone. In addition, aging induces oxidative stress in lymphatic vessels and facilitates the spread of pathogens from these vessels into perilymphatic tissues. Aging causes the basal activation of perilymphatic mast cells, which, in turn, restricts recruitment/activation of immune cells in perilymphatic tissues. This aging-associated basal activation of mast cells limits proper functioning of the mast cell/histamine/NF-κB axis that is essential for the regulation of lymphatic vessel transport and barrier functions as well as for both the interaction and trafficking of immune cells near and within lymphatic collecting vessels. Cumulatively, these changes play important roles in the pathogenesis of alterations in inflammation and immunity associated with aging.

  5. Cytotoxicity of activated monocytes on endothelial cells.

    Science.gov (United States)

    Peri, G; Chiaffarino, F; Bernasconi, S; Padura, I M; Mantovani, A

    1990-02-15

    Unstimulated human monocytes did not express appreciable levels of cytotoxicity on normal human umbilical vein endothelial cells (EC) in a 24-48 hr TdR release assay. On activation with IFN-gamma and LPS, monocytes had appreciable cytotoxicity on EC. Monocyte cytotoxicity on EC was not dependent on the presence of contaminating lymphoid cells. Recombinant TNF, IL-1, and IL-6 as well as monocyte supernatants did not exert a cytotoxic effect on EC. Moreover, anti-TNF, anti-IL-1, and anti-IL-6 antibodies, as well as scavengers of reactive oxygen intermediates, did not affect the cytotoxicity of activated monocytes on EC. Antibodies against the beta-chain (CD18) of leukocyte integrins inhibited the adhesion and cytotoxicity of activated monocytes on EC. Pretreatment of EC with IL-1 augmented the adhesion of monocytes on EC. Normal monocytes were not cytotoxic on IL-1-pretreated EC and IL-1 treatment did not increase the susceptibility of EC to activated monocytes. Thus adhesion is necessary but not sufficient for monocyte killing of EC. Anti-alpha L (LFA-1) antibodies markedly reduced monocyte cytotoxicity on EC, although anti-alpha X (p150) antibodies had only a modest effect. Anti-alpha M (Mac-1/CR3) antibodies were intermediate inhibitors of EC killing by activated monocytes. Thus, alpha L, beta 2 (LFA-1), and, to a lesser extent, alpha M, beta 2 (Mac-1/CR3) and alpha X, beta 2 (p 150, 95) integrins are the main adhesive structures involved in the cytotoxic interaction of activated monocytes with EC. Monocyte-mediated damage of EC could play a role as a mechanism of tissue injury under conditions of local or systemic activation of mononuclear phagocytes.

  6. Gas6 stimulates angiogenesis of human retinal endothelial cells and of zebrafish embryos via ERK1/2 signaling.

    Directory of Open Access Journals (Sweden)

    Young Sook Kim

    Full Text Available AIM: To determine if growth arrest-specific 6 (Gas6 plays an important role in the regulation of angiogenesis in human retinal microvascular endothelial cells (HRMECs and in vessel development of zebrafish. METHODS: Proliferation, wound-healing cell migration, and tube formation were measured in HRMECs treated with recombinant human Gas6 (rhGas6. Sprague-Dawley rat aortas in Matrigels were treated with rhGas6, and microvessel sprouting emanating from arterial rings was analyzed. Transgenic zebrafish embryos (flk:GFP were microinjected with rhGas6 at 50 hours post-fertilization (hpf, and ectopic sprouting of subintestinal vessels (SIVs was observed under a confocal microscope. Morpholino oligonucleotides (MOs were microinjected to knockdown gas6 in zebrafish embryos, and intersegmental vessel impairment was observed. The effect of the extracellular signal-regulated kinase (ERK1/2 inhibitor on the migration of HRMECs and on vessel development in zebrafish embryos was tested. RESULTS: rhGas6 stimulated proliferation, migration, and tube formation in HRMECs in a dose-dependent manner. In rat aortas, rhGas6 induced vessel outgrowth, and the sprouting length was longer than that of controls. The rhGas6-microinjected zebrafish embryos had significantly increased vessel outgrowth in the SIVs. Recombinant human vascular endothelial growth factor (rhVEGF served as a positive control. Knockdown of gas6 inhibited angiogenesis in the developing vessels of zebrafish. The ERK1/2 inhibitor inhibited HRMEC migration and intersegmental vessel formation in zebrafish embryos. CONCLUSIONS/INTERPRETATIONS: These data suggest that Gas6 plays a pivotal role in proliferation, migration, and sprouting of angiogenic endothelial cells in the retina and in zebrafish embryos. Furthermore, Gas6 induced angiogenic processes are induced via phosphorylation of ERK1/2.

  7. Circulating endothelial progenitor cells in obese children and adolescents.

    Science.gov (United States)

    Pires, António; Martins, Paula; Paiva, Artur; Pereira, Ana Margarida; Marques, Margarida; Castela, Eduardo; Sena, Cristina; Seiça, Raquel

    2015-01-01

    This study aimed to investigate the relationship between circulating endothelial progenitor cell count and endothelial activation in a pediatric population with obesity. Observational and transversal study, including 120 children and adolescents with primary obesity of both sexes, aged 6-17 years, who were recruited at this Cardiovascular Risk Clinic. The control group was made up of 41 children and adolescents with normal body mass index. The variables analyzed were: age, gender, body mass index, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance, monocyte chemoattractant protein-1, E-selectin, asymmetric dimethylarginine and circulating progenitor endothelial cell count. Insulin resistance was correlated to asymmetric dimethylarginine (ρ=0.340; p=0.003), which was directly, but weakly correlated to E-selectin (ρ=0.252; p=0.046). High sensitivity C-reactive protein was not found to be correlated to markers of endothelial activation. Systolic blood pressure was directly correlated to body mass index (ρ=0.471; p<0.001) and the homeostasis model assessment-insulin resistance (ρ=0.230; p=0.012), and inversely correlated to adiponectin (ρ=-0.331; p<0.001) and high-density lipoprotein cholesterol (ρ=-0.319; p<0.001). Circulating endothelial progenitor cell count was directly, but weakly correlated, to body mass index (r=0.211; p=0.016), leptin (ρ=0.245; p=0.006), triglyceride levels (r=0.241; p=0.031), and E-selectin (ρ=0.297; p=0.004). Circulating endothelial progenitor cell count is elevated in obese children and adolescents with evidence of endothelial activation, suggesting that, during infancy, endothelial repairing mechanisms are present in the context of endothelial activation. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  8. Single-cell imaging of mechanotransduction in endothelial cells.

    Science.gov (United States)

    Lu, Shaoying; Wang, Yingxiao

    2014-01-01

    Endothelial cells (ECs) are constantly exposed to chemical and mechanical microenvironment in vivo. In mechanotransduction, cells can sense and translate the extracellular mechanical cues into intracellular biochemical signals, to regulate cellular processes. This regulation is crucial for many physiological functions, such as cell adhesion, migration, proliferation, and survival, as well as the progression of disease such as atherosclerosis. Here, we overview the current molecular understanding of mechanotransduction in ECs associated with atherosclerosis, especially those in response to physiological shear stress. The enabling technology of live-cell imaging has allowed the study of spatiotemporal molecular events and unprecedented understanding of intracellular signaling responses in mechanotransduction. Hence, we also introduce recent studies on mechanotransduction using single-cell imaging technologies. © 2014 Elsevier Inc. All rights reserved.

  9. Potential manipulation of endothelial progenitor cells in diabetes and its complications.

    Science.gov (United States)

    Fadini, G P; Avogaro, A

    2010-07-01

    Diabetes mellitus increases cardiovascular risk through its negative impact on vascular endothelium. Although glucotoxicity and lipotoxicity account for endothelial cell damage, endothelial repair is also affected by diabetes. Endothelial progenitor cells (EPCs) are involved in the maintenance of endothelial homoeostasis and in the process of new vessel formation. For these reasons, EPCs are thought to have a protective impact within the cardiovascular system. In addition, EPCs appear to modulate the functioning of other organs, providing neurotropic signals and promoting repair of the glomerular endothelium. The exact mechanisms by which EPCs provide cardiovascular protection are unknown and the definition of EPCs is not standardized. Notwithstanding these limitations, the literature consistently indicates that EPCs are altered in type 1 and type 2 diabetes and in virtually all diabetic complications. Moreover, experimental models suggest that EPC-based therapies might help prevent or reverse the features of end-organ complications. This identifies EPCs as having a novel pathogenic role in diabetes and being a potential therapeutic target. Several ways of favourably modulating EPCs have been identified, including lifestyle intervention, commonly used medications and cell-based approaches. Herein, we provide a comprehensive overview of EPC pathophysiology and the potential for EPC modulation in diabetes.

  10. Difference in protective effects of GIP and GLP-1 on endothelial cells according to cyclic adenosine monophosphate response.

    Science.gov (United States)

    Lim, Dong-Mee; Park, Keun-Young; Hwang, Won-Min; Kim, Ju-Young; Kim, Byung-Joon

    2017-05-01

    Receptors for glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are present in vascular endothelial cells. Previous studies investigating euglycemic status have demonstrated that GIP is directly involved in the physiology of blood vessels by controlling the blood flow rate of portal veins and that GLP-1 has a protective effect on blood vessels by acting on endothelial cells. However, to the best of our knowledge, the effects of GIP and GLP-1 on endothelial cells in patients with hyperglycemia remain unknown. Therefore, the present study investigated whether the effect of the incretin hormones GLP-1 and GIP differed with regards to the reversal of endothelial cell dysfunction caused by hyperglycemia. The production of nitric oxide (NO) was measured using the Griess reagent system kit and the expression of cyclic adenosine monophosphate (cAMP) in the cell was measured at a wavelength of 405 nm with the ELISA reader using the cyclic AMP EIA kit. Exposure of human umbilical vein endothelial cells (HUVEC) to a high glucose concentration decreased NO and endothelial nitric oxide synthase (eNOS) levels but increased inducible NOS (iNOS) levels. However, when HUVECs were pretreated with GLP-1, a reduction of iNOS expression was observed and the expression of eNOS and NO were increased, as opposed to pretreatment with GIP. The results differed according to the response of cAMP, the second messenger of incretin hormones: The GIP pretreatment group did not exhibit an increase in cAMP levels while the GLP-1 pretreatment group did. The results of the present study provide evidence that GLP-1, but not GIP, has a protective effect on endothelial function associated with cardiovascular disease, as it is associated with increased eNOS expression and the levels of NO. This effect may be due to an increase in the cAMP concentration during hyperglycemic events.

  11. In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

    Science.gov (United States)

    Zilla, P; Fasol, R; Dudeck, U; Siedler, S; Preiss, P; Fischlein, T; Müller-Glauser, W; Baitella, G; Sanan, D; Odell, J

    1990-08-01

    A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

  12. Cultured bovine aortic endothelial cells show increased histamine metabolism when exposed to oscillatory shear stress.

    Science.gov (United States)

    Skarlatos, S I; Hollis, T M

    1987-03-01

    Oscillatory shear stress applied to the lining of blood vessels causes endothelial cell injury, one of the essential postulated prerequisites to the development of atherosclerosis. The purpose of this investigation was to study effects of shear stress on bovine aortic endothelial cells (BAEC), in vitro, for varying lengths of time (6 h, 12 h, 24 h) on BAEC histamine content (HC) and histidine decarboxylase activity (HD). Low intensity stress (1.6 dynes/cm2) as well as intermediate and high intensity shear stresses (3.5 dynes/cm2 and 7.6 dynes/cm2) resulted in an accelerated HD (281%) and elevated HC (144%). These data indicate that oscillatory shear stress produces increases in histamine metabolism.

  13. In vivo imaging of tumor vascular endothelial cells

    Science.gov (United States)

    Zhao, Dawen; Stafford, Jason H.; Zhou, Heling; Thorpe, Philip E.

    2013-02-01

    Phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, becomes exposed on the outer surface of viable (non-apoptotic) endothelial cells in tumor blood vessels, probably in response to oxidative stresses present in the tumor microenvironment. In the present study, we optically imaged exposed PS on tumor vasculature in vivo using PGN635, a novel human monoclonal antibody that targets PS. PGN635 F(ab')2 was labeled with the near infrared (NIR) dye, IRDye 800CW. Human glioma U87 cells or breast cancer MDA-MB-231 cells were implanted subcutaneously or orthotopically into nude mice. When the tumors reached ~5 mm in diameter, 800CW- PGN635 was injected via a tail vein and in vivo dynamic NIR imaging was performed. For U87 gliomas, NIR imaging allowed clear detection of tumors as early as 4 h later, which improved over time to give a maximal tumor/normal ratio (TNR = 2.9 +/- 0.5) 24 h later. Similar results were observed for orthotopic MDA-MB-231 breast tumors. Localization of 800CW-PGN635 to tumors was antigen specific since 800CW-Aurexis, a control probe of irrelevant specificity, did not localize to the tumors, and pre-administration of unlabeled PGN635 blocked the uptake of 800CW-PGN635. Fluorescence microscopy confirmed that 800CW-PGN635 was binding to PS-positive tumor vascular endothelium. Our studies suggest that tumor vasculature can be successfully imaged in vivo to provide sensitive tumor detection.

  14. Blood flow reprograms lymphatic vessels to blood vessels.

    Science.gov (United States)

    Chen, Chiu-Yu; Bertozzi, Cara; Zou, Zhiying; Yuan, Lijun; Lee, John S; Lu, MinMin; Stachelek, Stan J; Srinivasan, Sathish; Guo, Lili; Vicente, Andres; Vincente, Andres; Mericko, Patricia; Levy, Robert J; Makinen, Taija; Oliver, Guillermo; Kahn, Mark L

    2012-06-01

    Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic forces. Although the genetic mutations that underlie the formation of many human vascular malformations are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program and natural history is not. Loss of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) resulted in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice. Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic identity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter vessel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly lost expression of PROX1, a lymphatic fate-specifying transcription factor. These findings reveal that blood flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.

  15. CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

    NARCIS (Netherlands)

    Siemerink, Martin J.; Klaassen, Ingeborg; Vogels, Ilse M. C.; Griffioen, Arjan W.; van Noorden, Cornelis J. F.; Schlingemann, Reinier O.

    2012-01-01

    The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend

  16. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-01-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

  17. Interleukin-33 induces urokinase in human endothelial cells--possible impact on angiogenesis.

    Science.gov (United States)

    Stojkovic, S; Kaun, C; Heinz, M; Krychtiuk, K A; Rauscher, S; Lemberger, C E; de Martin, R; Gröger, M; Petzelbauer, P; Huk, I; Huber, K; Wojta, J; Demyanets, S

    2014-06-01

    Urokinase-type plasminogen activator (u-PA) plays a pivotal role in extracellular proteolysis and is thought to be critically involved in the modulation of angiogenesis. Interleukin (IL)-33 is a member of the IL-1 cytokine family, which is thought to act as danger signal that is released from cells after injury. IL-33 is involved in the pathogenesis of various inflammatory diseases and previously was shown to induce angiogenesis and inflammatory activation of endothelial cells. We investigated the impact of IL-33 on u-PA in endothelial cells as a new possible function for IL-33. We could demonstrate that IL-33 upregulated u-PA mRNA expression and protein production in human coronary artery and human umbilical vein endothelial cells in a time- and concentration-dependent manner via interaction with its receptor ST2 and activation of the nuclear factor-κB pathway but independent of autocrine IL-1-induced effects. The hydroxymethylglutaryl-coenzyme A reductase inhibitor simvastatin abrogated the IL-33-induced increase in u-PA, thus providing further evidence for pleiotropic effects of statins. IL-33 induced u-PA-dependent capillary-like tube formation and vessel sprouting. In human carotid atherosclerotic plaques (n = 16), u-PA mRNA positively correlated with IL-33 mRNA expression (r = 0.780, P < 0.001). Furthermore, IL-33 and u-PA protein were detected in endothelial cells in these samples using fluorescence immunohistochemistry. We hypothesize that IL-33, representing a danger signal that is released after tissue damage, in addition to its role in the inflammatory activation of endothelial cells, is involved in u-PA-driven angiogenesis, a process that has been shown before to be linked to inflammation in various pathologies. © 2014 International Society on Thrombosis and Haemostasis.

  18. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells.

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-11-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1'-dioctadecyl-3,3,3',3'-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration.

  19. Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes.

    Science.gov (United States)

    Edwards, Sarah; Lalor, Patricia F; Nash, Gerard B; Rainger, G Ed; Adams, David H

    2005-03-01

    Crosstalk between hepatic sinusoidal ECs and closely juxtaposed hepatocytes via vascular endothelial growth factor is essential for the maintenance of sinusoidal endothelial growth and differentiation. We propose that paracrine interactions between endothelial cells and hepatocytes also may be responsible for the unique complement of adhesion receptors expressed on sinusoidal endothelium that regulate the recruitment of lymphocytes into the liver. To address this hypothesis, we developed an in vitro model of the hepatic sinusoid in which flowing lymphocytes could interact with hepatic endothelium conditioned by the presence of hepatocytes. Human hepatic sinusoidal endothelial cells cocultured with hepatocytes were activated so that they supported the adhesion of lymphocytes at levels equivalent to those seen on endothelium stimulated with the inflammatory cytokine tumour necrosis factor-beta. Lymphocyte adhesion was supported by intracellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, with an additional contribution from the novel adhesion receptor VAP-1. In conclusion, we show that interactions between hepatocytes and endothelial cells amplify leukocyte recruitment through the sinusoids by regulating the expression and function of endothelial adhesion molecules. These paracrine interactions may be responsible for the induction of the adhesion molecules that support constitutive lymphocyte recruitment to the liver as well as contributing significantly to the patterns of leukocyte adhesion seen during episodes of hepatic inflammation.

  20. The influence of biomaterials on endothelial cell thrombogenicity

    Science.gov (United States)

    McGuigan, Alison P.; Sefton, Michael V.

    2007-01-01

    Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thromobgenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions. PMID:17316788

  1. Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

    Science.gov (United States)

    Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

    2013-01-01

    Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

  2. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

    Directory of Open Access Journals (Sweden)

    Maskomani Silambarasan

    2016-04-01

    Full Text Available Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose and at various time intervals (6, 12, 24 and 48 h. miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis.

  3. A comparison of the tube forming potentials of early and late endothelial progenitor cells.

    Science.gov (United States)

    Mukai, Nana; Akahori, Taichi; Komaki, Motohiro; Li, Qin; Kanayasu-Toyoda, Toshie; Ishii-Watabe, Akiko; Kobayashi, Akiko; Yamaguchi, Teruhide; Abe, Mayumi; Amagasa, Teruo; Morita, Ikuo

    2008-02-01

    The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 mum wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.

  4. Blood Outgrowth Endothelial Cells Increase Tumor Growth Rates and Modify Tumor Physiology: Relevance for Therapeutic Targeting

    Directory of Open Access Journals (Sweden)

    Kalpna Gupta

    2013-02-01

    Full Text Available Endothelial cell precursors from human peripheral blood have been shown to home to areas of neovascularization and may assist tumor growth by increasing or fortifying blood vessel growth. In the present study, the influence of these cells on tumor growth and physiology was investigated and the role of these cells as a therapeutic target or in determining treatment sensitivity was tested. After isolation from human blood and expansion in vitro, actively growing cells with verified endothelial phenotype (Blood Outgrowth Endothelial Cell, BOEC were injected i.v. into tumor bearing mice for three consecutive days. The growth rate was significantly enhanced in relatively small RERF human lung tumors (i.e., less than 150 mm3 grown in immunocompromised mice by an average of 1.5-fold while it had no effect when injections were given to animals bearing larger tumors. There were no signs of toxicity or unwanted systemic effects. We also observed evidence of increased perfusion, vessel number, response to 15 Gy radiation and oxygenation in RERF tumors of animals injected with BOECs compared to control tumors. In addition, FSaII murine fibrosarcoma tumors were found to grow faster upon injection of BOECs. When FSaII tumors were subjected to a partial thermal ablation treatment using high intensity focused ultrasound (HIFU there was consistently elevated detection of fluorescently labeled and i.v. injected endothelial precursors in the tumor when analyzed with optical imaging and/or histological preparations. Importantly, we also observed that BOECs treated with the novel anti-angiogenic peptide anginex in-vitro, show decreased proliferation and increased sensitivity to radiation. In vivo, the normal increase in FSaII tumor growth induced by injected BOECs was blunted by the addition of anginex treatment. It appears that endothelial precursors may significantly contribute to tumor vessel growth, tumor progression and/or repair of tumor damage and may improve

  5. Blood Outgrowth Endothelial Cells Increase Tumor Growth Rates and Modify Tumor Physiology: Relevance for Therapeutic Targeting

    Energy Technology Data Exchange (ETDEWEB)

    Pagan, Jonathan, E-mail: jdpagan@uams.edu; Przybyla, Beata; Jamshidi-Parsian, Azemat [Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Gupta, Kalpna [Vascular Biology Center and Division of Hematology-Oncology Transplantation, Department of Medicine, University of Minnesota Medical School, MN 72223 (United States); Griffin, Robert J. [Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2013-02-18

    Endothelial cell precursors from human peripheral blood have been shown to home to areas of neovascularization and may assist tumor growth by increasing or fortifying blood vessel growth. In the present study, the influence of these cells on tumor growth and physiology was investigated and the role of these cells as a therapeutic target or in determining treatment sensitivity was tested. After isolation from human blood and expansion in vitro, actively growing cells with verified endothelial phenotype (Blood Outgrowth Endothelial Cell, BOEC) were injected i.v. into tumor bearing mice for three consecutive days. The growth rate was significantly enhanced in relatively small RERF human lung tumors (i.e., less than 150 mm{sup 3}) grown in immunocompromised mice by an average of 1.5-fold while it had no effect when injections were given to animals bearing larger tumors. There were no signs of toxicity or unwanted systemic effects. We also observed evidence of increased perfusion, vessel number, response to 15 Gy radiation and oxygenation in RERF tumors of animals injected with BOECs compared to control tumors. In addition, FSaII murine fibrosarcoma tumors were found to grow faster upon injection of BOECs. When FSaII tumors were subjected to a partial thermal ablation treatment using high intensity focused ultrasound (HIFU) there was consistently elevated detection of fluorescently labeled and i.v. injected endothelial precursors in the tumor when analyzed with optical imaging and/or histological preparations. Importantly, we also observed that BOECs treated with the novel anti-angiogenic peptide anginex in-vitro, show decreased proliferation and increased sensitivity to radiation. In vivo, the normal increase in FSaII tumor growth induced by injected BOECs was blunted by the addition of anginex treatment. It appears that endothelial precursors may significantly contribute to tumor vessel growth, tumor progression and/or repair of tumor damage and may improve the

  6. Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress.

    Science.gov (United States)

    Ranjan, V; Xiao, Z; Diamond, S L

    1995-08-01

    The role of chronic fluid shear stress on endothelial constitutive nitric oxide synthase (cNOS) levels may have an important role in vessel diameter control. We subjected primary human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC, passages 2-14) to steady laminar shear stress. In both cell types, the intracellular level of cNOS was elevated within 3 h of flow exposure at 25 dyn/cm2 and remained elevated at 6 and 12 h of flow exposure, compared with stationary controls, as indicated by digital immunofluorescence microscopy. Shear stress exposure for 6 h caused a 2.2 +/- 0.3- and 2.8 +/- 0.3-fold elevation of cNOS protein levels in BAEC (n = 3, P shear stress. Flow exposure at 4 dyn/cm2 caused no enhancement of cNOS levels in either cell type. The flow induction of the cNOS protein levels was not blocked by preincubation of BAEC with 100-400 microM of NG-nitro-L-arginine methyl ester, indicating that flow-induced NO (or guanosine 3',5'-cyclic monophosphate) was not involved in the elevation of cNOS levels. Protein kinase C inhibitor H-7 (10 microM) had no effect on induction of NOS protein in BAEC exposed to 25 dyn/cm2. The cNOS mRNA levels were found to be elevated by two- to threefold in BAEC after 6 or 12 h of flow exposure at either 4 or 25 dyn/cm2, and this induction of NOS mRNA occurred in the presence of dexamethasone. The elevation of cNOS levels by chronic flow exposure may provide a mechanism for chronic regulation of vessel diameter by endothelial response to prevailing blood flow.

  7. Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells.

    Science.gov (United States)

    Gao, Qi; Chen, Kexin; Gao, Lu; Zheng, Yang; Yang, Yong-Guang

    2016-09-08

    CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.

  8. Does corneal hysteresis correlate with endothelial cell density?

    Science.gov (United States)

    Akova-Budak, Berna; Kıvanç, Sertaç Argun

    2015-05-21

    Our aim was to determine if there is a correlation between corneal biomechanical properties, endothelial cell count, and corneal pachymetry in healthy corneas. Ninety-two eyes of all subjects underwent complete ocular examination, including intraocular pressure measurement by Goldmann applanation tonometer, objective refraction, and slit-lamp biomicroscopy. Topographic measurements and corneal pachymetry were performed using a Scheimpflug-based (Pentacam, Oculus, Germany) corneal topographer. Corneal hysteresis (CH) and corneal resistance factor (CRF) were measured with an Ocular Response Analyzer (ORA, Reichert Ophthalmic Instruments, Buffalo, NY). Endothelial cell count measurement was done using a specular microscope (CellChek, Konan, USA). Right eye values of the subjects were taken for the study. The mean CH was 11.5±1.7 mmHg and the mean CRF was 11.2±1.4 mmHg. Mean intraocular pressure was 15.3±2.3 mmHg. The mean endothelial cell count was 2754±205 cells/mm2. No correlation was found between biomechanical properties of cornea and endothelial cell count. There was a significant positive correlation between CH, CRF, and corneal thickness (pcorneal biomechanical properties significantly correlated with corneal thickness. We found no correlation between CH and CRF with the endothelial cell density in normal subjects.

  9. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  10. Low immunogenic endothelial cells maintain morphofunctional properties needed for tissue engineering.

    Science.gov (United States)

    Lau, Skadi; Eicke, Dorothee; Carvalho Oliveira, Marco; Wiegmann, Bettina; Schrimpf, Claudia; Haverich, Axel; Blasczyk, Rainer; Wilhelmi, Mathias; Figueiredo, Constanςa; Böer, Ulrike

    2017-10-05

    The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue engineered vascular grafts containing autologous cells are considered as ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leucocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood, umbilical cord blood- and vein. EC from all three sources were transduced by 93.1 ± 4.8% and effectively HLA I-silenced by up to 67% compared to non-transduced cells or transduced with a non-specific short hairpin RNA (shRNA), respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low density lipoprotein (ac-LDL), and to form capillary-like tube structures in 3D fibrin gels similarly to non-transduced cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC towards long and widely branched vascular networks necessary for pre-vascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological

  11. Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

    2014-01-01

    electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

  12. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... However, troglitazone had protective effects of EPCR on injured cells. Key words: Endothelial protein C receptor, renal tubular epithelial cell, troglitazone, tumor necrosis factor-α, interleukin-1β; high glucose. ..... induce apoptosis of vascular smooth muscle cells through an extracellular signal- regulated ...

  13. Differential Gene Expression of Primary Cultured Lymphatic and Blood Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Gregory M. Nelson

    2007-12-01

    Full Text Available Blood vascular endothelial cells (BECs and the developmentally related lymphatic endothelial cells (LECs create complementary, yet distinct vascular networks. Each endothelial cell type interacts with flowing fluid and circulating cells, yet each vascular system has evolved specialized gene expression programs and thus both cell types display different phenotypes. BECs and LECs express distinct genes that are unique to their specific vascular microenvironment. Tumors also take advantage of the molecules that are expressed in these vascular systems to enhance their metastatic potential. We completed transcriptome analyses on primary cultured LECs and BECs, where each comparative set was isolated from the same individual. Differences were resolved in the expression of several major categories, such as cell adhesion molecules (CAMs, cytokines, cytokine receptors. We have identified new molecules that are associated with BECs (e.g., claudin-9, CXCL11, neurexin-1, neurexin-2, the neuronal growth factor regulator-1 and LECs (e.g., claudin-7, CD58, hyaluronan and proteoglycan link protein 1 (HAPLN1, the poliovirus receptor-related 3 molecule that may lead to novel therapeutic treatments for diseases of lymphatic or blood vessels, including metastasis of cancer to lymph nodes or distant organs.

  14. Primary Phenomenon in the Network Formation of Endothelial Cells: Effect of Charge.

    Science.gov (United States)

    Arai, Shunto

    2015-12-07

    Blood vessels are essential organs that are involved in the supply of nutrients and oxygen and play an important role in regulating the body's internal environment, including pH, body temperature, and water homeostasis. Many studies have examined the formation of networks of endothelial cells. The results of these studies have revealed that vascular endothelial growth factor (VEGF) affects the interactions of these cells and modulates the network structure. Though almost all previous simulation studies have assumed that the chemoattractant VEGF is present before network formation, vascular endothelial cells secrete VEGF only after the cells bind to the substrate. This suggests VEGF is not essential for vasculogenesis especially at the early stage. Using a simple experiment, we find chain-like structures which last quite longer than it is expected, unless the energetically stable cluster should be compact. Using a purely physical model and simulation, we find that the hydrodynamic interaction retard the compaction of clusters and that the chains are stabilized through the effects of charge. The charge at the surface of the cells affect the interparticle potential, and the resulting repulsive forces prevent the chains from folding. The ions surrounding the cells may also be involved in this process.

  15. Primary Phenomenon in the Network Formation of Endothelial Cells: Effect of Charge

    Directory of Open Access Journals (Sweden)

    Shunto Arai

    2015-12-01

    Full Text Available Blood vessels are essential organs that are involved in the supply of nutrients and oxygen and play an important role in regulating the body’s internal environment, including pH, body temperature, and water homeostasis. Many studies have examined the formation of networks of endothelial cells. The results of these studies have revealed that vascular endothelial growth factor (VEGF affects the interactions of these cells and modulates the network structure. Though almost all previous simulation studies have assumed that the chemoattractant VEGF is present before network formation, vascular endothelial cells secrete VEGF only after the cells bind to the substrate. This suggests VEGF is not essential for vasculogenesis especially at the early stage. Using a simple experiment, we find chain-like structures which last quite longer than it is expected, unless the energetically stable cluster should be compact. Using a purely physical model and simulation, we find that the hydrodynamic interaction retard the compaction of clusters and that the chains are stabilized through the effects of charge. The charge at the surface of the cells affect the interparticle potential, and the resulting repulsive forces prevent the chains from folding. The ions surrounding the cells may also be involved in this process.

  16. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells

    OpenAIRE

    Abbasian, Nima; Burton, James O.; Herbert, Karl E.; Tregunna, Barbara-Emily; Brown, Jeremy R.; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J.; Goodall, Alison H.; Bevington, Alan

    2015-01-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intra...

  17. Differentiation of monocyte-derived dendritic cells on endothelial cells as feeding layer

    Directory of Open Access Journals (Sweden)

    Gholami K

    2011-06-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: The innate and adaptive immune responses are dependent on the migration of leukocytes across endothelial cells. Dendritic cells (DCs play an important role in the initiation of cellular immune responses during their migration from tissues into the lymph nodes where they interact with endothelial cells of lymphatic vessels. We investigated the effects of surface-adherent and non-activated endothelial cells on phenotypic and functional characteristics of dendritic cells."n"nMethods: Immature dendritic cells were generated from the isolation of peripheral blood mononuclear cells and their subsequent culture in DC-RPMI 1640 medium containing 10% FCS, interleukin-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF for five days. On day five, a maturation factor (composed of monocyte-conditioned medium, tumor necrosis factor-α (TNF-α and poly I:C was added to the RPMI medium where immature DCs were co-cultured with endothelial cell monolayer for 24 h. The maturation of harvested DCs on day seven was evaluated via flow cytometry, a beta-counter and an ELISA kit."n"nResults: This study showed that the endothelial cells interact with dendritic cells generated from peripheral blood monocytes via cell-to-cell

  18. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    Science.gov (United States)

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (Pmetformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (Pmetformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.

    Science.gov (United States)

    Okumura, Naoki; Ishida, Naoya; Kakutani, Kazuya; Hongo, Akane; Hiwa, Satoru; Hiroyasu, Tomoyuki; Koizumi, Noriko

    2017-11-01

    To develop analysis software for cultured human corneal endothelial cells (HCECs). Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.

  20. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities.

    Science.gov (United States)

    Aman, Jurjan; Weijers, Ester M; van Nieuw Amerongen, Geerten P; Malik, Asrar B; van Hinsbergh, Victor W M

    2016-08-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches. Copyright © 2016 the American Physiological Society.

  1. Knockdown of ezrin suppresses the migration and angiogenesis of human umbilical vein endothelial cells in vitro.

    Science.gov (United States)

    Zhao, Liang-ping; Huang, Lei; Tian, Xun; Liang, Feng-qi; Wei, Jun-cheng; Zhang, Xian; Li, Sha; Zhang, Qing-hua

    2016-04-01

    Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.

  2. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    Science.gov (United States)

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Endothelial progenitor cells in vascular health: focus on lifestyle.

    Science.gov (United States)

    Van Craenenbroeck, Emeline M; Conraads, Viviane M

    2010-05-01

    Endothelial dysfunction, which is considered the functional equivalent of a disrupted balance between endothelial injury and repair, precedes overt atherosclerosis by many years. Although this phenomenon is part of the normal aging process, prevention of early and progressive endothelial dysfunction has become an important therapeutic target. Evidence has accumulated to show that endothelial progenitor cells (EPC), contribute substantially to preservation of a structurally and functionally intact endothelium. There has been considerable progress in our understanding of the various cell types that were in the past all covered by the term "EPC." EPC home to sites of endothelial injury and ischemia, where they proliferate, differentiate and integrate into the endothelial layer or exert a paracrine function by producing vascular growth factors. Although more emphasis has been put on the pharmacological approach of endothelial dysfunction, the effect of a healthy lifestyle, via mobilization and functional improvement of EPC, is increasingly recognized. This review will focus on successful lifestyle interventions that aim to maintain vascular health through beneficial actions on cell populations with vasculogenic potential ("EPC"). The role of physical activity and dietary recommendations, which are considered essential elements of a healthy lifestyle, will be particularly emphasized. A thorough understanding of the physiology of endothelial benefits, derived from such interventions, may help to implement these measures on top of classical drug therapy, but also provides a solid basis for primary prevention. The effects of additional elements of a comprehensive lifestyle advice, such as smoking cessation, weight and stress reduction, also comprise a modulation of EPC function and circulating numbers and are therefore included in this review as well. Copyright 2009 Elsevier Inc. All rights reserved.

  4. Circulating endothelial cell number and markers of endothelial dysfunction in previously preeclamptic women.

    Science.gov (United States)

    Tuzcu, Zeyneb Baspehlivan; Asicioglu, Ebru; Sunbul, Murat; Ozben, Beste; Arikan, Hakki; Koc, Mehmet

    2015-10-01

    Patients with preeclampsia (PE) have endothelial dysfunction and an increased future risk of cardiovascular (CV) mortality. The number of circulating endothelial cells (CECs) is markedly increased in conditions associated with a high degree of endothelial cell activation/injury including PE. We hypothesized that the number of CECs continues to be increased in women with a history of PE, reflecting ongoing endothelial cell activation/injury. CECs, flow-mediated vasodilation, levels of adhesion molecules and soluble vascular endothelial growth factor receptor-1 (sVEGFR1), and urine albumin/creatinine ratio were determined in 21 healthy women with ongoing normal pregnancy, 24 healthy currently nonpregnant women with a history of normal pregnancy, a total of 17 women with currently active mild (n = 11) or severe (n = 6) PE without hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, and 16 currently nonpregnant women with a history of mild (n = 10) or severe (n = 6) PE. Blood samples from women with active preeclampsia had higher CECs (9.9 ± 7.9 cells/mL) than healthy pregnant women (3.0 ± 4.1 cells/mL; P < .001), healthy nonpregnant women with a history of normal pregnancy (3.4 ± 4.0 cells/mL; P < .001), or women with a history of preeclampsia (2.4 ± 2.0 cells/mL; P < .001). The number of CECs were similar between women with a history of preeclampsia and healthy nonpregnant women with a history of normal pregnancy. Patients with active preeclampsia had significantly higher soluble vascular cell adhesion molecule-1, soluble E-selectin, sVEGFR1, and urinary albumin/creatinine ratio than healthy pregnant women. However, soluble vascular cell adhesion molecule-1, soluble E-selectin, urinary albumin/creatinine ratio were similar in women with a history of preeclampsia and healthy nonpregnant women with a history of normal pregnancy. However, women with a history of preeclampsia had higher sVEGFR1 levels than women with a history of normal

  5. High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Iwata, Yasuhiro; Klaren, William D; Lebakken, Connie S; Grimm, Fabian A; Rusyn, Ivan

    Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Constitutive Expression of Adiponectin in Endothelial Progenitor Cells Protects a Rat Model of Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Renwei Zhang

    2017-01-01

    Full Text Available Endothelial progenitor cells (EPCs, as precursors to endothelial cells, play a significant part in the process of endogenous blood vessel repair and maintenance of endothelial integrity. Adiponectin (APN is an adipocyte-specific adipocytokine. In this study, we aim to test whether we transplant a combined graft of EPCs transfected with the adiponectin gene into a rat model of cerebral ischemia could improve functional recovery after middle cerebral artery occlusion (MCAO. Sprague-Dawley (SD rats were randomly divided into a MCAO control group, a MCAO EPC treatment group, and a MCAO LV-APN-EPC treatment group. A focal cerebral ischemia and reperfusion model was induced by the intraluminal suture method. After 2 h of reperfusion, EPCs were transplanted by injection through the tail vein. A rotarod test was conducted to assess behavioral function before MCAO and on days 1, 7, and 14 after MCAO. After 14 d, TTC staining, CD31 immunofluorescence, and TUNEL staining were used to evaluate infarct volume, microvessel density, and cell apoptosis. Results revealed that behavioral function, infarct area percentage, microvessel density, and cell apoptosis rates were more favorable in the LV-APN-EPC treatment group than in the EPC treatment group. These data suggested that gene-modified cell therapy may be a useful approach for the treatment of ischemic stroke.

  8. Interplay between Inflammatory Responses and Lymphatic Vessels.

    Science.gov (United States)

    Shin, Kihyuk; Lee, Seung-Hyo

    2014-08-01

    Lymphatic vessels are routes for leukocyte migration and fluid drainage. In addition to their passive roles in migration of leukocytes, increasing evidence indicates their active roles in immune regulation. Tissue inflammation rapidly induces lymphatic endothelial cell proliferation and chemokine production, thereby resulting in lymphangiogenesis. Furthermore, lymphatic endothelial cells induce T cell tolerance through various mechanisms. In this review, we focus on the current knowledge on how inflammatory cytokines affect lymphangiogenesis and the roles of lymphatic vessels in modulating immune responses.

  9. Characterization of angiogenin receptors on bovine brain capillary endothelial cells.

    Science.gov (United States)

    Chamoux, M; Dehouck, M P; Fruchart, J C; Spik, G; Montreuil, J; Cecchelli, R

    1991-04-30

    The mitogenic effect of bovine milk angiogenin was studied on bovine brain capillary and aortic endothelial cells, smooth muscle cells and fibroblasts. The proliferation of only bovine brain capillary endothelial cells was detected at concentrations ranging from 10 to 1,000 ng/ml, with a maximum effect at 100 ng/ml. This mitogenic activity may be correlated with a specific binding of angiogenin which was demonstrated only to bovine brain capillary endothelial cells. [125I]-labeled angiogenin binding was time and concentration dependent and saturable. Scatchard analyses of binding data showed evidence of a single class of binding sites with an apparent dissociation constant of 5.10(-10)M. The molecular mass of the angiogenin receptor (49 kDa) was determined by ligand blotting.

  10. Ponatinib reduces viability, migration, and functionality of human endothelial cells.

    Science.gov (United States)

    Gover-Proaktor, Ayala; Granot, Galit; Shapira, Saar; Raz, Oshrat; Pasvolsky, Oren; Nagler, Arnon; Lev, Dorit L; Inbal, Aida; Lubin, Ido; Raanani, Pia; Leader, Avi

    2017-06-01

    Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.

  11. Focus on biological identity of endothelial progenitors cells.

    Science.gov (United States)

    Zaccone, V; Flore, R; Santoro, L; De Matteis, G; Giupponi, B; Li Puma, D D; Santoliquido, A

    2015-11-01

    Circulating Endothelial Progenitor Cells (EPCs) were discovered by Asahara et al in 1997 and defined as bone marrow CD34+/KDR+ cells endowed with angiogenic potentialities in vitro and in vivo. The most likely assumption is that EPCs consist of several cell subpopulations with functions targeted at accomplishing the post-natal neovascularization process in a synergic and complementary fashion. Indeed, the subsequent identification of numerous and differentiated hematic populations, characterized by the capacity to develop an endothelial phenotype, has posed a number of questions as to the real identity of EPCs. This concept does not represent a sterile speculation but rather it suggests important implications for the future practice of stem cell therapy. The aim of this report was to explore through a critical analysis the two main experimental methodologies, in vitro culture and flow cytometry, applied to EPCs, followed by a brief revaluation of the endothelial progenitors employing a globally functional approach.

  12. Rapid flow-induced responses in endothelial cells

    Science.gov (United States)

    Stamatas, G. N.; McIntire, L. V.

    2001-01-01

    Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

  13. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2013-12-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

  14. LGN Directs Interphase Endothelial Cell Behavior via the Microtubule Network.

    Directory of Open Access Journals (Sweden)

    Catherine E Wright

    Full Text Available Angiogenic sprouts require coordination of endothelial cell (EC behaviors as they extend and branch. Microtubules influence behaviors such as cell migration and cell-cell interactions via regulated growth and shrinkage. Here we investigated the role of the mitotic polarity protein LGN in EC behaviors and sprouting angiogenesis. Surprisingly, reduced levels of LGN did not affect oriented division of EC within a sprout, but knockdown perturbed overall sprouting. At the cell level, LGN knockdown compromised cell-cell adhesion and migration. EC with reduced LGN levels also showed enhanced growth and stabilization of microtubules that correlated with perturbed migration. These results fit a model whereby LGN influences interphase microtubule dynamics in endothelial cells to regulate migration, cell adhesion, and sprout extension, and reveal a novel non-mitotic role for LGN in sprouting angiogenesis.

  15. 3D study of capillary network derived from human cord blood mesenchymal stem cells and differentiated into endothelial cell with VEGFR2 protein expression

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Tahmasbi

    2013-09-01

    Full Text Available New blood forming vessels are produced by differentiation of mesodermal precursor cells to angioblasts that become endothelial cells (ECs which in turn give rise to primitive capillary network. Human cord blood (HCB contains large subsets of mononuclear cells (MNCs that can be differentiated into endothelial-like cells in vitro. Human mononuclear progenitor cells were purified from fresh umbilical cord blood by the expression of CD34 and FLK-1 antigens expressed in both angioblasts and hematopoetic stem cells. The HCB derived mesenchymal stem cells (MSCs can be differentiated into adipocyte, osteocyte, chondrocyte and ECs. In this study, the differentiation of human cord blood mesenchymal stem cells (hCBMSCs into endothelial-like cells was induced in the presence of vascular endothelial growth factor (VEGF and insulin-like growth factor (IGF-1. The differentiated ECs were then examined for their ability to express VEGF receptor-2 (VEGFR2 and von Willebrand factor (vWF. These cells were adopted to grow, proliferate and develop into a capillary network in a semisolid gel matrix in vitro. The capillary network formation in each well of 24-well plate was found to be 80% in presence of VEGF (40 ng/ml and IGF-1 (20 ng/ml of culture media, suggesting that the capillary network formation is associated with endothelial-like cells derived from hCBMSCs by expression of their markers.

  16. Magnetizable stent-grafts enable endothelial cell capture

    Science.gov (United States)

    Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

  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. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation.

    Science.gov (United States)

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-03-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), Pcytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

  19. Direct Reprogramming of Human Dermal Fibroblasts Into Endothelial Cells Using ER71/ETV2.

    Science.gov (United States)

    Lee, Sangho; Park, Changwon; Han, Ji Woong; Kim, Ju Young; Cho, Kyuwon; Kim, Eun Jae; Kim, Sangsung; Lee, Shin-Jeong; Oh, Se Yeong; Tanaka, Yoshiaki; Park, In-Hyun; An, Hyo Jae; Shin, Claire Min; Sharma, Shraya; Yoon, Young-Sup

    2017-03-03

    Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process. © 2016 American Heart Association, Inc.

  20. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation

    Science.gov (United States)

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-01-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte–endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), Pstretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution

  1. Endothelial cells are essential for ovarian stromal tissue restructuring after xenotransplantation of isolated ovarian stromal cells

    National Research Council Canada - National Science Library

    Dath, C; Dethy, A; Van Langendonckt, A; Van Eyck, A S; Amorim, C A; Luyckx, V; Donnez, J; Dolmans, M M

    2011-01-01

    .... To help improve the grafting technique, we investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival...

  2. Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling

    Directory of Open Access Journals (Sweden)

    Kristy A. Warner

    2008-02-01

    Full Text Available Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3 and Kaposi's sarcoma (SLK show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2, compared to cocultures with empty vector controls (HDMEC-LXSN. Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF, as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.

  3. TNFα-Damaged-HUVECs Microparticles Modify Endothelial Progenitor Cell Functional Activity

    Science.gov (United States)

    Luna, Carlos; Carmona, Andrés; Alique, Matilde; Carracedo, Julia; Ramirez, Rafael

    2015-01-01

    Endothelial progenitor cells (EPCs) have an important role in the maintenance of vascular integrity and homeostasis. While there are many studies that explain EPCs mechanisms action, there are few studies that demonstrate how they interact with other emerging physiological elements such as Endothelial Microparticles (EMPs). EMPs are membranous structures with a size between 100 and 1000 nm that act as molecular information transporter in biological systems and are known as an important elements in develop different pathologies; moreover a lot of works explains that are novel biomarkers. To elucidate these interactions, we proposed an in vitro model of endothelial damage mediated by TNFalpha, in which damaged EMPs and EPCs are in contact to assess EPCs functional effects. We have observed that damaged EMPs can modulate several EPCs classic factors as colony forming units (CFUs), contribution to repair a physically damaged endothelium (wound healing), binding to mature endothelium, and co-adjuvants to the formation of new vessels in vitro (angiogenesis). All of these in a dose-dependent manner. Damaged EMPs at a concentration of 103 MPs/ml have an activating effect of these capabilities, while at concentrations of 105 MPs/ml these effects are attenuated or reduced. This in vitro model helps explain that in diseases where there is an imbalance between these two elements (EPCs and damaged EMPs), the key cellular elements in the regeneration and maintenance of vascular homeostasis (EPCs) are not fully functional, and could explain, at least in part, endothelial dysfunction associated in various pathologies. PMID:26733886

  4. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    Science.gov (United States)

    Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

    1988-05-01

    The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

  5. Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

    Directory of Open Access Journals (Sweden)

    Shuei-Kuen Tseng

    2014-09-01

    Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

  6. 3D map of the human corneal endothelial cell

    OpenAIRE

    Zhiguo He; Fabien Forest; Philippe Gain; Damien Rageade; Aurélien Bernard; Sophie Acquart; Michel Peoc’h; Dennis M. Defoe; Gilles Thuret

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexa...

  7. Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chien Shu [Departments of Bioengineering and Medicine and Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California 92093-0427 (United States)

    2011-01-01

    Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

  8. High glucose induced endothelial to mesenchymal transition in human umbilical vein endothelial cell.

    Science.gov (United States)

    Yu, Chun-Hong; Suriguga; Gong, Meng; Liu, Wen-Juan; Cui, Ning-Xuan; Wang, Ying; Du, Xin; Yi, Zong-Chun

    2017-06-01

    Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11mM, 30mM, 60mM and 120mM glucose for 0, 24, 48, 72 and 168h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers α-SMA and collagen I, and transforming growth factor TGF-β1. Immunofluorescence staining was performed to detect the expression of CD31 and α-SMA. The concentration of TGF-β1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. High glucose induced EndMT and increased the TGF-β1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of α-SMA and collagen I, while lost CD31 staining and acquired α-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-β1 expression and attenuated EndMT marker protein synthesis. High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-β1 biosynthesis during high glucose-induced EndMT. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Conditional ablation of LYVE-1+ cells unveils defensive roles of lymphatic vessels in intestine and lymph nodes.

    Science.gov (United States)

    Jang, Jeon Yeob; Koh, Young Jun; Lee, Seung-Hun; Lee, Junyeop; Kim, Kyoo Hyun; Kim, Daesoo; Koh, Gou Young; Yoo, Ook Joon

    2013-09-26

    To unveil the organotypic role and vulnerability of lymphatic vessels, we generated a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-Cre/iDTR double-transgenic mouse and ablated LYVE-1-expressing lymphatic vessels in adult mice in a diphtheria toxin (DT)-inducible manner based on selective expression of LYVE-1 in most lymphatic vessels. Strikingly, lymphatic vessels in the small intestine and lymph nodes were rapidly ablated, but lymphatic vessels in the other organs were relatively intact at 24 hours after DT administration. Unexpectedly, LYVE-1-Cre/iDTR mice died of sepsis without visible edema at 24 and 60 hours after DT administration. The cause of death appeared to be related to acute failure of immune surveillance systems in the small intestine and draining lymph nodes. Of note, acute loss of lymphatic lacteals in intestinal villi appeared to trigger distortion of blood capillaries and the whole architecture of the villi, whereas acute loss of lymphatic vessels in lymph nodes caused dysfunction of lymph drainage and abnormal distribution of dendritic cells and macrophages. Thus, intact lymphatic vessels are required for structural and functional maintenance of surrounding tissues in an organotypic manner, at least in the intestine and lymph nodes.

  10. Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

    Science.gov (United States)

    Stratmann, Bernd; Engelbrecht, Britta; Espelage, Britta C; Klusmeier, Nadine; Tiemann, Janina; Gawlowski, Thomas; Mattern, Yvonne; Eisenacher, Martin; Meyer, Helmut E; Rabbani, Naila; Thornalley, Paul J; Tschoepe, Diethelm; Poschmann, Gereon; Stühler, Kai

    2016-11-29

    Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

  11. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R.; Grosso, Mariela F. del [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Behar, Moni [Instituto de Física, UFRGS, Porto Alegre, RS (Brazil); García Bermúdez, Gerardo, E-mail: ggb@tandar.cnea.gov.ar [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnología, UNSAM (Argentina)

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  12. Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts.

    Science.gov (United States)

    Zakharova, I S; Zhiven', M K; Saaya, Sh B; Shevchenko, A I; Smirnova, A M; Strunov, A; Karpenko, A A; Pokushalov, E A; Ivanova, L N; Makarevich, P I; Parfyonova, Y V; Aboian, E; Zakian, S M

    2017-03-03

    Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function. Endothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate

  13. Serglycin in Quiescent and Proliferating Primary Endothelial Cells

    Science.gov (United States)

    Reine, Trine M.; Vuong, Tram T.; Rutkovskiy, Arkady; Meen, Astri J.; Vaage, Jarle; Jenssen, Trond G.; Kolset, Svein O.

    2015-01-01

    Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development. PMID:26694746

  14. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC.

    Science.gov (United States)

    Girard, J P; Springer, T A

    1995-01-01

    High endothelial venules (HEV) in lymphoid tissues support high levels of lymphocyte extravasion from the blood. We purified high endothelial cells from human tonsils by immunomagnetic selection with MECA-79 MAb to construct an HEV cDNA library. Differential screening of this library using cDNA probes from HEV (plus) or flat-walled vessel (minus) endothelial cells allowed us to characterize a novel human cDNA expressed to high levels in HEV. The cDNA encodes a secreted acidic calcium-binding glycoprotein of 664 aa residues, designated hevin, exhibiting 62% identity with the antiadhesive extracellular matrix protein SPARC, over a region of 232 aa spanning more than four fifths of the SPARC coding sequence. The primary structure and sequence of hevin and similar to SPARC-like proteins from rat and quail, called SC1 or QR1. Hevin could contribute to the induction or maintenance of features of the HEV endothelium that facilitate lymphocyte migration.

  15. H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules

    Science.gov (United States)

    Zhou, Xueping; Yuan, Dong; Wang, Mingxia

    2013-01-01

    Although elevated levels of H2O2 have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H2O2 on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H2O2-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca2+]i was measured on fura-2-loaded vessels. Perfusion of H2O2 (10 μM) caused a delayed and progressively increased endothelial [Ca2+]i and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H2O2 induced two phases of NO production followed by caspase activation, intracellular Ca2+ accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser1177 phosphorylation in the absence of elevated endothelial [Ca2+]i, whereas the second phase of NO depended on increased [Ca2+]i and was associated with Thr495 dephosphorylation without increased Ser1177 phosphorylation. Inhibition of NOS prevented H2O2-induced caspase activation, cell apoptosis, and increases in endothelial [Ca2+]i and Lp. Our results indicate that H2O2 at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca2+ accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases. PMID:23086988

  16. Tumor-endothelium cross talk blocks recruitment of neutrophils to endothelial cells: a novel mechanism of endothelial cell anergy.

    Science.gov (United States)

    Blaheta, Roman A; Powerski, Maciej; Hudak, Lukasz; Juengel, Eva; Jonas, Dietger; von Knethen, Andreas; Doerr, Hans Willhelm; Cinatl, Jindrich

    2009-10-01

    Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule 1 (VCAM-1) adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC) subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCalpha activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.

  17. Tumor-Endothelium Cross Talk Blocks Recruitment of Neutrophils to Endothelial Cells: A Novel Mechanism of Endothelial Cell Anergy

    Directory of Open Access Journals (Sweden)

    Roman A. Blaheta

    2009-10-01

    Full Text Available Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs and/or peripheral blood lymphocytes (PBLs. Human umbilical vein endothelial cells (HUVECs and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1, E-selectin, and vascular cell adhesion molecule 1 (VCAM-1 adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCα activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.

  18. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells.

    Science.gov (United States)

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay; Smith, Joseph D

    2016-07-12

    Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. Cerebral malaria is a severe neurological complication of P. falciparum infection associated with infected erythrocyte (IE) binding in cerebral vessels. Yet little is known about the mechanisms by which parasites adhere in the brain or other microvascular sites. Here, we studied parasite lines

  19. Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

    KAUST Repository

    Malara, N.M.

    2015-03-01

    Aim: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods and results: Isolated primary fragment of different vessel types was expanded in Ham\\'s F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusion: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications. © 2015 The Authors. Published by Elsevier Ireland Ltd.

  20. Monocytes initiate a cycle of leukocyte recruitment when cocultured with endothelial cells.

    Science.gov (United States)

    Tsouknos, Andreas; Nash, Gerard B; Rainger, G Ed

    2003-09-01

    During the development of atherosclerotic plaque, monocytes and T-lymphocytes are recruited to the arterial intima by endothelial cells (EC) lining the vessel. This process is associated with chronic arterial inflammation and requires the activation-dependent expression of adhesion receptors and chemokines on EC. Here we show that monocytes can activate cocultured EC so that they support the adhesion, activation and transmigration of a secondary bolus of flowing peripheral blood monocytes or lymphocytes. The number of adherent leukocytes and their behaviour was comparable to that seen on EC activated with tumour necrosis factor-alpha (TNF-alpha). Depending upon the duration of endothelial cell/monocyte coculture different patterns of adhesion receptors were utilised by leukocytes. After 4 h coculture, antibodies against E-selectin, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) reduced mononuclear leukocyte adhesion. After 24 h coculture, antibodies against E-selectin and VCAM-1 but not P-selectin were effective. Immunofluorescence analysis confirmed that monocyte coculture induced endothelial expression of E-selectin and VCAM-1, while P-selectin was at the limit of detection. We conclude that EC stimulated by monocytes can support the adhesion of flowing mononuclear leukocytes. We hypothesise that this mode of EC activation and leukocyte recruitment could initiate a self-perpetuating cycle of inflammation that could be relevant to atherogenesis and other chronic inflammatory disease states.

  1. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology.

    Science.gov (United States)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje; van Rooijen, Karlijn L; Dijstelbloem, Hilde M; Rasmussen, Jan T; de Vooght, Karen M K; Schiffelers, Raymond M; Gaillard, Carlo A J M; van Solinge, Wouter W

    2012-04-01

    Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and α(v)-integrin. Phagocytosis via the phosphatidylserine-lactadherin-α(v)-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes. Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity. Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape ('rounding up'). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects. Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell

  2. Allogeneic Mesenchymal Stem Cells Restore Endothelial Function in Heart Failure by Stimulating Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Courtney Premer

    2015-05-01

    Interpretation: These findings reveal a novel mechanism whereby allogeneic, but not autologous, MSC administration results in the proliferation of functional EPCs and improvement in vascular reactivity, which in turn restores endothelial function towards normal in patients with HF. These findings have significant clinical and biological implications for the use of MSCs in HF and other disorders associated with endothelial dysfunction.

  3. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  4. HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Pasquinelli Gianandrea

    2011-05-01

    Full Text Available Abstract Background HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs. Results MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. Conclusions The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

  5. Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC)

    Science.gov (United States)

    Stein, Daniel J.; Nelson, Victoria M.; Otterson, Mary F.; Shaker, Reza; Binion, David G.

    2010-01-01

    The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-α but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-α/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-α/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-κB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-α/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-κB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD. PMID:19926820

  6. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    Science.gov (United States)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy.

  7. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Infante, Teresa [SDN-Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, 80143 Naples (Italy); Cesario, Elena [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy); Gallo, Michele; Fazioli, Flavio [Division of Skeletal Muscles Oncology Surgery, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); De Chiara, Annarosaria [Anatomic Pathology Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Tutucci, Cristina; Apice, Gaetano [Medical Oncology of Bone and Soft Sarcoma tissues Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Nigris, Filomena de, E-mail: filomena.denigris@unina2.it [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy)

    2013-04-11

    Cooperation between endothelial cells and bone in bone remodelling is well established. In contrast, bone microvasculature supporting the growth of primary tumors and metastasis is poorly understood. Several antiangiogenic agents have recently been undergoing trials, although an extensive body of clinical data and experimental research have proved that angiogenic pathways differ in each tumor type and stage. Here, for the first time, we characterize at the molecular and functional level tumor endothelial cells from human bone sarcomas at different stages of disease and with different histotypes. We selected a CD31{sup +} subpopulation from biopsies that displayed the capability to grow as adherent cell lines without vascular endothelial growth factor (VEGF). Our findings show the existence in human primary bone sarcomas of highly proliferative endothelial cells expressing CD31, CD44, CD105, CD146 and CD90 markers. These cells are committed to develop capillary-like structures and colony formation units, and to produce nitric oxide. We believe that a better understanding of tumor vasculature could be a valid tool for the design of an efficacious antiangiogenic therapy as adjuvant treatment of sarcomas.

  8. Post-transcriptional control of Amblyomin-X on secretion of vascular endothelial growth factor and expression of adhesion molecules in endothelial cells.

    Science.gov (United States)

    Drewes, C C; Dias, R Y; Branco, V G; Cavalcante, M F; Souza, J G; Abdalla, D S P; Chudzinski-Tavassi, A M; Farsky, S H P

    2015-07-01

    Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 μL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and β3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Influence of Levamisole and Other Angiogenesis Inhibitors on Angiogenesis and Endothelial Cell Morphology in Vitro

    Energy Technology Data Exchange (ETDEWEB)

    Friis, Tina; Engel, Anne-Marie; Bendiksen, Christine D.; Larsen, Line S.; Houen, Gunnar, E-mail: gh@ssi.dk [Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen (Denmark)

    2013-06-24

    Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity

  10. Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

    Science.gov (United States)

    Hellberg, Hege; Bjerkås, Inge; Vågnes, Øyvind B; Noga, Edward J

    2013-12-01

    . Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

  11. Reelin expression in brain endothelial cells: an electron microscopy study.

    Science.gov (United States)

    Perez-Costas, Emma; Fenton, Erin Y; Caruncho, Hector J

    2015-03-24

    Reelin expression and function have been extensively studied in the brain, although its expression has been also reported in other tissues including blood. This raises the possibility that reelin might be able to cross the blood-brain barrier, which could be functionally relevant. Up-to-date no studies have been conducted to assess if reelin is present in the blood-brain barrier, which is mainly constituted by tightly packed endothelial cells. In this report we assessed the expression of reelin in brain capillaries using immunocytochemistry and electron microscopy. At the light microscope, reelin immunolabeling appeared in specific endothelial cells in brain areas that presented abundant diffuse labeling for this protein (e.g., layer I of the cortex, or the stratum lacunosum moleculare of the hippocampus), while it was mostly absent from capillaries in other brain areas (e.g., deeper cortical layers, or the CA1 layer of the hippocampus). As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum. Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope. In these cells, reelin labeling was located primarily in caveolae (i.e., vesicles of transcytosis), and associated with the plasma membrane of the luminal side of endothelial cells. In addition, some scarce labeling was observed in the nuclear membrane. The presence of reelin immunolabeling in brain endothelial cells, and particularly in caveolar vesicles within these cells, suggests that reelin and/or reelin peptides may be able to cross the blood-brain barrier, which could have important physiological, pathological, and therapeutic implications.

  12. Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

    Directory of Open Access Journals (Sweden)

    Martina Schleicher

    2012-01-01

    Full Text Available In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2 and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P<0.05. Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization.

  13. Nature's rheologists: Lymphatic endothelial cells control migration in response to shear stress

    Science.gov (United States)

    Fuller, Gerald; Dunn, Alex; Surya, Vinay

    2015-03-01

    Endothelial cells (ECs) line the inner surface of blood and lymphatic vessels and are sensitive to fluid flow as part of their physiological function. EC organization, migration and vessel development are profoundly influenced by shear stresses, with important implications in cardiovascular disease and tumor metastasis. How ECs sense fluid flow is a central and unanswered question in cardiovascular biology. We developed a high-throughput live-cell flow chamber that models the gradients in wall shear stress experienced by ECs in vivo. Live-cell imaging allows us to probe cellular responses to flow, most notably EC migration, which has a key role in vessel remodeling. We find that most EC subtypes, including ECs from the venous, arterial, and microvascular systems, migrate in the flow direction. In contrast, human lymphatic microvascular ECs (hLMVECs) migrate against flow and up spatial gradients in wall shear stress. Further experiments reveal that hLMVECs are sensitive to the magnitude, direction, and the local spatial gradients in wall shear stress. Lastly, recent efforts have aimed to link this directional migration to spatial gradients in cell-mediated small molecule emission that may be linked to the gradient in wall shear stress.

  14. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells

    NARCIS (Netherlands)

    Gremmels, Hendrik; Bevers, Lonneke M.; Fledderus, Joost O.; Braam, Branko; Jan Van Zonneveld, Anton; Verhaar, Marianne C.; Joles, Jaap A.

    2015-01-01

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric

  15. Effect of propionyl-L-carnitine on human endothelial cells

    NARCIS (Netherlands)

    Hinsbergh, V.W.M. van; Scheffer, M.A.

    1991-01-01

    A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no

  16. Dabigatran abrogates brain endothelial cell permeability in response to thrombin.

    Science.gov (United States)

    Hawkins, Brian Thomas; Gu, Yu-Huan; Izawa, Yoshikane; del Zoppo, Gregory John

    2015-06-01

    Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

  17. Store-operated calcium entry and increased endothelial cell permeability.

    Science.gov (United States)

    Norwood, N; Moore, T M; Dean, D A; Bhattacharjee, R; Li, M; Stevens, T

    2000-11-01

    We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.

  18. Comparison of Endothelial Cell Loss by Specular Microscopy ...

    African Journals Online (AJOL)

    life restored.[1,2] Providing Early Childhood Care and. Education (ECCE) to 95% of those who need it (95% coverage level) would avert more than 3.5 million disability‑adjusted life years per year globally.[2]. Some degree of endothelial cell loss is inevitable after any type of cataract surgery.[3,4]. The number and.

  19. Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells.

    Science.gov (United States)

    Tancharoen, Waleephan; Aungsuchawan, Sirinda; Pothacharoen, Peraphan; Markmee, Runchana; Narakornsak, Suteera; Kieodee, Junjira; Boonma, Nonglak; Tasuya, Witoon

    2017-03-01

    Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering. Copyright © 2016 Elsevier GmbH. All rights reserved.

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  18. File list: Pol.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: His.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: DNS.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.CDV.20.AllAg.Brachiocephalic_endothelial_cells hg19 DNase-seq Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.CDV.20.AllAg.Brachiocephalic_endothelial_cells.bed ...

  2. DMPD: Lipopolysaccharide signaling in endothelial cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16357866 Lipopolysaccharide signaling in endothelial cells. Dauphinee SM, Karsan A.... Lab Invest. 2006 Jan;86(1):9-22. (.png) (.svg) (.html) (.csml) Show Lipopolysaccharide signaling in endothe...lial cells. PubmedID 16357866 Title Lipopolysaccharide signaling in endothelial cells. Authors Dauphinee SM,

  3. File list: DNS.CDV.05.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: DNS.CDV.50.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.CDV.05.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Oth.CDV.50.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  12. File list: Pol.CDV.50.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Oth.CDV.20.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: DNS.CDV.20.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: DNS.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

    Science.gov (United States)

    Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

    2014-01-01

    Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Figulla Hans R

    2004-05-01

    Full Text Available Abstract Background Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. Methods We measured the effects of statins on the intracellular free calcium concentration ([Ca2+]i in human umbilical vein endothelial cells (HUVEC after acute application and 24-h-preincubation of statins. Results Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca2+]i. For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca2+]i in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca2+ release induced by histamine was not affected. Conclusions The increase of resting [Ca2+]i after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo.

  5. The Cell Surface Structure of Tumor Endothelial Marker 8 (TEM8) is Regulated by the Actin Cytoskeleton

    OpenAIRE

    Yang, Mi Young; Chaudhary, Amit; Seaman, Steven; Dunty, Jill; Stevens, Janine; Elzarrad, Mohammed K.; Frankel, Arthur E.; St. Croix, Brad

    2010-01-01

    Tumor Endothelial Marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5’s ability to distinguish different forms of cell-surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding p...

  6. Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5.

    Science.gov (United States)

    Kim, Young; Abplanalp, William A; Jung, Andrew D; Schuster, Rebecca M; Lentsch, Alex B; Gulbins, Erich; Caldwell, Charles C; Pritts, Timothy A

    2018-03-01

    Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 min up to 24 h, suggesting endocytosis of the microparticles by endothelial cells. Colocalization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared with nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response seems to be mediated, in part, by the Rab5 signaling protein.

  7. Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish

    OpenAIRE

    Kanada, Masamitsu; Zhang, Jinyan; Libo YAN; Sakurai, Takashi; Terakawa, Susumu

    2014-01-01

    The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility re...

  8. Changes in Zn homeostasis during long term culture of primary endothelial cells and effects of Zn on endothelial cell senescence.

    Science.gov (United States)

    Malavolta, Marco; Costarelli, Laura; Giacconi, Robertina; Basso, Andrea; Piacenza, Francesco; Pierpaoli, Elisa; Provinciali, Mauro; Ogo, Ogo A; Ford, Dianne

    2017-12-01

    Endothelial cell senescence and Zn nutritional status influence cardiovascular disease. The influence of Zn appears dichotomous, hence it is imperative to understand the relationship with cellular senescence to improve knowledge about the molecular and cellular basis of the disease. Here we aimed to determine: 1) the impact of chronic exposure to a moderately high dose of Zn on senescence of endothelial cells; 2) the changes in Zn homeostasis during the lifespan of primary cultured endothelial cells; and 3) the susceptibility of proliferating and senescent endothelial cells to cell death after short term exposure to increasing doses of Zn and of the Zn chelator TPEN. Chronic exposure to Zn accelerated senescence and untreated cells at later passages, where doubling time had increased, displayed relocation of labile Zn and altered expression of genes involved in the response to Zn toxicity, including SLC30A1, SLC39A6, SLC30A5, SLC30A10 and metallothioneins, indicating that senescent cells have altered zinc homeostasis. Most Zn-dependent genes that were expressed differently between early and late passages were correlated with changes in the expression of anti-apoptotic genes. Short-term treatment with a high dose of Zn leads to cell death, but only in the population of cells at both earlier and later passages that had already entered senescence. In contrast, Zn depletion led to death of cells at earlier but not later passages, which suggests that there are sub-populations of senescent cells that are resistant to Zn depletion. This resistant senescent cell population may accumulate under conditions of Zn deficiency and contribute to vascular pathology. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Regulation of T Helper Cell Responses During Antigen Presentation by Norepinephrine-exposed Endothelial Cells.

    Science.gov (United States)

    Xu, Linghui; Ding, Wanhong; Stohl, Lori L; Zhou, Xi K; Azizi, Shayan; Chuang, Ethan; Lam, Jimmy; Wagner, John A; Granstein, Richard D

    2017-11-21

    Dermal blood vessels and regional lymph nodes are innervated by sympathetic nerves and, under stress, sympathetic nerves release norepinephrine (NE). Exposure of primary murine dermal microvascular endothelial cells (pDMECs) to NE followed by co-culture with Langerhans cells (LCs), responsive CD4+ T cells and antigen resulted in modulation of CD4+ T cell responses. NE-treatment of pDMECs induced increased production of interleukin (IL)-6 and IL-17A while downregulating interferon (IFN)-γ and IL-22 release. This effect did not require contact between pDMECs and LCs or T cells and depended on pDMEC production of IL-6. The presence of NE-treated pDMECs increased the proportion of CD4+ T cells expressing intracellular IL-17A and increased IL-17A mRNA while decreasing the proportion of IFN-γ- or IL-22-expressing CD4+ T cells and mRNA levels for those cytokines. Retinoic acid receptor-related orphan receptor gamma (RORγt) mRNA was significantly increased in CD4+ T cells while T-box transcription factor (T-bet) mRNA was decreased. Intradermal administration of NE prior to hapten immunization at the injection site produced a similar bias in draining lymph node CD4+ T cells toward IL-17A and away from IFN-γ and IL-22 production. Under stress, release of NE may have significant regulatory effects on the outcome of antigen presentation through actions on ECs as well as enhancement of inflammatory skin disorders involving IL-17/Th17 cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

    Science.gov (United States)

    Abrahimi, Parwiz; Chang, William G; Kluger, Martin S; Qyang, Yibing; Tellides, George; Saltzman, W Mark; Pober, Jordan S

    2015-07-03

    The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering. © 2015 American Heart Association, Inc.

  11. Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways.

    Science.gov (United States)

    Harding, Aja; Cortez-Toledo, Elizabeth; Magner, Nataly L; Beegle, Julie R; Coleal-Bergum, Dane P; Hao, Dake; Wang, Aijun; Nolta, Jan A; Zhou, Ping

    2017-04-01

    Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31 + and 78%-83% VE-cadherin + ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31 + and 96.8% VE-cadherin + ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  12. Endothelial progenitor cells from human fetal aorta cure diabetic foot in a rat model.

    Science.gov (United States)

    Zhao, Wan-Ni; Xu, Shi-Qing; Liang, Jian-Feng; Peng, Liang; Liu, Hong-Lin; Wang, Zai; Fang, Qing; Wang, Meng; Yin, Wei-Qin; Zhang, Wen-Jian; Lou, Jin-Ning

    2016-12-01

    preferentially and durably engrafted into the blood vessels. In addition, anti-human CD31+-AMA+-vWF+ microvasculars were detected after transplantation of EPCs. Early fetal aorta-derived EPCs possess strong self-renewal ability and can differentiate into endothelial cells. We demonstrated for the first time that transplanting HFA-derived EPCs could ameliorate DF prognosis in a rat model. These findings suggest that the transplantation of HFA-derived EPCs could serve as an innovative therapeutic strategy for managing DF. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Corneal endothelial cell density and morphology in normal Iranian eyes

    Directory of Open Access Journals (Sweden)

    Fallah Mohammad

    2006-03-01

    Full Text Available Abstract Background We describe corneal endothelial cell density and morphology in normal Iranian eyes and compare endothelial cell characteristics in the Iranian population with data available in the literature for American and Indian populations. Methods Specular microscopy was performed in 525 eyes of normal Iranian people aged 20 to 85 years old. The studied parameters including mean endothelial cell density (MCD, mean cell area (MCA and coefficient of variation (CV in cell area were analyzed in all of the 525 eyes. Results MCD was 1961 ± 457 cell/mm2 and MCA was 537.0 ± 137.4 μm2. There was no statistically significant difference in MCD, MCA and CV between genders (Student t-test, P = 0.85, P = 0.97 and P = 0.15 respectively. There was a statistically significant decrease in MCD with age (P r = -0.64. The rate of cell loss was 0.6% per year. There was also a statistically significant increase in MCA (P r = 0.56 and CV (P r = 0.30 from 20 to 85 years of age. Conclusion The first normative data for the endothelium of Iranian eyes seems to confirm that there are no differences in MCD, MCA and CV between genders. Nevertheless, the values obtained in Iranian eyes seem to be different to those reported by the literature in Indian and American populations.

  14. Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

    DEFF Research Database (Denmark)

    Wang, X; Häring, M-F; Rathjen, Thomas

    2017-01-01

    did not change intestinal tumour number or size distribution on either a low or high-fat diet. We therefore asked whether cells in the tumour stroma might explain the association between tumour formation and insulin resistance. To this end, we generated Apc(Min/+) mice with loss of insulin receptors......The risk of several cancers, including colorectal cancer, is increased in patients with obesity and type 2 diabetes, conditions characterised by hyperinsulinaemia and insulin resistance. Because hyperinsulinaemia itself is an independent risk factor for cancer development, we examined tissue...... in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...

  15. Effect of cytokines on tumour cell-endothelial interactions.

    Science.gov (United States)

    Cohen, M C; Bereta, M; Bereta, J

    1997-01-01

    The adherence of tumour cells to microvascular endothelium is believed to be a necessary step in their migration to sites of metastasis. It has been proposed that this process occurs when cell surface molecules on tumour cells bind to complementary sites on endothelial cells. The expression of these endothelial-derived cell adhesion molecules appears to be modulated by cytokines, a broad class of protein mediators which play important roles in immune and inflammatory reactions. It has been found by ourselves and others that exposure of endothelium to some cytokines augments the adhesion of inflammatory cells as well as tumour cells in in vitro assays. We used a murine model consisting of P815 mastocytoma cells and microvascular endothelium and found that pretreatment of endothelial monolayers with TNF-alpha, IL-1, LPS or PMA augmented the number of tumour cells that attach in a dose-dependent fashion. FACS analysis showed that the change in binding was due to an increase in the expression of VCAM-1 on the surface of the endothelial cell. Methylxanthines (caffeine and theophylline) as well as "classical" calcium-mobilizing agents (ionomycin and thapsigargin) inhibited the expression of VCAM-1 in MME. We also studied the possible mechanisms of TNF-alpha signal transduction in endothelial cells. We examined the involvement of protein kinases in the TNF-alpha effect. Although we found that inhibitors of PKC could inhibit the TNF-alpha effect, our studies suggest that the "classical" PKC pathway is not completely responsible for signaling since TNF-alpha did not cause translocation of PKC to the cell membrane and its effect could not be completely mimicked by PMA. We also studied the effect of TGF-beta on the binding of tumour cells to endothelium. Exposure of endothelium to TGF-beta led to the inhibition of both basal and TNF-alpha enhanced binding of P815 cells. Inhibitors of G-proteins do not abolish TGF-beta action, and PKC and PKA activators elicit an opposite

  16. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2,

  17. Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells

    Science.gov (United States)

    Solomon, Ioana; O’Reilly, Megan; Ionescu, Lavinia; Alphonse, Rajesh S.; Rajabali, Saima; Zhong, Shumei; Vadivel, Arul; Shelley, W. Chris; Yoder, Mervin C.

    2016-01-01

    Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31+, CD105+, CD144+, CD146+, CD14−, and CD45−, took up 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications. Significance This research confirms that resident endothelial colony

  18. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  19. 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

  20. Methylene blue modulates adhesion molecule expression on microvascular endothelial cells.

    Science.gov (United States)

    Werner, Isabella; Guo, Fengwei; Stock, Ulrich A; Lupinski, Michèle; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

    2014-08-01

    As methylene blue (MB) has been recently proposed to preserve blood pressure in case of vasoplegic syndrome and shock, an entity directly related to systemic inflammation, we aimed to elucidate the effect of MB on the expression of adhesion-molecules in endothelial-cells. Human microvascular endothelial-cells (HuMEC-1) were treated with 10, 30 or 60 µM MB for 30 min and 2 h each. Additionally, the treated HuMEC-1 were co-cultured with either human peripheral blood mononuclear cells (PBMCs) or Jurkat cells (human T-lymphocytes) for 2 h. HuMEC-1 were analyzed after MB treatment and after co-culture experiments for expression of different adhesion-molecules (ICAM-1, VCAM-1, L-selectin, E-selectin) via FACS measurement and western blot analysis. The supernatants of the experiments were analyzed with regard to the soluble forms of the adhesion molecules. We found that MB is able to modulate the expression of adhesion-molecules on EC. Administration of MB increases the expression of E-selectin and VCAM-1 depending on the dosage and time of exposure. ICAM-1 measurements provide evidence that different circulating blood cells can differently alter the adhesion-molecule expression on EC after MB exposure. Our results provide evidence regarding the immunomodulatory effect of MB upon endothelial-cells after inflammation.

  1. Endothelial progenitor cells in sudden sensorineural hearing loss.

    Science.gov (United States)

    Quaranta, Nicola; Ramunni, Alfonso; De Luca, Concetta; Brescia, Paola; Dambra, Porzia; De Tullio, Giacomina; Vacca, Angelo; Quaranta, Antonio

    2011-04-01

    Endothelial progenitor cells (EPCs) are a unique subtype of circulating cells with properties similar to those of embryonal angioblasts. They have the potential to proliferate and to differentiate into mature endothelial cells. EPCs are reduced in patients with vascular risk factors due to a decreased mobilization, an increased consumption at the site of damage or a reduced half-life. The results of this study confirm the existence of an endothelial dysfunction in patients with sudden sensorineural hearing loss (SSHL) and support the vascular involvement in the pathogenesis of the disease. The aim of this study was to evaluate the concentration of EPCs in patients affected by SSHL. Twenty-one patients affected by SSHL were evaluated. The number of EPCs was analyzed by flow cytometry analysis of peripheral blood CD34+KDR+CD133+ cells. Circulating levels of EPCs were significantly lower in SSHL patients compared with controls. In particular, CD34+KDR+ cells and CD34+CD133+KDR+ cells were significantly reduced (p < 0.05).

  2. Microvasculature on a chip: study of the Endothelial Surface Layer and the flow structure of Red Blood Cells

    Science.gov (United States)

    Tsvirkun, Daria; Grichine, Alexei; Duperray, Alain; Misbah, Chaouqi; Bureau, Lionel

    2017-03-01

    Microvasculatures-on-a-chip, i.e. in vitro models that mimic important features of microvessel networks, have gained increasing interest in recent years. Such devices have allowed investigating pathophysiological situations involving abnormal biophysical interactions between blood cells and vessel walls. Still, a central question remains regarding the presence, in such biomimetic systems, of the endothelial glycocalyx. The latter is a glycosaminoglycans-rich surface layer exposed to blood flow, which plays a crucial role in regulating the interactions between circulating cells and the endothelium. Here, we use confocal microscopy to characterize the layer expressed by endothelial cells cultured in microfluidic channels. We show that, under our culture conditions, endothelial cells form a confluent layer on all the walls of the circuit and display a glycocalyx that fully lines the lumen of the microchannels. Moreover, the thickness of this surface layer is found to be on the order of 600 nm, which compares well with measurements performed ex or in vivo on microcapillaries. Furthermore, we investigate how the presence of endothelial cells in the microchannels affects their hydrodynamic resistance and the near-wall motion of red blood cells. Our study thus provides an important insight into the physiological relevance of in vitro microvasculatures.

  3. Erectile dysfunction precedes other systemic vascular diseases due to incompetent cavernous endothelial cell-cell junctions.

    Science.gov (United States)

    Ryu, Ji-Kan; Jin, Hai-Rong; Yin, Guo Nan; Kwon, Mi-Hye; Song, Kang-Moon; Choi, Min Ji; Park, Jin-Mi; Das, Nando Dulal; Kwon, Ki-Dong; Batbold, Dulguun; Lee, Tack; Gao, Zhen Li; Kim, Kyu-Won; Kim, Woo Jean; Suh, Jun-Kyu

    2013-08-01

    Erectile dysfunction is often a harbinger of cardiovascular disease. We sought to gain mechanistic insight at the cellular and molecular levels into why erectile dysfunction precedes the clinical consequences of cardiovascular disease. Diabetes was induced by intraperitoneal streptozotocin injection in 8-week-old C57BL/6J mice. At 8 weeks after diabetes induction, we determined the expression of endothelial cell-cell junction proteins and vascular endothelial permeability in the penis, heart and hind limb by systemic injection of various vascular space markers (350 Da to 2,000 kDa) or by immunohistochemical staining with antibody to oxidized low density lipoprotein. We also investigated the effect of recombinant Ang1 protein on cavernous endothelial permeability. Alterations in the integrity of the endothelial cell-cell junction, including a decrease in endothelial cell-cell junction proteins and an increase in vascular permeability to fluorescent tracers or oxidized low density lipoprotein, were prominent in the cavernous tissue of diabetic mice. In contrast, no significant changes in endothelial cell-cell junction proteins or vascular permeability were noted in heart or hind limb tissue according to the diabetic condition. Intracavernous injection of Ang1 protein, an anti-permeability factor, significantly decreased cavernous endothelial permeability to oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins in diabetic mice. The incompetent cavernous endothelial cell-cell junction in the diabetic condition provides an important clue to why erectile dysfunction is highly prevalent and often precedes other systemic vascular diseases. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  4. Young's modulus of elasticity of Schlemm's canal endothelial cells.

    Science.gov (United States)

    Zeng, Dehong; Juzkiw, Taras; Read, A Thomas; Chan, Darren W-H; Glucksberg, Matthew R; Ethier, C Ross; Johnson, Mark

    2010-02-01

    Schlemm's canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young's modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young's modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8-15 mm Hg). However, increasing intraocular pressure (22-30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.

  5. Electrical Impedance Study of Cultured Endothelial Cells Under Fluid Shear Stress

    Science.gov (United States)

    Dong, Chunzhi; Depaola, Natacha; Keese, Charles R.; Giaever, Ivar

    2004-03-01

    The lumen of blood vessels is lined with a monolayer of endothelial cells (EC). In this work, electric cell-substrate impedance sensing (ECIS) was used to monitor the effect of fluid shear stress (FSS) on the morphology and function of cultured EC layers. Confluent layers of bovine aortic endothelial cells (BAEC) were grown on small gold electrodes and exposed to different flow conditions, while the impedance of the system was monitored. When the cells are subjected to FSS, the impedance rapidly increases 5-10%, and if the FSS is removed after a few minutes duration, the impedance returns back to the initial level in about 10 minutes. If the FSS remains for a long duration, the impedance will decrease 20-30% over a 10-15 hour period but ultimately returns to the original value, as the cells apparently accommodate to the FSS condition. These results suggest that ECIS may provide a sensitive means to study the response of EC to shear stress in vitro.

  6. Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

    Science.gov (United States)

    Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

    2013-01-01

    A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Hold Me, but Not Too Tight-Endothelial Cell-Cell Junctions in Angiogenesis.

    Science.gov (United States)

    Szymborska, Anna; Gerhardt, Holger

    2017-09-08

    Endothelial cell-cell junctions must perform seemingly incompatible tasks during vascular development-providing stable connections that prevent leakage, while allowing dynamic cellular rearrangements during sprouting, anastomosis, lumen formation, and functional remodeling of the vascular network. This review aims to highlight recent insights into the molecular mechanisms governing endothelial cell-cell adhesion in the context of vascular development. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. Grateloupia longifolia polysaccharide inhibits angiogenesis by downregulating tissue factor expression in HMEC-1 endothelial cells.

    Science.gov (United States)

    Zhang, Chao; Yang, Fan; Zhang, Xiong-Wen; Wang, Shun-Chun; Li, Mei-Hong; Lin, Li-Ping; Ding, Jian

    2006-07-01

    1. The antiangiogenic and antitumor properties of Grateloupia longifolia polysaccharide (GLP), a new type of polysaccharide isolated from the marine alga, were investigated with several in vitro and in vivo models. Possible mechanisms underlying its antiangiogenic activity were also assessed. 2. GLP dose-dependently inhibited proliferation of human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC), with IC50 values of 0.86 and 0.64 mg ml(-1), respectively. In tube formation and cell migration assays using HMEC-1 cells, noncytotoxic doses of GLP significantly inhibited formation of intact tube networks and reduced the number of migratory cells. Inhibition by GLP was VEGF-independent. 3. In the chick chorioallantoic membrane (CAM) assay, GLP (2.5 microg egg(-1)) reduced new vessel formation compared with the vehicle control. GLP (0.1 mg plug(-1)) also reduced the vessel density in Matrigel plugs implanted in mice. 4. The levels of pan and phosphorylated receptors for VEGF, VEGFR-1 (flt-1) and VEGFR-2 (KDR) were not significantly altered by 5 mg ml(-1) GLP treatment of HMEC-1, although tissue factor (TF) showed significant decreases at both mRNA and protein levels following GLP treatment. 5. In mice bearing sarcoma-180 cells, intravenous administration of GLP (200 mg kg(-1)) decreased tumor weight by 52% without obvious toxicity. Vascular density in sections of the tumor was reduced by 64% after GLP treatment. 6. Collectively, these results indicate that GLP has antitumor properties, associated at least, in part, with the antiangiogenesis induced by downregulation of TF.

  10. Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hilde Kvestad

    2014-01-01

    Full Text Available The combined use of the histone deacetylase inhibitor valproic acid (VPA, the retinoic acid receptor-α agonist all-trans retinoic acid (ATRA, and the deoxyribonucleic acid polymerase-α inhibitor cytarabine (Ara-C is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML. Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

  11. F3-targeted cisplatin-hydrogel nanoparticles as an effective therapeutic that targets both murine and human ovarian tumor endothelial cells in vivo.

    Science.gov (United States)

    Winer, Ira; Wang, Shouyan; Lee, Yong-Eun Koo; Lee, Youg-Eun Koo; Fan, Wenzhe; Gong, Yusong; Burgos-Ojeda, Daniela; Spahlinger, Greg; Kopelman, R; Buckanovich, Ronald J

    2010-11-01

    Recent studies indicate that ovarian cancer may be highly responsive to antivascular therapeutics. We have developed an antivascular tumor therapeutic using the F3 peptide to target cisplatin-loaded nanoparticles (F3-Cis-Np) to tumor vessels. We show that although F3-Cis-Np bind with high specificity to both human ovarian tumor cells and tumor endothelial cells in vitro, they only show cytotoxic activity against the tumor endothelial cells. In vivo these nanoparticles bind primarily to tumor endothelial cells. Therapeutic studies in both flank and orthotopic i.p. murine ovarian tumor models, as well as human tumor xenograft models, show rapid tumor regression with treatment. Treatment was associated with significant vascular necrosis consistent with an antivascular effect. Furthermore, treatment was active in both platinum-sensitive and platinum-resistant cell lines. Importantly, we show that F3-Cis-Np bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo. Therapy targeting human vasculature in vivo with F3-Cis-Np led to near complete loss of all human tumor vessels in a murine model of human tumor vasculature. Our studies indicate that F3-targeted vascular therapeutics may be an effective treatment modality in human ovarian cancer. ©2010 AACR.

  12. Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

    Science.gov (United States)

    Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J

    2017-11-01

    Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r  = -0.49, P = 0.003), p21 ( r  = -0.38, P = 0.03), and p16 ( r  = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function. Copyright © 2017 the American Physiological Society.

  13. Coniferyl Aldehyde Ameliorates Radiation Intestine Injury via Endothelial Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Ye Ji; Jung, Myung Gu; Lee, Yoonjin; Lee, Haejune [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yunsil [Ewha Woman' s Univ., Seoul (Korea, Republic of); Ko, Younggyu [Korea Univ., Seoul (Korea, Republic of)

    2014-05-15

    Cancer treatments related gastrointestinal toxicity has also been recognized as a significant economic burden. Especially, extensive apoptosis of microvascular endothelial cell of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Coniferyl aldehyde (CA) is phenolic compounds isolated from cork stoppers, and one of the major pyrolysis products of lignin. Shi H. was support for the empirical use of CA as a medicinal food for cardiovascular diseases. CA has positive effect in broad way but there is no consequence in radiation induced intestine damage. Here, we investigate effect of CA on small intestine after abdominal IR to mice in this study. In this study, CA increased the survival rate in C3H mice against 13.5 Gy abdominal IR. We found CA protects small intestine via preventing endothelial cell apoptosis and enhancing their angiogenic activity. CA also showed protective effect on crypt cell survival. Endothelial cell survival may affect crypt cell protection against IR. From this data, we concluded that CA is effective for protection against abdominal radiation injury. CA could ameliorate side-effect of radiation therapy.

  14. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

    Science.gov (United States)

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  15. Pharmacologically active microcarriers for endothelial progenitor cell support and survival.

    Science.gov (United States)

    Musilli, Claudia; Karam, Jean-Pierre; Paccosi, Sara; Muscari, Claudio; Mugelli, Alessandro; Montero-Menei, Claudia N; Parenti, Astrid

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase.

    Science.gov (United States)

    Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

    2014-01-01

    Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

  17. Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation.

    Science.gov (United States)

    Lanier, Viola; Gillespie, Corey; Leffers, Merle; Daley-Brown, Danielle; Milner, Joy; Lipsey, Crystal; Webb, Nia; Anderson, Leonard M; Newman, Gale; Waltenberger, Johannes; Gonzalez-Perez, Ruben Rene

    2016-10-01

    Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Effect of Mitomycin-C augmented trabeculectomy on corneal endothelial cells

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    Reza Zarei

    2015-01-01

    Conclusion: MMC application in trabeculectomy seems to cause a small but significant corneal endothelial loss. Most of the damage occurs intraoperatively, or in the early postoperative period, however progressive endothelial cell loss is not a major concern.

  19. Stability of vision, topography, and endothelial cell density from 1 year to 2 years after deep lamellar endothelial keratoplasty surgery.

    Science.gov (United States)

    Ousley, Paula J; Terry, Mark A

    2005-01-01

    To evaluate whether the visual, topographic, and endothelial cell count results observed 1 year after deep lamellar endothelial keratoplasty (DLEK) surgery remain stable up to 2 years after surgery. Prospective, noncomparative, interventional case series. Twenty eyes of 20 patients with corneal edema from Fuchs' endothelial dystrophy. Deep lamellar endothelial keratoplasty endothelial replacement surgery, with a 9.0-mm or 9.5-mm scleral access incision and a specialized intrastromal trephine, was performed. Snellen visual acuities, corneal topography, and endothelial cell counts were prospectively measured preoperatively and 1 year and 2 years after DLEK. Uncorrected and best spectacle-corrected visual acuity (BSCVA), refractive and topographic astigmatism, mean corneal curvature, topographic regularity and symmetry, and endothelial cell density. At 1 year postoperatively, BSCVA averaged 20/50 (range, 20/25-20/200), spherical equivalents (SE) averaged -0.194+/-1.521 diopters (D), manifest refraction (MR) astigmatism averaged 2.04+/-1.05 D (range, 0.0-4.0 D), topographic astigmatism averaged 2.3+/-1.1 D, mean corneal curvature was 43.2+/-1.8 D, the surface regularity index (SRI) averaged 1.16+/-0.41, and the surface asymmetry index (SAI) averaged 1.05+/-1.09. At 2 years postoperatively, BSCVA averaged 20/48 (range, 20/25-20/200), SE averaged -0.369+/-1.267 D, MR astigmatism averaged 1.76+/-0.66 D (range, 0.75-3.0 D), topographic astigmatism averaged 2.4+/-1.1 D, mean corneal curvature was 43.6+/-1.8 D, the SRI averaged 1.13+/-0.44, and the SAI averaged 0.76+/-0.59. There was no significant change in visual or topographic parameters between 1 year and 2 years postoperatively (P>0.05). Endothelial cell counts averaged 2335+/-468 cells/mm(2) at 1 year and 2151+/-457 cells/mm(2) at 2 years postoperatively (P = 0.041). Deep lamellar endothelial keratoplasty provides stable refractions, corneal topography, and endothelial cell densities as long as 2 years after surgery

  20. miR-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting VEGF Signaling

    Science.gov (United States)

    Hassel, David; Cheng, Paul; White, Mark P.; Ivey, Kathryn N.; Kroll, Jens; Augustin, Hellmut G.; Katus, Hugo A.; Stainier, Didier Y.R.; Srivastava, Deepak

    2012-01-01

    Rationale Formation and remodeling of the vasculature during development and disease involves a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their post-transcriptional dose-titration by miRNAs is poorly understood. Objective To identify miRNAs that regulate angiogenesis. Methods and Results We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating pro-angiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth (ISV) in the trunk of zebrafish larvae, while overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells (HUVECs). We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters VEGF, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in HUVECs inhibited the angiogenic behavior of endothelial cells largely by antagonizing VEGF receptor-2 signaling. Conclusion Our study provides insights into how FLT1 and VEGF receptor-2 signaling is titrated in a miRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis. PMID:22955733

  1. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P muscle cells....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P cells in culture alone did not cause extracellular accumulation of adenosine. 4. Skeletal muscle cells were...

  2. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    DEFF Research Database (Denmark)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

    2012-01-01

    Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells gener...... cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.......Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells...... generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine...

  3. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jingshan Zhao

    Full Text Available The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL, a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF signaling and angiogenesis in endothelial cells (ECs. We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.

  4. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

    Science.gov (United States)

    Zhao, Jingshan; Niu, Honglin; Li, Aiying; Nie, Lei

    2016-01-01

    The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.

  5. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    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...

  6. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

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    A Barkhordari

    2014-09-01

    Full Text Available [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists. Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP compared to those found in normal tissue. Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system. Results: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (β1,4GlcNAc and Wisteria floribunda (GalNAc as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue. Conclusion: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

  7. Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass*

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Chen, Zean; El-Gohary, Yousef; Wiersch, John; Gaffar, Iljana; Prasadan, Krishna; Shiota, Chiyo; Gittes, George K.

    2013-01-01

    VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need. PMID:23378532

  8. Hypoglycemia reduces vascular endothelial growth factor A production by pancreatic beta cells as a regulator of beta cell mass.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Chen, Zean; El-Gohary, Yousef; Wiersch, John; Gaffar, Iljana; Prasadan, Krishna; Shiota, Chiyo; Gittes, George K

    2013-03-22

    VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

  9. Cellular Cytotoxicity of Antiglaucoma Drugs in Cultured Corneal Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Kwou-Yeung Wu

    2007-03-01

    Full Text Available In this study, the various antiglaucoma drugs including betaxolol, timolol, levobunolol, carteolol, brimonidine, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine were used to investigate the effects of cellular cytotoxicity in cultured bovine corneal endothelial cells. After exposure to the drugs in three dilutions, 1/100, 1/1,000, and 1/10,000, for 100 minutes, cells were estimated based on the release assay of lactate dehydrogenase (LDH enzyme. It was found that cellular LDH was significantly released in the medium only at 1/100th dilution of betaxolol, brimonidine, dorzolamide, dipivefrin, latanoprost and unoprostone to 130%, 123%, 145%, 157%, 128% and 237%, respectively, compared with controls upon exposure to drugs for 100 minutes. Moreover, benzalkonium chloride preservative at the concentrations ranging from 0.001 to 0.00001mg/mL did not affect cellular LDH release in bovine corneal endothelial cells. These results indicate that high concentrations of antiglaucoma drugs may induce cytotoxicity in corneal endothelial cells.

  10. Extended incubation times improve corneal endothelial cell transplantation success.

    Science.gov (United States)

    Insler, M S; Lopez, J G

    1991-05-01

    To investigate the ability of extended incubation times to improve the success of endothelial cell transplantation, eight human donor corneas were denuded of their native endothelium, seeded twice during a 1-hr interval with a suspension of cultured infant human corneal endothelial cells, and then incubated for 144 hr under standard conditions. Subsequently the corneas were transplanted into African green monkeys using routine penetrating keratoplasty techniques. Rotational autografts and corneas devoid of endothelial cells served as controls. The seeded corneas appeared hazy at the time of surgery (mean pachymetry 48 hr postoperatively, 0.794 mm). Six corneas (75%) subsequently cleared, yielding a mean corneal thickness of 0.541 +/- 0.040 and 0.554 +/- 0.040 at 6 and 12 postoperative months, respectively. All control eyes showed advanced edema (thickness, greater than 1.0 mm) and developed extensive neovascularization. Clinically, the extended postseeding incubation corneas were observed to clear more rapidly and stabilize their thickness earlier than corneas incubated for only 24-48 hr. Scanning electron microscopy of extended postseeding incubation corneas revealed an intact monolayer of contact-inhibited cells with the hexagonal mosaic typical of corneal endothelium in vivo and improved intercellular contact compared with corneas incubated for only 24-48 hr.

  11. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    Science.gov (United States)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P pathophysiology associated with diabetic microangiopathy.

  12. 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.

  13. Novel role of immature myeloid cells in formation of new lymphatic vessels associated with inflammation and tumors.

    Science.gov (United States)

    Ran, Sophia; Wilber, Andrew

    2017-08-01

    Inflammation triggers an immune cell-driven program committed to restoring homeostasis to injured tissue. Central to this process is vasculature restoration, which includes both blood and lymphatic networks. Generation of new vessels or remodeling of existing vessels are also important steps in metastasis-the major cause of death for cancer patients. Although roles of the lymphatic system in regulation of inflammation and cancer metastasis are firmly established, the mechanisms underlying the formation of new lymphatic vessels remain a subject of debate. Until recently, generation of new lymphatics in adults was thought to occur exclusively through sprouting of existing vessels without help from recruited progenitors. However, emerging findings from clinical and experimental studies show that lymphoendothelial progenitors, particularly those derived from immature myeloid cells, play an important role in this process. This review summarizes current evidence for the existence and significant roles of myeloid-derived lymphatic endothelial cell progenitors (M-LECPs) in generation of new lymphatics. We describe specific markers of M-LECPs and discuss their biologic behavior in culture and in vivo, as well as currently known molecular mechanisms of myeloid-lymphatic transition (MLT). We also discuss the implications of M-LECPs for promoting adaptive immunity, as well as cancer metastasis. We conclude that improved mechanistic understanding of M-LECP differentiation and its role in adult lymphangiogenesis may lead to new therapeutic approaches for correcting lymphatic insufficiency or excessive formation of lymphatic vessels in human disorders. © Society for Leukocyte Biology.

  14. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  15. Conversion of vascular endothelial cells into multipotent stem-like cells

    Science.gov (United States)

    Medici, Damian; Shore, Eileen M.; Lounev, Vitali Y.; Kaplan, Frederick S.; Kalluri, Raghu; Olsen, Bjorn R.

    2011-01-01

    Mesenchymal stem cells can give rise to several cell types, but variations depending on isolation method and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an ALK2 receptor-dependent mechanism. In lesions from patients with Fibrodysplasia Ossificans Progressiva, a disease where heterotopic ossification occurs as a result of activating ALK2 mutations, or from a mutant ALK2 transgenic mouse model, chondrocytes and osteoblasts express endothelial markers. Tie2-Cre lineage tracing also suggests an endothelial origin of these cells. Expressing mutant ALK2 in endothelial cells, or treatment with the ALK2 ligands TGF-β2 or BMP4, causes endothelial-mesenchymal transition and acquisition of a stem cell-like phenotype. In selective media, these cells differentiate into osteoblasts, chondrocytes, or adipocytes. The process is inhibited by ALK2-specific siRNA. Conversion of endothelial cells to stem-like cells may provide a novel approach to tissue engineering. PMID:21102460

  16. Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Three-Dimensional Microphysiological Systems.

    Science.gov (United States)

    Kurokawa, Yosuke K; Yin, Rose T; Shang, Michael R; Shirure, Venktesh S; Moya, Monica L; George, Steven C

    2017-08-01

    Microphysiological systems (MPS), or "organ-on-a-chip" platforms, aim to recapitulate in vivo physiology using small-scale in vitro tissue models of human physiology. While significant efforts have been made to create vascularized tissues, most reports utilize primary endothelial cells that hinder reproducibility. In this study, we report the use of human induced pluripotent stem cell-derived endothelial cells (iPS-ECs) in developing three-dimensional (3D) microvascular networks. We established a CDH5-mCherry reporter iPS cell line, which expresses the vascular endothelial (VE)-cadherin fused to mCherry. The iPS-ECs demonstrate physiological functions characteristic of primary endothelial cells in a series of in vitro assays, including permeability, response to shear stress, and the expression of endothelial markers (CD31, von Willibrand factor, and endothelial nitric oxide synthase). The iPS-ECs form stable, perfusable microvessels over the course of 14 days when cultured within 3D microfluidic devices. We also demonstrate that inhibition of TGF-β signaling improves vascular network formation by the iPS-ECs. We conclude that iPS-ECs can be a source of endothelial cells in MPS providing opportunities for human disease modeling and improving the reproducibility of 3D vascular networks.

  17. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy......, nano-scale 3D-imaging using focused ion beam/scanning electron microscopy (FIB/SEM), and single particle inductively coupled plasma-mass spectrometry (spICP-MS). HUVECs were cultured for 3 h or 24 h in medium with AuNPs in a concentration range of 1.25–10 μg ml−1. There was a concentration...

  18. Influence of Endothelial Cells on Vascular Smooth Muscle Cells Phenotype after Irradiation

    Science.gov (United States)

    Milliat, Fabien; François, Agnès; Isoir, Muriel; Deutsch, Eric; Tamarat, Radia; Tarlet, Georges; Atfi, Azeddine; Validire, Pierre; Bourhis, Jean; Sabourin, Jean-Christophe; Benderitter, Marc

    2006-01-01

    Damage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. We undertook a study in patients treated with preoperative radiotherapy and demonstrated in vivo the importance of proliferation, migration, and fibrogenic phenotype of vascular smooth muscle cells (VSMCs) in radiation-induced vascular damage. These lesions may result from imbalance in the cross talk between endothelial cells (ECs) and VSMCs. Using co-culture models, we examined whether ECs influence proliferation, migration, and fibrogenic phenotype of VSMCs. In the presence of irradiated ECs, proliferation and migration of VSMCs were increased. Moreover, expressions of α-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor type 1, heat shock protein 27, and collagen type III, alpha 1 were up-regulated in VSMCs exposed to irradiated ECs. Secretion of transforming growth factor (TGF)-β1 was increased after irradiation of ECs, and irradiated ECs activated the Smad pathway in VSMCs by inducing Smad3/4 nuclear translocation and Smad-dependent promoter activation. Using small interferring RNA targeting Smad3 and a TGFβ-RII neutralizing antibody, we demonstrate that a TGF-β1/TGF-β-RII/Smad3 pathway is involved in the fibrogenic phenotype of VSMCs induced by irradiated ECs. In conclusion, we show the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients. Moreover, we demonstrate in vitro that ECs influence these fundamental mechanisms involved in radiation-induced vascular damages. PMID:17003501

  19. Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity.

    Directory of Open Access Journals (Sweden)

    Justin Wai-Chung Leung

    Full Text Available Endothelin-1 (ET-1 is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1 with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A receptor (BQ123 but not by ET(B receptor (BQ788 antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.

  20. Histone Deacetylase (HDAC Inhibitors Down-Regulate Endothelial Lineage Commitment of Umbilical Cord Blood Derived Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Horia Maniu

    2012-11-01

    Full Text Available To test the involvement of histone deacetylases (HDACs activity in endothelial lineage progression, we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs derived from umbilical cord blood (UCB. Adherent EPCs, that expressed the endothelial marker proteins (PCAM-1, CD105, CD133, and VEGFR2 revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA, Trichostatin A (TSA, and Valproic acid (VPA. RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore, flow cytometry analysis illustrated that HDAC inhibitors selectively reduce the expression of VEGFR2, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD34 and CD45 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR demonstrated that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. The balance of acethylation/deacethylation kept in control by the activity of HAT (histone acetyltransferases/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.

  1. PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7

    Science.gov (United States)

    Martin, Maud; Geudens, Ilse; Bruyr, Jonathan; Potente, Michael; Bleuart, Anouk; Lebrun, Marielle; Simonis, Nicolas; Deroanne, Christophe; Twizere, Jean-Claude; Soubeyran, Philippe; Peixoto, Paul; Mottet, Denis; Janssens, Veerle; Hofmann, Wolf-Karsten; Claes, Filip; Carmeliet, Peter; Kettmann, Richard; Gerhardt, Holger; Dequiedt, Franck

    2013-01-01

    To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell–extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell–matrix adhesion. PMID:23955003

  2. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

    Directory of Open Access Journals (Sweden)

    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  3. Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

    Science.gov (United States)

    Wagner, Andreas H.; Schwarz, Manuel; König, Gerd; Hecker, Markus

    2014-11-01

    CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

  4. A Case of Abnormal Lymphatic-Like Differentiation and Endothelial Progenitor Cell Activation in Neovascularization Associated with Hemi-Retinal Vein Occlusion

    Directory of Open Access Journals (Sweden)

    Sirpa Loukovaara

    2015-07-01

    Full Text Available Purpose: Pathological vascular differentiation in retinal vein occlusion (RVO-related neovessel formation remains poorly characterized. The role of intraocular lymphatic-like differentiation or endothelial progenitor cell activity has not been studied in this disease. Methods: Vitrectomy was performed in an eye with hemi-RVO; the neovessel membrane located at the optic nerve head was removed and subjected to immunohistochemistry. Characterization of the neovascular tissue was performed using hematoxylin and eosin, α-smooth muscle actin, and the pan-endothelial cell (EC adhesion molecule CD31. The expression of lymphatic EC markers was studied by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1, podoplanin (PDPN, and prospero-related homeobox protein 1 (Prox-1. Potential vascular stem/progenitor cells were identified by active cellular proliferation (Ki67 and expression of the stem cell marker CD117. Results: The specimen contained blood vessels lined by ECs and surrounded by pericytes. Immunoreactivity for LYVE-1 and Prox-1 was detected, with Prox-1 being more widely expressed in the active Ki67-positive lumen-lining cells. PDPN expression was instead found in the cells residing in the extravascular tissue. Expression of the stem cell markers CD117 and Ki67 suggested vascular endothelial progenitor cell activity. Conclusions: Intraocular lymphatic-like differentiation coupled with progenitor cell activation may be involved in the pathology of neovessel formation in ischemia-induced human hemi-RVO.

  5. Aberrant mural cell recruitment to lymphatic vessels and impaired lymphatic drainage in a murine model of pulmonary fibrosis.

    Science.gov (United States)

    Meinecke, Anna-Katharina; Nagy, Nadine; Lago, Gabriela D'Amico; Kirmse, Santina; Klose, Ralph; Schrödter, Katrin; Zimmermann, Annika; Helfrich, Iris; Rundqvist, Helene; Theegarten, Dirk; Anhenn, Olaf; Orian-Rousseau, Véronique; Johnson, Randall S; Alitalo, Kari; Fischer, Jens W; Fandrey, Joachim; Stockmann, Christian

    2012-06-14

    Pulmonary fibrosis is a progressive disease with unknown etiology that is characterized by extensive remodeling of the lung parenchyma, ultimately resulting in respiratory failure. Lymphatic vessels have been implicated with the development of pulmonary fibrosis, but the role of the lymphatic vasculature in the pathogenesis of pulmonary fibrosis remains enigmatic. Here we show in a murine model of pulmonary fibrosis that lymphatic vessels exhibit ectopic mural coverage and that this occurs early during the disease. The abnormal lymphatic vascular patterning in fibrotic lungs was driven by expression of platelet-derived growth factor B (PDGF-B) in lymphatic endothelial cells and signaling through platelet-derived growth factor receptor (PDGFR)-β in associated mural cells. Because of impaired lymphatic drainage, aberrant mural cell coverage fostered the accumulation of fibrogenic molecules and the attraction of fibroblasts to the perilymphatic space. Pharmacologic inhibition of the PDGF-B/PDGFR-β signaling axis disrupted the association of mural cells and lymphatic vessels, improved lymphatic drainage of the lung, and prevented the attraction of fibroblasts to the perilymphatic space. Our results implicate aberrant mural cell recruitment to lymphatic vessels in the pathogenesis of pulmonary fibrosis and that the drainage capacity of pulmonary lymphatics is a critical mediator of fibroproliferative changes.

  6. Toxicity of fatty acids on ECV-304 endothelial cells.

    Science.gov (United States)

    Masi, Laureane Nunes; Portioli-Sanches, Erica Paula; Lima-Salgado, Thaís Martins; Curi, Rui

    2011-12-01

    The effects of stearic (saturated) or oleic (monounsaturated) acids and their combination with ω-3 and ω-6 polyunsaturated fatty acids (PUFA) on death of endothelial cells (ECV-304 cell line) were investigated. We examined: loss of plasma membrane integrity, DNA fragmentation, accumulation of neutral lipids (NL) and release of reactive oxygen species (ROS). The fatty acids studied were: stearic (SA), oleic (OA), docosahexaenoic (DHA), eicosapentaenoic (EPA), linoleic (LA) and gamma-linolenic (γA) acids. SA at 150 μM induced cell death, did not lead to accumulation of NL and raised the release of ROS. ω-3 PUFA decreased ROS production, increased NL content but did not protect against ECV-304 cell death induced by SA. ω-6 PUFA inhibited SA-induced cell death, increased NL content and decreased ROS production. OA caused cell death but did not increase NL content and ROS production even at 300 μM. ω-3 and ω-6 FA associated with OA further increased cell death with no change in ROS production and NL content. Concluding, ω-6 PUFA had a greater protective effect than ω-3 PUFA on the deleterious effects caused by SA whereas OA had low cytotoxicity but, when associated with PUFA, presented marked toxic effects on ECV-304 endothelial cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. In vitro analysis of human periodontal microvascular endothelial cells.

    Science.gov (United States)

    Tsubokawa, Mizuki; Sato, Soh

    2014-08-01

    Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites

  8. Quantitative study of the microvasculature and its endothelial cells in the porcine iris.

    Science.gov (United States)

    Yang, Hongfang; Yu, Paula K; Cringle, Stephen J; Sun, Xinghuai; Yu, Dao-Yi

    2015-03-01

    The roles of the iris microvasculature have been increasingly recognised in the pathogenesis of glaucoma and cataract; however limited information exists regarding the iris microvasculature and its endothelium. This study quantitatively assessed the iris microvascular network and its endothelium using intra-luminal micro-perfusion, fixation, and staining of the porcine iris. The temporal long posterior ciliary artery of 11 isolated porcine eyes was cannulated, perfusion-fixed and labelled using silver nitrate. The iris microvasculature was studied for its distribution, orders and endothelial morphometrics. The density of three layers of microvasculature was measured. Endothelial cell length and width were measured for each vessel order. The iris has an unusual vascular distribution which consisted of abundant large vessels in the middle of the iris stroma, branching over a relatively short distance to the microvasculature located in the superficial and deep stroma as well as the pupil edge. The average vascular density of the middle, superficial, and deep layers were 38.9 ± 1.93%, 10.9 ± 1.61% and 8.0 ± 0.79% respectively. Multiple orders of iris vessels (capillary, 6 orders of arteries, and 4 orders of veins) with relatively large capillary and input arteries (319.5 ± 25.6 μm) were found. Significant heterogeneity of vascular diameter and shape of the endothelia was revealed in different orders of the iris vasculature. Detailed information of topography and endothelium of the iris microvasculature combined with unique structural features of the iris may help us to further understand the physiological and pathogenic roles of the iris in relevant ocular diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  10. Slits and Roundabouts in cancer, tumour angiogenesis and endothelial cell migration.

    Science.gov (United States)

    Legg, John A; Herbert, John M J; Clissold, Patricia; Bicknell, Roy

    2008-01-01

    Angiogenesis describes the development of new blood vessels from pre-existing vessels. The hijacking of this physiological process by tumours allows them to develop their own supplies of nutrients and oxygen, enabling their growth and metastasis. A large body of literature has accumulated over the last 20 years relating to angiogenesis, including signalling pathways involved in this process. One such pathway uses Slit-Roundabout proteins that are implicated in the development of cancers and tumour angiogenesis. The Roundabout family of receptors are large, single-pass transmembrane cell surface receptors involved in directing cell migration in response to their cognate Slit ligands. Although best known for their role in neuronal development, Slits and Roundabouts have now been implicated in myogenesis, leukocyte chemotaxis and tumour angiogenesis, confirming that the Robo signalling pathway functions across multiple cell types. We review here the evidence for a role for Slits and Roundabouts in cancer. In particular, we focus on the role of Robo1 and Robo4 in tumour angiogenesis and discuss the signalling pathways downstream of these proteins mediating endothelial cell migration.

  11. Endothelial Side Population Cells Contribute to Tumor Angiogenesis and Antiangiogenic Drug Resistance.

    Science.gov (United States)

    Naito, Hisamichi; Wakabayashi, Taku; Kidoya, Hiroyasu; Muramatsu, Fumitaka; Takara, Kazuhiro; Eino, Daisuke; Yamane, Keitaro; Iba, Tomohiro; Takakura, Nobuyuki

    2016-06-01

    Angiogenesis plays a crucial role in tumor growth, with an undisputed contribution of resident endothelial cells (EC) to new blood vessels in the tumor. Here, we report the definition of a small population of vascular-resident stem/progenitor-like EC that contributes predominantly to new blood vessel formation in the tumor. Although the surface markers of this population are similar to other ECs, those from the lung vasculature possess colony-forming ability in vitro and contribute to angiogenesis in vivo These specific ECs actively proliferate in lung tumors, and the percentage of this population significantly increases in the tumor vasculature relative to normal lung tissue. Using genetic recombination and bone marrow transplant models, we show that these cells are phenotypically true ECs and do not originate from hematopoietic cells. After treatment of tumors with antiangiogenic drugs, these specific ECs selectively survived and remained in the tumor. Together, our results established that ECs in the peripheral vasculature are heterogeneous and that stem/progenitor-like ECs play an indispensable role in tumor angiogenesis as EC-supplying cells. The lack of susceptibility of these ECs to antiangiogenic drugs may account for resistance of the tumor to this drug type. Thus, inhibiting these ECs might provide a promising strategy to overcome antiangiogenic drug resistance. Cancer Res; 76(11); 3200-10. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty versus penetrating keratoplasty in Asian eyes

    Science.gov (United States)

    Ang, Marcus; Mehta, Jodhbir S; Anshu, Arundhati; Wong, Hon Kiat; Htoon, Hla M; Tan, Donald

    2012-01-01

    Background The purpose of this study was to compare endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty in Asian eyes. Methods This was a retrospective study of patients from our prospective Singapore Corneal Transplant Study cohort who received corneal transplantation in 2006–2008. We compared eyes that underwent DSAEK or penetrating keratoplasty for Fuchs’ endothelial dystrophy or pseudophakic and aphakic bullous keratopathy. Clinical data, and donor and recipient characteristics were recorded. Of 241 patients who met our inclusion criteria, 68 underwent DSAEK and 173 underwent penetrating keratoplasty. The main outcome measure was endothelial cell loss at 1 year. Secondary outcome measures were graft survival and visual outcomes at 1-year follow-up. Results There were no significant differences in baseline characteristics of patients between the treatment groups. Percent endothelial cell loss at 1-year follow-up was greater in penetrating keratoplasty eyes (40.9% ± 2.9%) compared with DSAEK eyes (22.4% ± 2.3%; P keratoplasty-treated eyes. Penetrating keratoplasty-treated eyes had worse astigmatism as compared with DSAEK-treated eyes (−3.0 ± 2.1 versus −1.7 ± 0.8; P keratoplasty-treated eyes had clear grafts (P = 0.479). Conclusion We report lower percent endothelial cell loss comparing DSAEK and penetrating keratoplasty at 1-year follow-up in Asian eyes, with comparable graft survival rates in both groups. PMID:22536049

  13. On the Normal Force Mechanotransduction of Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Vahabikashi, Amir; Wang, Qiuyun; Wilson, James; Wu, Qianhong; Vucbmss Team

    2016-11-01

    In this paper, we report a cellular biomechanics study to examine the normal force mechanotransduction of Human Umbilical Vein Endothelial Cells (HUVECs) with their implications on hypertension. Endothelial cells sense mechanical forces and adjust their structure and function accordingly. The mechanotransduction of normal forces plays a vital role in hypertension due to the higher pressure buildup inside blood vessels. Herein, HUVECs were cultured to full confluency and then exposed to different mechanical loadings using a novel microfluidic flow chamber. One various pressure levels while keeps the shear stress constant inside the flow chamber. Three groups of cells were examined, the control group (neither shear nor normal stresses), the normal pressure group (10 dyne/cm2 of shear stress and 95 mmHg of pressure), and the hypertensive group (10 dyne/cm2 of shear stress and 142 mmHg of pressure). Cellular response characterized by RT-PCR method indicates that, COX-2 expressed under normal pressure but not high pressure; Mn-SOD expressed under both normal and high pressure while this response was stronger for normal pressure; FOS and e-NOS did not respond under any condition. The differential behavior of COX-2 and Mn-SOD in response to changes in pressure, is instrumental for better understanding the pathogenesis of hypertensive cardiovascular diseases. This research was supported by the National Science Foundation under Award #1511096.

  14. Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

    2014-12-15

    The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

  15. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Science.gov (United States)

    Strobel, Claudia; Oehring, Hartmut; Herrmann, Rudolf; Förster, Martin; Reller, Armin; Hilger, Ingrid

    2015-05-01

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO2) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO2 nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO2 nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  16. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  17. Combined transplantation of mesenchymal stem cells and endothelial progenitor cells for tissue engineering: a systematic review and meta-analysis.

    Science.gov (United States)

    Sun, Kunming; Zhou, Zheng; Ju, Xinxin; Zhou, Yang; Lan, Jiaojiao; Chen, Dongdong; Chen, Hongzhi; Liu, Manli; Pang, Lijuan

    2016-10-10

    Combined cell implantation has been widely applied in tissue engineering in recent years. In this meta-analysis, we aimed to establish whether the combined transplantation of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) promotes angiogenesis and tissue repair, compared with transplantation of a single cell type, following tissue injury or during tissue regeneration. The electronic databases PubMed, EMBASE, MEDLINE, Chinese Biomedical Literature, and China National Knowledge Infrastructure were searched in this systematic review and meta-analysis. Eighteen controlled preclinical studies involving MSC and EPC transplantation in animal models of disease, or in coculture in vitro, were included in this review. The vessel density and other functional indexes, which were classified according to the organ source, were used to evaluate the efficiency of cotransplantation. Publication bias was assessed. There was no obvious difference in angiogenesis following combined cell transplantation (EPCs and MSCs) and transplantation of EPCs alone; however, an improvement in the function of damaged organs was observed following cotransplantation. In addition, combined cell transplantation significantly promoted tissue recovery in cardiovascular disease, cerebrovascular disease, and during bone regeneration. Compared with combined transplantation (EPCs and MSCs) and transplantation of MSCs alone, cotransplantation significantly promoted angiogenesis and bone regeneration, as well as vessel revascularization and tissue repair in cerebrovascular disease; however, no obvious effects on cardiovascular disease were observed. As an exploratory field in the discipline of tissue engineering, MSC and EPC cotransplantation offers advantages, although it is essential to assess the feasibility of this approach before clinical trials can be performed.

  18. Outcome of rotational keratoplasty: comparison of endothelial cell loss in autografts vs allografts.

    Science.gov (United States)

    Bertelmann, Eckart; Hartmann, Christian; Scherer, Matthias; Rieck, Peter

    2004-10-01

    The nature of chronic endothelial cell loss in homologous corneal grafts is still unclear. Possible causes are cell migration to the recipient bed and chronic subclinical immune reaction. To compare endothelial cell loss after autologous rotational keratoplasty and homologous keratoplasty and present the clinical outcome of patients after rotational keratoplasty. In this open prospective study, we included 7 consecutive patients who underwent rotational keratoplasty between 1998 and 2000 in our hospital. Patients were examined clinically every 3 months, and visual acuity, astigmatism, and endothelial cell density were evaluated. Endothelial cell densities were compared with endothelial cell counts of 293 homologous keratoplasties. Mean follow-up for autologous grafts was 39 months. Mean increase in visual acuity was 3.5 lines. Mean astigmatism was 4.75 diopters in the autologous graft group. Mean preoperative endothelial cell density was 2058 (637 cells/mm(2)). Mean endothelial cell density after 1 year was 1865 (639 cells/mm(2)), which represents a mean +/- SD cell loss of 15% +/- 7.19%. At the end of follow-up, endothelial cell number after autologous grafting was 1630 +/- 622 cells/mm(2). Endothelial cell loss after 1 year in homologous grafts was 40% +/- 21.34%. There was 1 decompensation of autologous graft in the follow-up period. Main Outcome Measure Comparison of endothelial cell count at different postoperative time points using nonpaired t test. Endothelial cell loss in autologous grafts is significantly lower than in homologous grafts, which supports the hypothesis that chronic endothelial cell loss is due to chronic subclinical immune reactions in homologous grafts. Autologous keratoplasties can lead to good functional results and can be superior to homologous corneal grafting in suitable situations.

  19. ADAM17 Promotes Motility, Invasion, and Sprouting of Lymphatic Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Renata Mężyk-Kopeć

    Full Text Available Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC. When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i single-cell motility, (ii cell migration through a 3D Matrigel/collagen type I matrix, and (iii their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.

  20. ADAM17 Promotes Motility, Invasion, and Sprouting of Lymphatic Endothelial Cells.

    Science.gov (United States)

    Mężyk-Kopeć, Renata; Wyroba, Barbara; Stalińska, Krystyna; Próchnicki, Tomasz; Wiatrowska, Karolina; Kilarski, Witold W; Swartz, Melody A; Bereta, Joanna

    2015-01-01

    Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.

  1. Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

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    Valgimigli Simond

    2010-01-01

    Full Text Available Abstract The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs and bone marrow (BMMCs. Mesenchymal stem cells (MSCs were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.

  2. Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

    Directory of Open Access Journals (Sweden)

    Muscari Claudio

    2010-02-01

    Full Text Available Abstract The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs and bone marrow (BMMCs. Mesenchymal stem cells (MSCs were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.

  3. Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

    Science.gov (United States)

    Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

    2017-11-01

    Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

  4. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

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    Efthalia Kerasioti

    2016-01-01

    Full Text Available Excessive production of reactive oxygen species (ROS may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP from tert-butyl hydroperoxide- (tBHP- induced oxidative stress in endothelial cells (EA.hy926 were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS, protein carbonyls (CARB, and oxidized glutathione (GSSG were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

  5. Pulmonary endothelial cell DNA methylation signature in pulmonary arterial hypertension.

    Science.gov (United States)

    Hautefort, Aurélie; Chesné, Julie; Preussner, Jens; Pullamsetti, Soni S; Tost, Jorg; Looso, Mario; Antigny, Fabrice; Girerd, Barbara; Riou, Marianne; Eddahibi, Saadia; Deleuze, Jean-François; Seeger, Werner; Fadel, Elie; Simonneau, Gerald; Montani, David; Humbert, Marc; Perros, Frédéric

    2017-08-08

    Pulmonary arterial hypertension (PAH) is a severe and incurable pulmonary vascular disease. One of the primary origins of PAH is pulmonary endothelial dysfunction leading to vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Our objective was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. DNA was extracted from cultured PEC from idiopathic PAH ( n = 11), heritable PAH ( n = 10) and controls ( n = 18). DNA methylation was assessed using the Illumina HumanMethylation450 Assay. After normalization, samples and probes were clustered according to their methylation profile. Differential clusters were functionally analyzed using bioinformatics tools. Unsupervised hierarchical clustering allowed the identification of two clusters of probes that discriminates controls and PAH patients. Among 147 differential methylated promoters, 46 promoters coding for proteins or miRNAs were related to lipid metabolism. Top 10 up and down-regulated genes were involved in lipid transport including ABCA1, ABCB4, ADIPOQ, miR-26A, BCL2L11. NextBio meta-analysis suggested a contribution of ABCA1 in PAH. We confirmed ABCA1 mRNA and protein downregulation specifically in PAH PEC by qPCR and immunohistochemistry and made the proof-of-concept in an experimental model of the disease that its targeting may offer novel therapeutic options. In conclusion, DNA methylation analysis identifies a set of genes mainly involved in lipid transport pathway which could be relevant to PAH pathophysiology.

  6. Endothelial progenitor cells as a therapeutic option in intracerebral hemorrhage

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    Juan Pías-Peleteiro

    2017-01-01

    Full Text Available Intracerebral hemorrhage (ICH is the most severe cerebrovascular disease, which represents a leading cause of death and disability in developed countries. However, therapeutic options are limited, so is mandatory to investigate repairing processes after stroke in order to develop new therapeutic strategies able to promote brain repair processes. Therapeutic angiogenesis and vasculogenesis hold promise to improve outcome of ICH patients. In this regard, circulating endothelial progenitor cells (EPCs have recently been suggested to be a marker of vascular risk and endothelial function. Moreover, EPC levels have been associated with good neurological and functional outcome as well as reduced residual hematoma volume in ICH patients. Finally, experimental and clinical studies indicate that EPC might mediate endothelial cell regeneration and neovascularization. Therefore, EPC-based therapy could be an excellent therapeutic option in ICH. In this mini-review, we discuss the present status of knowledge about the possible therapeutic role of EPCs in ICH, molecular mechanisms, and the future perspectives and strategies for their use in clinical practice.

  7. Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny

    NARCIS (Netherlands)

    Moonen, Jan-Renier A. J.; Krenning, Guido; Brinker, Marja G. L.; Koerts, Jasper A.; van Luyn, Marja J. A.; Harmsen, Martin C.

    2010-01-01

    Reciprocal plasticity exists between endothelial and mesenchymal lineages. For instance, mature endothelial cells adopt a smooth muscle-like phenotype through transforming growth factor beta-1 (TGF beta 1)-driven endothelial-to-mesenchymal transdifferentiation (EndMT). Peripheral blood contains

  8. Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions

    Science.gov (United States)

    Qiu, Yongzhi; Tong, Sheng; Zhang, Linlin; Sakurai, Yumiko; Myers, David R.; Hong, Lin; Lam, Wilbur A.; Bao, Gang

    2017-06-01

    The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.

  9. Changes of junctions of endothelial cells in coronary sclerosis: A review

    Directory of Open Access Journals (Sweden)

    Li-Zi Zhang

    2016-03-01

    Full Text Available Atherosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States and it has been on the rise globally. Endothelial cell–cell junctions are critical for vascular integrity and maintenance of vascular function. Endothelial cell junctions dysfunction is the onset step of future coronary events and coronary artery disease. Keywords: Coronary atherosclerosis, Junctions, Endothelial cells

  10. Effects of pulsatile flow on cultured vascular endothelial cell morphology.

    Science.gov (United States)

    Helmlinger, G; Geiger, R V; Schreck, S; Nerem, R M

    1991-05-01

    Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Effects of Surface Viscoelasticity on Cellular Responses of Endothelial Cells

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    Motahare-Sadat Hosseini

    2014-10-01

    Full Text Available Background: One area of nanoscience deals with nanoscopic interactions between nanostructured materials and biological systems. To elucidate the effects of the substrate surface morphology and viscoelasticity on cell proliferation, fractal analysis was performed on endothelial cells cultured on nanocomposite samples based on silicone rubber (SR and various concentrations of organomodified nanoclay (OC. Methods: The nanoclay/SR ratio was tailored to enhance cell behavior via changes in sample substrate surface roughness and viscoelasticity. Results: Surface roughness of the cured SR filled with negatively-charged nanosilicate layers had a greater effect than elasticity on cell growth. The surface roughness of SR nanocomposite samples increased with increasing the OC content, leading to enhanced cell growth and extracellular matrix (ECM remodeling. This was consistent with the decrease in SR segmental motions and damping factor as the primary viscoelastic parameters by the nanosilicate layers with increasing clay concentrations. Conclusions: The inclusion of clay nanolayers affected the growth and behavior of endothelial cells on microtextured SR.

  12. Mesenchymal Stromal Cells as Active Regulators of Lymphocyte Recruitment to Blood Vascular Endothelial Cells.

    Science.gov (United States)

    Mcgettrick, Helen M; Ward, Lewis S C; Rainger, George Edward; Nash, Gerard B

    2017-01-01

    Methods are described for analyzing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been cocultured with different mesenchymal stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0 or 0.4 μm pore filters, depending on whether migration through the whole construct is to be analyzed, or adhesion to the endothelial cells alone. Migration away from the sub-endothelial space and through the stromal layer can also be assessed by culturing mesenchymal stromal cells within a 3-D collagen gel overlaid with endothelial cells. Assays may be "static" or the filter-based constructs can be incorporated into flow chambers so that cell behavior can be directly observed under conditions simulating those in vivo. In general, by choice of method, one can evaluate efficiency of attachment, and ability of cells to migrate across the endothelial monolayer, through the filter and through the stromal cell layer in 2-D or 3-D. Fluorescence microscopic examination of fixed filters can be used, e.g., to ascertain whether lymphocytes are retained by stromal cells. In general, static assays have the higher throughput and greatest ease of use, while the flow-based assays are more physiologically relevant and allow detailed recording of cell behavior in real time.

  13. Endothelial cell contraction increases Candida adherence to exposed extracellular matrix.

    Science.gov (United States)

    Klotz, S A; Maca, R D

    1988-01-01

    Bovine vascular endothelial cells treated with EDTA, urea, or thrombin underwent a marked, reversible contraction resulting in exposure of the subendothelial extracellular matrix (ECM). Candida yeasts adhered more to contracted monolayers than to confluent monolayers (P less than 0.01) by preferentially adhering to the ECM. Two strains of Candida albicans and one strain of Candida tropicalis bound avidly to exposed ECM, but Pseudomonas aeruginosa did not. However, treatment of endothelium with forskolin, which induces cell shape changes without exposure of the ECM, did not cause an increase in adherence. Images PMID:3137171

  14. From glioblastoma to endothelial cells through extracellular vesicles: messages for angiogenesis.

    Science.gov (United States)

    Giusti, Ilaria; Delle Monache, Simona; Di Francesco, Marianna; Sanità, Patrizia; D'Ascenzo, Sandra; Gravina, Giovanni Luca; Festuccia, Claudio; Dolo, Vincenza

    2016-09-01

    Glioblastoma has one of the highest mortality rates among cancers, and it is the most common and malignant form of brain cancer. Among the typical features of glioblastoma tumors, there is an aberrant vascularization: all gliomas are among the most vascularized/angiogenic tumors. In recent years, it has become clear that glioblastoma cells can secrete extracellular vesicles which are spherical and membrane-enclosed particles released, in vitro or in vivo, by both normal and tumor cells; they are involved in the regulation of both physiological and pathological processes; among the latter, cancer is the most widely studied. Extracellular vesicles from tumor cells convey messages to other tumor cells, but also to normal stromal cells in order to create a microenvironment that supports cancer growth and progression and are implicated in drug resistance, escape from immunosurveillance and from apoptosis, as well as in metastasis formation; they are also involved in angiogenesis stimulation, inducing endothelial cells proliferation, and other pro-angiogenic activities. To this aim, the present paper assesses in detail the extracellular vesicles phenomenon in the human glioblastoma cell line U251 and evaluates extracellular vesicles ability to promote the processes required to achieve the formation of new blood vessels in human brain microvascular endothelial cells, highlighting that they stimulate proliferation, motility, and tube formation in a dose-response manner. Moreover, a molecular characterization shows that extracellular vesicles are fully equipped for angiogenesis stimulation in terms of proteolytic enzymes (gelatinases and plasminogen activators), pro-angiogenic growth factors (VEGF and TGFβ), and the promoting-angiogenic CXCR4 chemokine receptor.

  15. Liver sinusoidal endothelial cells induce immunosuppressive IL-10-producing Th1 cells via the Notch pathway

    NARCIS (Netherlands)

    Neumann, Katrin; Rudolph, Christine; Neumann, Christian; Janke, Marko; Amsen, Derk; Scheffold, Alexander

    2015-01-01

    Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T-cell responses towards tolerance. However, the role of LSECs in the regulation of T-cell-induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro-inflammatory Th1-cell

  16. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

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    Chia-Wei Huang

    2015-01-01

    Full Text Available Neonatal hypoxic-ischemic (HI brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs from adipose-derived stem cells (ASCs and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2 was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

  17. Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

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    Parth Thakor

    2017-03-01

    Full Text Available Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC remains a treatment-re-sistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1, endothelial cells (human umbilical vein endothelial cell line [HUVEC], and primary cultures of kidney proximal tubular epithelial cells (PTEC were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  18. Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro

    NARCIS (Netherlands)

    Schaefer, Antje; van Duijn, Trynette J.; Majolee, Jisca; Burridge, Keith; Hordijk, Peter L.

    2017-01-01

    Inflammation is driven by excessive transmigration (diapedesis) of leukocytes from the blood to the tissue across the endothelial cell monolayer that lines blood vessels. Leukocyte adhesion, crawling, and transmigration are regulated by clustering of the endothelial mechanosensitive receptor

  19. Evidence that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits angiogenesis by inducing vascular endothelial cell apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Pei-Lin, E-mail: pchen@dal.ca [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Easton, Alexander S., E-mail: alexander.easton@dal.ca [Department of Pathology, Dalhousie University, Halifax, Nova Scotia (Canada); Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia (Canada); Division of Neurosurgery, Department of Surgery, Dalhousie University, Halifax, Nova Scotia (Canada)

    2010-01-01

    Tumor necrosis factor (TNF) and its related ligands TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) play roles in the regulation of vascular responses, but their effect on the formation of new blood vessels (angiogenesis) is unclear. Therefore, we have examined the effects of these ligands on angiogenesis modeled with primary cultures of human umbilical vein endothelial cells (HUVEC). To examine angiogenesis in the context of the central nervous system, we have also modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3. Parameters studied were bromodeoxyuridine (BrdU) incorporation and cell number (MTT) assay (to assess endothelial proliferation), scratch assay (migration) and networks on Matrigel (tube formation). In our hands, neither TRAIL nor FasL (1, 10, and 100 ng/ml) had an effect on parameters of angiogenesis in the HUVEC model. In hCMEC/D3 cells by contrast, TRAIL inhibited all parameters (10-100 ng/ml, 24 h). This was due to apoptosis, since its action was blocked by the pan-caspase inhibitor zVADfmk (5 x 10{sup -5} mol/l) and TRAIL increased caspase-3 activity 1 h after application. However FasL (100 ng/ml) increased BrdU uptake without other effects. We conclude that TRAIL has different effects on in vitro angiogenesis depending on which model is used, but that FasL is generally ineffective when applied in vitro. The data suggest that TRAIL primarily influences angiogenesis by the induction of vascular endothelial apoptosis, leading to vessel regression.

  20. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

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    Rui-Li Zhang

    Full Text Available The recombinant Treponema pallidum protein Tp0965 (rTp0965, one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

  1. Knock-down of CD44 regulates endothelial cell differentiation via NFκB-mediated chemokine production.

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    Berit Olofsson

    Full Text Available A striking feature of microvascular endothelial cells is their capacity to fuse and differentiate into tubular structures when grown in three-dimensional (3D extracellular matrices, in collagen or Matrigel, mimicking the in vivo blood vessel formation. In this study we demonstrate that human telomerase-immortalised foreskin microvascular endothelial (TIME cells express high levels of the hyaluronan receptor CD44 and the hyaluronidase HYAL2. Knock-down of CD44 or HYAL2 resulted in an inability of TIME cells to form a tubular network, suggesting a key regulatory role of hyaluronan in controlling TIME cell tubulogenesis in 3D matrices. Knock-down of CD44 resulted in an upregulation of mRNA expression of the chemokines CXCL9 and CXCL12, as well as their receptors CXCR3 and CXCR4. This was accompanied by a defect maturation of the tubular structure network and increased phosphorylation of the inhibitor of NFκB kinase (IKK complex and thus translocation of NFκB into the nucleus and activation of chemokine targed genes. Furthermore, the interaction between CD44 and hyaluronan determines the adhesion of breast cancer cells. In summary, our observations support the notion that the interaction between CD44 and hyaluronan regulates microvascular endothelial cell tubulogenesis by affecting the expression of cytokines and their receptors, as well as breast cancer dissemination.

  2. Levamisole induced apoptosis in cultured vascular endothelial cells

    Science.gov (United States)

    Artwohl, Michaela; Hölzenbein, Thomas; Wagner, Ludwig; Freudenthaler, Angelika; Waldhäusl, Werner; Baumgartner-Parzer, Sabina M

    2000-01-01

    To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5–2 mmol l−1) alone or in combination with antioxidants (10 mmol l−1 glutathione or 5 mmol l−1 N-Acetylcysteine or 0.1 mmol l−1 Tocopherol) were evaluated for apoptosis (3H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation (3H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (−70%), reduced expression of survival factors such as clusterin (−30%), endothelin-1 (−43%), bcl-2 (−34%), endothelial NO-synthase (−32%) and pRb (Retinoblastoma protein: −89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l−1)-induced apoptosis was inhibited by glutathione (−50%) and N-Acetylcysteine (−36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity. PMID:11139434

  3. 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

  4. Functional and gene expression analysis of hTERT overexpressed endothelial cells

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    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  5. A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

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    Stephen W Waldo

    Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

  6. Effects of space mission factors on the morphology and function of endothelial cells.

    Science.gov (United States)

    Kapitonova, M Yu; Kuznetsov, S L; Froemming, G R A; Muid, S; Nor-Ashikin, M N K; Otman, S; Shahir, A R M; Nawawi, H

    2013-04-01

    The structure and functions of endothelial cells after space mission were studied by electron and laser confocal microscopy, image analysis, and MTT test. The endothelial cells changed significantly (proliferative activity, size, contours, shape, distribution of mitochondria and microtubules) in comparison with controls on the Earth. These changes indicated injuries in the cytoskeleton and impairment of the barrier function of the cells, which presumably contributed to the development of endothelial dysfunction.

  7. Interplay between Inflammatory Responses and Lymphatic Vessels

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    Shin, Kihyuk; Lee, Seung-Hyo

    2014-01-01

    Lymphatic vessels are routes for leukocyte migration and fluid drainage. In addition to their passive roles in migration of leukocytes, increasing evidence indicates their active roles in immune regulation. Tissue inflammation rapidly induces lymphatic endothelial cell proliferation and chemokine production, thereby resulting in lymphangiogenesis. Furthermore, lymphatic endothelial cells induce T cell tolerance through various mechanisms. In this review, we focus on the current knowledge on h...

  8. The influence of C-ions and X-rays on human umbilical vein endothelial cells

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    Alexander eHelm

    2016-01-01

    Full Text Available Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells (HUVEC as a model system to examine the effect of low and high linear energy transfer (LET radiation. Cells were exposed to 250 kV X-rays or C-ions with the energies of either 9.8 MeV/u (LET=170 keV/µm or 91 MeV/u (LET=28 keV/µm. Subculture of cells was performed regularly up to 49 days (~22 population doublings post-irradiation. Immediately after exposure cells were seeded for the colony forming assay. Additionally, at regular intervals mitochondrial membrane potential (JC-1 staining and cellular senescence (senescence associated β-galactosidase staining were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution mFISH technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay as observed in other cell systems. For 9.8 and 91 MeV/u C-ions relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time-points the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the mitochondrial membrane potential was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time-point investigated. Altogether our data indicate that C-ions were LET-dependently more effective in damaging endothelial cells shortly after exposure. Late damage to endothelial cells, however, was not found for the applied conditions and endpoints.

  9. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

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    Ailar Nakhlband

    2011-09-01

    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  10. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development.

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    Chen, Jianan; Luo, Yongting; Hui, Hui; Cai, Tanxi; Huang, Hongxin; Yang, Fuquan; Feng, Jing; Zhang, Jingjing; Yan, Xiyun

    2017-09-05

    The blood-brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte-EC interaction during BBB development, the molecular mechanisms coordinating the pericyte-EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB.

  11. "Deep-media culture condition" promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro.

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    Sekiya, Sachiko; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2011-03-01

    In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a "deep-media culture conditions." Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.

  12. Exogenous endothelial cells as accelerators of hematopoietic reconstitution

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    Mizer J

    2012-11-01

    Full Text Available Abstract Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a ability to regulate secretion of cytokines based on biological need; b long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

  13. Effects of Parietaria judaica pollen extract on human microvascular endothelial cells.

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    Taverna, Simona; Flugy, Anna; Colomba, Paolo; Barranca, Marilisa; De Leo, Giacomo; Alessandro, Riccardo

    2008-08-08

    Pollinosis from Parietaria judaica is one of the main causes of allergy in the Mediterranean area. The present study is designed to assess if P. judaica pollens contain bioactive compounds able to elicit a functional response in endothelial cells. We have demonstrated that addition of pollen extract to human lung microvascular endothelial cells (HMVEC-L) induces a modification of cell morphology, actin cytoskeletal rearrangements and an increase in endothelial cell permeability. We further showed that the treatment of endothelial cells with pollen extract causes an increase of E-selectin and VCAM-1 protein levels as well as an increase of IL-8 production. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of polymorphonuclear cells (PMNs) to HMVEC-L monolayer. Our results suggest for the first time that pollen affect directly endothelial cells (EC) modulating critical functions related to the inflammatory response.

  14. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

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    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  15. Soluble THSD7A Is an N-Glycoprotein That Promotes Endothelial Cell Migration and Tube Formation in Angiogenesis

    Science.gov (United States)

    Kuo, Meng-Wei; Wang, Chian-Huei; Wu, Hsiao-Chun; Chang, Shing-Jyh; Chuang, Yung-Jen

    2011-01-01

    Background Thrombospondin type I domain containing 7A (THSD7A) is a novel neural protein that is known to affect endothelial migration and vascular patterning during development. To further understand the role of THSD7A in angiogenesis, we investigated the post-translational modification scheme of THS7DA and to reveal the underlying mechanisms by which this protein regulates blood vessel growth. Methodology/Principal Findings Full-length THSD7A was overexpressed in human embryonic kidney 293T (HEK293T) cells and was found to be membrane associated and N-glycosylated. The soluble form of THSD7A, which is released into the cultured medium, was harvested for further angiogenic assays. We found that soluble THSD7A promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation. HUVEC sprouts and zebrafish subintestinal vessel (SIV) angiogenic assays further revealed that soluble THSD7A increases the number of branching points of new vessels. Interestingly, we found that soluble THSD7A increased the formation of filopodia in HUVEC. The distribution patterns of vinculin and phosphorylated focal adhesion kinase (FAK) were also affected, which implies a role for THSD7A in focal adhesion assembly. Moreover, soluble THSD7A increased FAK phosphorylation in HUVEC, suggesting that THSD7A is involved in regulating cytoskeleton reorganization. Conclusions/Significance Taken together, our results indicate that THSD7A is a membrane-associated N-glycoprotein with a soluble form. Soluble THSD7A promotes endothelial cell migration during angiogenesis via a FAK-dependent mechanism and thus may be a novel neuroangiogenic factor. PMID:22194972

  16. Endothelial cells control pancreatic cell fate at defined stages through EGFL7 signaling.

    Science.gov (United States)

    Kao, Der-I; Lacko, Lauretta A; Ding, Bi-Sen; Huang, Chen; Phung, Kathleen; Gu, Guoqiang; Rafii, Shahin; Stuhlmann, Heidi; Chen, Shuibing

    2015-02-10

    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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

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    Julie Dwyer

    Full Text Available Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

  19. Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta.

    Science.gov (United States)

    Endres, Bradley T; Staruschenko, Alexander; Schulte, Marie; Geurts, Aron M; Palygin, Oleg

    2015-06-10

    Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca(2+)]i) and nitric oxide (NO). In order to understand how [Ca(2+)]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

  20. Circulating endothelial cells: a potential parameter of organ damage in sickle cell anemia?

    NARCIS (Netherlands)

    Strijbos, Michiel H.; Landburg, Precious P.; Nur, Erfan; Teerlink, Tom; Leebeek, Frank W. G.; Rijneveld, Anita W.; Biemond, Bart J.; Sleijfer, Stefan; Gratama, Jan W.; Duits, Ashley J.; Schnog, John-John B.

    2009-01-01

    Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count

  1. Circulating endothelial cells : a potential parameter of organ damage in sickle cell anemia?

    NARCIS (Netherlands)

    Strijbos, Michiel H; Landburg, Precious P; Nur, Erfan; Teerlink, Tom; Leebeek, Frank W G; Rijneveld, Anita W; Biemond, Bart J; Sleijfer, Stefan; Gratama, Jan W; Duits, Ashley J; Schnog, John-John B

    2009-01-01

    Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count

  2. Transcellular transport of CCL2 across brain microvascular endothelial cells.

    Science.gov (United States)

    Ge, Shujun; Song, Li; Serwanski, David R; Kuziel, William A; Pachter, Joel S

    2008-03-01

    The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood-brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal-to-luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous (125)I-labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin-1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double-label immuno-electronmicroscopy, which revealed co-localization of caveolin-1 with exogenous CCL2, during this chemokine's transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.

  3. 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.

  4. Behaviour of Endothelial Cells in a Tridimensional In Vitro Environment

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    Raif Eren Ayata

    2015-01-01

    Full Text Available Angiogenesis is a fundamental process in healing, tumor growth, and a variety of medical conditions. For this reason, in vitro angiogenesis is an area of interest for researchers. Additionally, in vitro angiogenesis is important for the survival of prevascularized tissue-engineering models. The aim of this study was to observe the self-tubular organization behaviour of endothelial cells in the self-assembly method. In this study, bilayered and dermal substitutes were prepared using the self-assembly method. Histological, immunostaining, and biochemical tests were performed. The behavioural dynamics of endothelial cells in this biological environment of supportive cells were observed, as were the steps of the in vitro angiogenic cascade with self-organizing capillary-like structures formation. The epidermal component of the substitutes was seen to promote network expansion and density. It also increased the quantity of angiogenic factors (VEGF and Ang-1 without increasing the proinflammatory factor (IL-8. In addition, the increased MMP activity contributed to matrix degradation, which facilitated capillary formation.

  5. Influence of stromal cells on lymphocyte adhesion and migration on endothelial cells

    OpenAIRE

    McGettrick, Helen M.; Buckley, Chris D.; Rainger, G.Ed; Nash, Gerard B

    2010-01-01

    Methods are described for analysing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been co-cultured with different stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0 or 0.4μm pore filters, depending on whether migration through the whole construct is to be analysed, or adhesion to the endothelial cells alone. Assays may be ‘static’ or filters can be incorporated into flow chambers...

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

    Science.gov (United States)

    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

  7. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials.

    Science.gov (United States)

    Rao, Rameshwar R; Peterson, Alexis W; Ceccarelli, Jacob; Putnam, Andrew J; Stegemann, Jan P

    2012-06-01

    Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

  8. Dendritic cells can turn CD4+ T lymphocytes into vascular endothelial growth factor-carrying cells by intercellular neuropilin-1 transfer.

    Science.gov (United States)

    Bourbié-Vaudaine, Sarah; Blanchard, Nicolas; Hivroz, Claire; Roméo, Paul-Henri

    2006-08-01

    Neuropilin-1 (NRP1) is a transmembrane protein expressed on neuronal and endothelial cells where it plays a crucial role in guiding axons and regulating angiogenesis. We have recently shown that NRP1 also is expressed on dendritic cells (DC) in the human immune system and have proposed a role for NRP1 in the first stages of the immune response. In these studies, we show that NRP1 can be transferred with a high efficiency from human DC to T lymphocytes by trogocytosis. The NRP1 transfer can occur independently of T lymphocyte activation; the amount of NRP1 transferred depends on the NRP1 expression level on APC and is enhanced when T cells are activated through the TCR. Moreover, the NRP1 transfer occurs between specific donor and recipient cells, because no NRP1 transfer is observed between endothelial cells and T lymphocytes or between APCs and CD34(+) hemopoietic cells. Finally, we show that a major NRP1 ligand, vascular endothelial growth factor (VEGF)(165), is secreted by mature human DCs and binds to NRP1 captured by T lymphocytes. These results show that NRP1 transfer to T lymphocytes during the immune synapse can convert T lymphocytes into VEGF(165)-carrying cells. Together with the enhanced signaling of VEGF-R2 on endothelial cells in the presence, in trans, of the NRP1-VEGF(165) complex, our results suggest that the intercellular transfer of NRP1 might participate in the Ag-independent remodelling of the endothelial vessels in secondary lymphoid organs during inflammation.

  9. Endothelial progenitor cells induce transplant arteriosclerosis via VEGFR-1/2 activity.

    Science.gov (United States)

    Yang, Zhaohua; Wang, Can; Yang, Shouguo; Hong, Tao; Wang, Fangshun; Xia, Limin; Wang, Chunsheng

    2015-01-01

    Acute rejection (AR) after organ transplantation results in transplant arteriosclerosis (TA). Endothelial progenitor cells (EPCs) are involved in tissue repair and blood vessel formation but are suspected to be a cause of TA. In this study, we introduced a syngeneic and allogeneic abdominal aortic transplant model with C57BL/6 and BALB/c mice. Syngeneic and allogeneic grafts were histopathologically analyzed after transplantation. Bone marrow-derived EPCs were injected into transplant model animals to observe their distribution and temporal concentration changes. Changes of vascular endothelial growth factor receptor 1 (VEGFR-1), phosphorylated VEGFR-1 (pVEGFR-1), VEGFR-2, pVEGFR-2, protein kinase B (Akt), pAkt, extracellular signal-regulated kinase 1 (Erk1), pErk1 levels in EPCs upon VEGF165 and the VEGFR inhibitor Vandetanib exposure were analyzed in vitro with western blotting. In the allogeneic transplant group, two weeks after transplantation, formations of new intima layers could be observed, and its proliferation gradually increased to four and six weeks post-transplantation (p arteriosclerosis induced by abdominal aorta transplantation by blocking VEGFRs in EPCs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  11. TH2 cells and their cytokines regulate formation and function of lymphatic vessels.

    Science.gov (United States)

    Shin, Kihyuk; Kataru, Raghu P; Park, Hyeung Ju; Kwon, Bo-In; Kim, Tae Woo; Hong, Young Kwon; Lee, Seung-Hyo

    2015-02-04

    Lymphatic vessels (LVs) are critical for immune surveillance and involved in the pathogenesis of diverse diseases. LV density is increased during inflammation; however, little is known about how the resolution of LVs is controlled in different inflammatory conditions. Here we show the negative effects of T helper type 2 (TH2) cells and their cytokines on LV formation. IL-4 and IL-13 downregulate essential transcription factors of lymphatic endothelial cells (LECs) and inhibit tube formation. Co-culture of LECs with TH2 cells also inhibits tube formation, but this effect is fully reversed by interleukin (IL)-4 and/or IL-13 neutralization. Furthermore, the in vivo blockade of IL-4 and/or IL-13 in an asthma model not only increases the density but also enhances the function of lung LVs. These results demonstrate an anti-lymphangiogenic function of TH2 cells and their cytokines, suggesting a potential usefulness of IL-4 and/or IL-13 antagonist as therapeutic agents for allergic asthma through expanding LV mediated-enhanced antigen clearance from the inflammatory sites.

  12. Exercise-induced mobilisation of endothelial progenitor cells in patients with premature coronary heart disease.