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Sample records for adjacent endothelial cells

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

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

  2. Endothelial cell-derived interleukin-6 regulates tumor growth

    Endothelial cells play a complex role in the pathobiology of cancer. This role is not limited to the making of blood vessels to allow for influx of oxygen and nutrients required for the high metabolic demands of tumor cells. Indeed, it has been recently shown that tumor-associated endothelial cells secrete molecules that enhance tumor cell survival and cancer stem cell self-renewal. The hypothesis underlying this work is that specific disruption of endothelial cell-initiated signaling inhibits tumor growth. Conditioned medium from primary human dermal microvascular endothelial cells (HDMEC) stably transduced with silencing RNA for IL-6 (or controls) was used to evaluate the role of endothelial-derived IL-6 on the activation of key signaling pathways in tumor cells. In addition, these endothelial cells were co-transplanted with tumor cells into immunodefficient mice to determine the impact of endothelial cell-derived IL-6 on tumor growth and angiogenesis. We observed that tumor cells adjacent to blood vessels show strong phosphorylation of STAT3, a key mediator of tumor progression. In search for a possible mechanism for the activation of the STAT3 signaling pathway, we observed that silencing interleukin (IL)-6 in tumor-associated endothelial cells inhibited STAT3 phosphorylation in tumor cells. Notably, tumors vascularized with IL-6-silenced endothelial cells showed lower intratumoral microvessel density, lower tumor cell proliferation, and slower growth than tumors vascularized with control endothelial cells. Collectively, these results demonstrate that IL-6 secreted by endothelial cells enhance tumor growth, and suggest that cancer patients might benefit from targeted approaches that block signaling events initiated by endothelial cells

  3. Endothelial progenitor cells in atherosclerosis

    Du, Fuyong; Zhou, Jun; Gong, Ren; Huang, Xiao; Pansuria, Meghana; Virtue, Anthony; Li, Xinyuan; Wang, Hong; Yang, Xiao-Feng

    2012-01-01

    Endothelial progenitor cells (EPCs) are involved in the maintenance of endothelial homoeostasis and in the process of new vessel formation. Experimental and clinical studies have shown that atherosclerosis is associated with reduced numbers and dysfunction of EPCs; and that medications alone are able to partially reverse the impairment of EPCs in patients with atherosclerosis. Therefore, novel EPC-based therapies may provide enhancement in restoring EPCs’ population and improvement of vascula...

  4. Endothelial potential of human embryonic stem cells

    Levenberg, Shulamit; Zoldan, Janet; Basevitch, Yaara; Langer, Robert

    2007-01-01

    Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realize...

  5. Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

    Bhatwadekar, Ashay D.; Glenn, Josephine V.; Curtis, Tim M.; Grant, Maria B.; Stitt, Alan W.; Gardiner, Tom A.

    2013-01-01

    Purpose Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair. Methods Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed. Results Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05– 0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-α when compared to control medium; SDF-1 remained unchanged. Conclusions The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment. PMID:19474402

  6. [Transplantation of corneal endothelial cells].

    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

  7. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  8. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.

    Klein, L M; Lavker, R M; Matis, W L; Murphy, G F

    1989-01-01

    To understand better the role of mast cell secretory products in the genesis of inflammation, a system was developed for in vitro degranulation of human mast cells in skin organ cultures. Within 2 hr after morphine sulfate-induced degranulation, endothelial cells lining microvessels adjacent to affected mast cells expressed an activation antigen important for endothelial-leukocyte adhesion. Identical results were obtained when other mast cell secretagogues (anti-IgE, compound 48/80, and calci...

  9. Signaling hierarchy regulating human endothelial cell development

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  10. Endothelial cell micropatterning: Methods, effects, and applications

    Anderson, Deirdre E.J.; Hinds, Monica T.

    2011-01-01

    The effects of flow on endothelial cells have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of endothelial cell morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and ...

  11. Microvascular endothelial cells of the corpus luteum

    Spanel-Borowski Katherina

    2003-11-01

    Full Text Available Abstract The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of

  12. Blood cells and endothelial barrier function.

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction. PMID:25838983

  13. Endothelial progenitor cells in cardiovascular diseases

    Poay; Sian; Sabrina; Lee; Kian; Keong; Poh

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.

  14. Optical Investigations of Endothelial Cell Motility

    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...... knowledge of endothelial cells and cell motility; Part 2 describes the projects conducted with twodimensional motility; and Part 3 describes the projects conducted with three-dimensional motility. The projects described in Part 2 all relate the endothelial cells' ability to maintain a barrier, both while...

  15. Radioprotection of human endothelial cells with amifostine

    Materials and methods: We studied the effect of amifostine on radiation sensitivity of human endothelial cells and several tumor cell lines (HeLa, MIA PaCa-2 and BxPC-3). The cells were incubated in medium with a concentration of 1 μg/μl amifostine and after 1 hour irradiated with 10 or 20 Gy single dose. Proliferation index was measured by BrdU assay after another 8 and 24 hours. Results: The results show a higher proliferation rate of endothelial cells following radiation plus amifostine, compared with radiation alone. Amifostine induced an increase of proliferation in the control-non-irradiated human endothelial cells. After irradiation with 10 Gy single dose the proliferation of amifostine treated human endothelial cells was still higher. Amifostine exerts no apparent proliferative effect on the tumor cells. Conclusions: The results presented indicate that amifostine acts as an activation of proliferation of the human endothelial cells in a simple in-vitro system and indicate that amifostine supplementation prior to radiation therapy might exert a radioprotective effect to healthy tissue without spurring tumor growth. (orig.)

  16. The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells

    Jinju Wang; Runmin Guo; Yi Yang; Bradley Jacobs; Suhong Chen; Ifeanyi Iwuchukwu; Gaines, Kenneth J.; Yanfang Chen; Richard Simman; Guiyuan Lv; Keng Wu; Bihl, Ji C.

    2016-01-01

    Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by ...

  17. Differentiation of Murine Embryonic Stem Cells into Endothelial Cells

    F. Fathi

    2008-01-01

    Full Text Available Objective: In this investigation Murine Embryonic Stem (ES cells were differentiatedinto endothelial cells.Materials and Methods: Murine ES cells (CCE cell line exposed to Alpha-MEM medium containing 10% FBS for 4 days. Then obtained Flk-1 (Flk-1:Vascular Endothelial Growth Factor Receptor 2 Positive cells were cultuted inEndothelial Growth Medium-2 (EGM-2 until the last day of experiment. Differentiatedcells were evaluated by immunocytochemistry, RT-PCR and Tube FormationAssays.Results: When the ES cells cultured in collagen coated dishes containingAlpha-MEM & FBS, Flk-1 positive cells were obtained. After transfering Flk-1positive cells into fibronectin coated dishes containing EGM2, the cells wereassumed a relatively uniform endothelial cell morphology and could be propagatedand expanded. Immunocytochemical and RT-PCR analysis of differentiatedcells showed that they take up acetylated low-density lipoprotein (LDL, express Flk-1, CD31 and bind the BS-l lectin. When placed in Matrigel, these MurineES cell–derived endothelial cells formed capillary-like structures characteristicof endothelial cellsConclusion: ES cell–derived endothelial cells provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.

  18. Blood cells and endothelial barrier function

    Rodrigues, Stephen F.; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of solub...

  19. Lymphatic endothelial differentiation in pulmonary lymphangioleiomyomatosis cells.

    Davis, Jennifer M; Hyjek, Elizabeth; Husain, Aliya N; Shen, Le; Jones, Jennifer; Schuger, Lucia A

    2013-08-01

    Pulmonary lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm affecting almost exclusively women of childbearing age. LAM belongs to the family of perivascular epithelioid cell tumors, characterized by spindle and epithelioid cells with smooth muscle and melanocytic differentiation. LAM cells infiltrate the lungs, producing multiple, bilateral lesions rich in lymphatic channels and forming cysts, leading to respiratory insufficiency. Here we used antibodies against four lymphatic endothelial markers-podoplanin (detected by D2-40), prospero homeobox 1 (PROX1), vascular endothelial growth factor receptor 3 (VEGFR-3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-to determine whether LAM cells show lymphatic differentiation. Twelve of 12 diagnostic biopsy specimens (early-stage LAM) and 19 of 19 explants (late-stage LAM) showed immunopositivity for D2-40 in most neoplastic cells. PROX1, VEGFR-3, and LYVE1 immunoreactivity varied from scarce in the early stage to abundant in the late stage. Lymphatic endothelial, smooth muscle, and melanocytic markers were partially co-localized. These findings indicate that lymphatic endothelial differentiation is a feature of LAM and provide evidence of a previously unidentified third lineage of differentiation in this neoplasm. This study has implications for the histological diagnosis of LAM, the origin of the neoplastic cells, and potential future treatment with drugs targeting lymphangiogenesis. PMID:23609227

  20. Endothelial Cell Response to Fusobacterium nucleatum.

    Mendes, Reila Tainá; Nguyen, Daniel; Stephens, Danielle; Pamuk, Ferda; Fernandes, Daniel; Van Dyke, Thomas E; Kantarci, Alpdogan

    2016-07-01

    Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescence-activated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation. PMID:27185790

  1. Endothelial cells derived from human embryonic stem cells

    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.

  2. Endothelial progenitor cells in hematologic malignancies.

    Testa, Ugo; Saulle, Ernestina; Castelli, Germana; Pelosi, Elvira

    2016-01-01

    Studies carried out in the last years have improved the understanding of the cellular and molecular mechanisms controlling angiogenesis during adult life in normal and pathological conditions. Some of these studies have led to the identification of some progenitor cells that sustain angiogenesis through indirect, paracrine mechanisms (hematopoietic angiogenic cells) and through direct mechanisms, i.e., through their capacity to generate a progeny of phenotypically and functionally competent endothelial cells [endothelial colony forming cells (ECFCs)]. The contribution of these progenitors to angiogenetic processes under physiological and pathological conditions is intensively investigated. Angiogenetic mechanisms are stimulated in various hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma, resulting in an increased angiogenesis that contributes to disease progression. In some of these conditions there is preliminary evidence that some endothelial cells could derive from the malignant clone, thus leading to the speculation that the leukemic cell derives from the malignant transformation of a hemangioblastic progenitor, i.e., of a cell capable of differentiation to the hematopoietic and to the endothelial cell lineages. Our understanding of the mechanisms underlying increased angiogenesis in these malignancies not only contributed to a better knowledge of the mechanisms responsible for tumor progression, but also offered the way for the discovery of new therapeutic targets. PMID:27583252

  3. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  4. Radioprotection of human endothelial cells with amifostine

    Andreopoulos, D.; Schleicher, U.M.; Ammon, J. [Technische Hochschule Aachen (Germany). Klinik fuer Radiotherapie - Onkologie; Cotarelo, C.L.; Hand, S. [Technische Hochschule Aachen (Germany). Inst. fuer Pathologie

    1999-11-01

    Materials and methods: We studied the effect of amifostine on radiation sensitivity of human endothelial cells and several tumor cell lines (HeLa, MIA PaCa-2 and BxPC-3). The cells were incubated in medium with a concentration of 1 {mu}g/{mu}l amifostine and after 1 hour irradiated with 10 or 20 Gy single dose. Proliferation index was measured by BrdU assay after another 8 and 24 hours. Results: The results show a higher proliferation rate of endothelial cells following radiation plus amifostine, compared with radiation alone. Amifostine induced an increase of proliferation in the control-non-irradiated human endothelial cells. After irradiation with 10 Gy single dose the proliferation of amifostine treated human endothelial cells was still higher. Amifostine exerts no apparent proliferative effect on the tumor cells. Conclusions: The results presented indicate that amifostine acts as an activation of proliferation of the human endothelial cells in a simple in-vitro system and indicate that amifostine supplementation prior to radiation therapy might exert a radioprotective effect to healthy tissue without spurring tumor growth. (orig.) [German] Material und Methode: Humane Endothelzellen und verschiedene Tumorzellinien (HeLa, MIA PaCa-2 and BxPC-3) wurden fuer eine Stunde mit 1 {mu}g/{mu}l Amifostin inkubiert und dann mit Dosen von 10 und 20 Gy bestrahlt. Die Proliferationsaktivitaet wurde mittels BrdU-Assay nach acht und 24 Stunden gemessen. Ergebnisse: Amifostin fuehrt zu einer verstaerkten Proliferation der unbestrahlten Endothelzellen. Nach der Bestrahlung mit 10 Gy Einzeitdosis zeigen die Endothelzellen mit Amifostin-Zusatz eine staerkere Proliferation als die Zellen ohne Amifostin. Ein protektiver Effekt auf die Tumorzellinien war nicht feststellbar. Schlussfolgerung: Die bisherigen Ergebnisse zeigen, dass Amifostin einen radioprotektiven Effekt auf humane Endothelzellen ausuebt und deren Proliferation stimuliert, ohne jedoch die Proliferation der Tumorzellen

  5. Circulating endothelial cells in cardiovascular disease.

    Boos, Christopher J; Lip, Gregory Y H; Blann, Andrew D

    2006-10-17

    Quantification of circulating endothelial cells (CECs) in peripheral blood is developing as a novel and reproducible method of assessing endothelial damage/dysfunction. The CECs are thought to be mature cells that have detached from the intimal monolayer in response to endothelial injury and are a different cell population to endothelial progenitor cells (EPCs). The EPCs are nonleukocytes derived from the bone marrow that are believed to have proliferative potential and may be important in vascular regeneration. Currently accepted methods of CEC quantification include the use of immunomagnetic bead separation (with cell counting under fluorescence microscopy) and flow cytometry. Several recent studies have shown increased numbers of CECs in cardiovascular disease and its risk factors, such as unstable angina, acute myocardial infarction, stroke, diabetes mellitus, and critical limb ischemia, but no change in stable intermittent claudication, essential hypertension, or atrial fibrillation. Furthermore, CEC quantification at 48 h after acute myocardial infarction has been shown to be an accurate predictor of major adverse coronary events and death at both 1 month and 1 year. This article presents an overview of the pathophysiology of CECs in the setting of cardiovascular disease and a brief comparison with EPCs. PMID:17045885

  6. Endothelial cell transfection of ex vivo arteries

    sprotocols

    2015-01-01

    Authors: Alexander Lohman, Adam Straub & Brant Isakson ### Abstract The vascular endothelium plays an essential role in regulating blood vessel tone, blood flow and blood pressure. Current vascular model systems for examination of endothelial cell biology and blood vessel physiology and pathology rely on cell culture and the generation of genetically modified animals. While these systems are advantageous for studying the endothelium, many cell culture models omit the contribution ...

  7. The control of vascular endothelial cell injury.

    Murota, S; Morita, I; Suda, N

    1990-01-01

    The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity. PMID:2248437

  8. Tissue factor expression by endothelial cells in sickle cell anemia.

    Solovey, A; Gui, L; Key, N. S.; Hebbel, R.P.

    1998-01-01

    The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the system's triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that s...

  9. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    Fang, Jennifer S; Gritz, Emily C; Marcelo, Kathrina L; Hirschi, Karen K

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  10. Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability.

    Ong, Siew Pei; Ng, Mah Lee; Chu, Justin Jang Hann

    2013-12-01

    Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation. Immunostaining of vascular endothelial cadherin (VE-cadherin) and zona occludin 1 (ZO-1) showed the absence of these endothelial junctional proteins at the cell-cell contact zones between adjacent cells. In addition, Ang1 that is required for protecting against endothelial hyperpermeability was found to be down-regulated during DV infection. Treatment with increasing concentrations of recombinant Ang1 was shown to prevent DV-induced endothelial hyperpermeability in a dose-dependent manner by preventing the down-regulation of VE-cadherin and ZO-1 at cell membrane. In contrast, the expression of Ang2, the natural antagonist of Ang1, was observed to be up-regulated during DV infection. Recombinant Ang2 added to HUVEC at non-toxic concentrations showed decreased in transendothelial electrical resistance reading and the down-regulation of VE-cadherin and ZO-1. These findings suggest that DV reduces the expression of Ang1 and enhances the expression of Ang2 in endothelial cells and that this imbalance of Ang 1 and Ang 2 may play a contributing role to the increased permeability of human primary endothelial cells during DV infection. PMID:23989887

  11. Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

    TAN YuZhen; ZHAO Yao; WANG HaiJie; ZHOU LiangFu; MAO Ying; LIU Rui; SHU Jia; WANG YongFei

    2008-01-01

    Human cerebral cavernous malformation (CM) is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type Ⅰ gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like struc-tures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to re-ceptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.

  12. Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

    Zhao, Jing; Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Morrell, Nicholas W; Lever, Andrew M L

    2016-01-01

    Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm(2). The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair. PMID:27413378

  13. Collective cell motion in endothelial monolayers

    Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior

  14. Cytokine production by endothelial cells infected with human T cell lymphotropic virus type I.

    H. Takashima; Eguchi, K.; Kawakami, A; Kawabe, Y; Migita, K; Sakai, M; Origuchi, T; Nagataki, S.

    1996-01-01

    OBJECTIVE: To investigate the ability of human T cell lymphotropic virus type I (HTLV-I) to infect endothelial cells and induce cytokine production by these cells. METHODS: Human umbilical vein endothelial cells (HUVEC) were cocultured with HTLV-I infected T cell line (MT-2 cells) or uninfected T cell line (CEM cells). RESULTS: Following coculture with MT-2 cells, endothelial cells expressed HTLV-I specific core antigens. Endothelial cells cocultured with MT-2 cells produced significant amoun...

  15. Endothelial progenitor cells with Alzheimer's disease

    KONG Xiao-dong; ZHANG Yun; LIU Li; SUN Ning; ZHANG Ming-yi; ZHANG Jian-ning

    2011-01-01

    Background Endothelial dysfunction is thought to be critical events in the pathogenesis of Alzheimer's disease (AD).Endothelial progenitor cells (EPCs) have provided insight into maintaining and repairing endothelial function. To study the relation between EPCs and AD, we explored the number of circulating EPCs in patients with AD.Methods A total of 104 patients were recruited from both the outpatients and inpatients of the geriatric neurology department at General Hospital, rianjin Medical University. Consecutive patients with newly diagnosed AD (n=30),patients with vascular dementia (VaD, n=34), and healthy elderly control subjects with normal cognition (n=40) were enrolled after matching for age, gender, body mass index, medical history, current medication and Mini Mental State Examination. Middle cerebral artery flow velocity was examined with transcranial Doppler. Endothelial function was evaluated according to the level of EPCs, and peripheral blood EPCs was counted by flow cytometry.Results There were no significant statistical differences of clinical data in AD, VaD and control groups (P >0.05). The patients with AD showed decreased CD34-positive (CD34+) or CD133-positive (CD133+) levels compared to the control subjects, but there were no significant statistical differences in patients with AD. The patients with AD had significantly lower CD34+CD133+ EPCs(CD34 and CD133 double positive endothelial progenitor cells) than the control subjects (P <0.05). In the patients with AD, a lower CD34+CD133+ EPCs count was independently associated with a lower Mini-Mental State Examination score (r=0.514, P=0.004). Patients with VaD also showed a significant decrease in CD34+CD133+ EPCs levels, but this was not evidently associated with the Mini-Mental State Examination score. The changes of middle cerebral artery flow velocity were similar between AD and VaD. Middle cerebral artery flow velocity was decreased in the AD and VaD groups and significantly lower than

  16. Heparin Binds Endothelial Cell Growth Factor, the Principal Endothelial Cell Mitogen in Bovine Brain

    Maciag, Thomas; Mehlman, Tevie; Friesel, Robert; Schreiber, Alain B.

    1984-08-01

    Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

  17. Endoderm Generates Endothelial Cells during Liver Development

    Orit Goldman

    2014-10-01

    Full Text Available Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1. Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.

  18. Production of soluble Neprilysin by endothelial cells

    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 IC50 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

  19. Production of soluble Neprilysin by endothelial cells

    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.

  20. Endothelial Progenitor Cells Enter the Aging Arena.

    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.

  1. Enhancing endothelial progenitor cell for clinical use

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) havebeen demonstrated to correlate negatively with vascularendothelial dysfunction and cardiovascular risk factors.However, translation of basic research into the clinicalpractice has been limited by the lack of unambiguousand consistent definitions of EPCs and reduced EPCcell number and function in subjects requiring them forclinical use. This article critically reviews the definitionof EPCs based on commonly used protocols, their valueas a biomarker of cardiovascular risk factor in subjectswith cardiovascular disease, and strategies to enhanceEPCs for treatment of ischemic diseases.

  2. Adjacent-cell preconditioners for accelerating multidimensional neutron transport methods

    The Adjacent-cell Preconditioner (AP) is derived for accelerating generic fixed-weight, Weighted Diamond Difference (WDD) neutron transport methods in multidimensional Cartesian geometry. The AP is determined by requiring: (a) the eigenvalue of the combined mesh sweep-AP iterations to vanish in the vicinity of the origin in Fourier space; and (b) the diagonal and off-diagonal elements of the preconditioner to satisfy a diffusion-like condition. The spectra of the resulting iterations for a wide range of problem parameters exhibit a spectral radius smaller than .25, that vanishes implying immediate convergence for very large computational cells. More importantly, unlike other unconditionally stable acceleration schemes, the AP is cell-centered and its spectral radius remains small when the cell aspect ratio approaches 0 or ∞. Testing of the AP and comparison of its rate of convergence to the standard Source Iterations (SI) for Burre's Suite of Test Problems (BSTeP) demonstrates its high efficiency in reducing the number of iterations required to achieve convergence, especially for optically thick cells where acceleration is most needed

  3. Differentiation state determines neural effects on microvascular endothelial cells

    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

  4. Immunological functions of liver sinusoidal endothelial cells.

    Knolle, Percy A; Wohlleber, Dirk

    2016-05-01

    Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and separate passenger leukocytes in the sinusoidal lumen from hepatocytes. LSECs further act as a platform for adhesion of various liver-resident immune cell populations such as Kupffer cells, innate lymphoid cells or liver dendritic cells. In addition to having an extraordinary scavenger function, LSECs possess potent immune functions, serving as sentinel cells to detect microbial infection through pattern recognition receptor activation and as antigen (cross)-presenting cells. LSECs cross-prime naive CD8 T cells, causing their rapid differentiation into memory T cells that relocate to secondary lymphoid tissues and provide protection when they re-encounter the antigen during microbial infection. Cross-presentation of viral antigens by LSECs derived from infected hepatocytes triggers local activation of effector CD8 T cells and thereby assures hepatic immune surveillance. The immune function of LSECs complements conventional immune-activating mechanisms to accommodate optimal immune surveillance against infectious microorganisms while preserving the integrity of the liver as a metabolic organ. PMID:27041636

  5. Endothelial progenitor cells and revascularization following stroke.

    Ma, Feifei; Morancho, Anna; Montaner, Joan; Rosell, Anna

    2015-10-14

    Brain injury after ischemia induces the mobilization of endothelial progenitor cells (EPCs), a population of bone marrow-derived cells with angio-vasculogenic capabilities. These cells have been also tested in pre-clinical models and proposed for neurorepair therapy aiming to treat patients in the delayed phases of stroke disease. Promising results in the pre-clinical field encourage the translation into a clinical therapeutic approach. In this review, we will describe EPCs actions for enhanced revascularization and neurorepair, which on one hand are by their direct incorporation into new vascular networks/structures or by direct cell-cell interactions with other brain cells, but also to indirect cell-cell communication thorough EPCs secreted growth factors. All these actions contribute to potentiate neurovascular remodeling and neurorepair. The data presented in this review encourages for a deep understanding of the mechanisms of the cross-talks between EPCs and other brain and progenitor cells, which deserves additional investigations and efforts that may lead to new EPCs-based therapies for stroke patients. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke. PMID:25725381

  6. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  7. DIFFERENT RESPONSES OF CHORIOCAPILLARY ENDOTHELIAL CELLS AND RETINALCAPILLARY ENDOTHELIAL CELLS TO MITOGENIC AND VASOACTIVE FACTORS

    李维业; 刘熙朴; MyronYanoff

    1994-01-01

    The reaponses of choriocapillary endothelial cells(CCE) and retinal capillary ondothelial cells (RCE) in cul-ture,in terms of phosphoinositide (PI) breakdown and cellular mitogenesis,to retinal pigment epithelial cell (RPE)-conditioned medium and vasoactive agents have been compared.RPE-conditioned medium did not induce PI breakdown in either type of cell.However,it stimulated DNA synthesis in CCE but not in RCE.Bradykinin (BDK)acted as both a fast signaling and a slow mitogenic factor on CCE,out BDK did not affect PI turnover or DNA synthesis in RCE.In contrast,thrombin stimulated PI turnover in RCE but not in CCE,though it did not in-duce 3H-thymidine incorporation into either type of cell.These differences in cellular functions between CCE and RCE following stimulation suggest that induction of DNA synthesis and recptor-mediated PI turnover by external factors is determined,at least in part,by the origin of the capillary endothelial cell.Therefore,extrapolation to CCE pathophysiology from experiments using endothelial cells from other capillary origins may not be valid.

  8. The Use of Mild Trypsinization Conditions in the Detachment of Endothelial Cells to Promote Subsequent Endothelialization on Synthetic Surfaces

    Brown, Melissa A.; Wallace, Charles S.; Anamelechi, Charles C.; Clermont, Edward; Reichert, William M.; Truskey, George A.

    2007-01-01

    A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for five minutes and seeded onto glass pretreated with fibronectin had close to 100%...

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

    Bjerregaard, Bolette; Holck, S.; Christensen, I. J.; Larsson, Lars-Inge

    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 specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to ex...

  10. Radioprotection of mouse CNS endothelial cells in vivo

    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

  11. Endothelial cell cultures as a tool in biomaterial research

    Kirkpatrick, CJ; Otto, M; Kooten, TV; 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)

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

    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

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

    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 pho...

  14. Obstructive sleep apnea and endothelial progenitor cells

    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

  15. Prolonged cyclic strain inhibits human endothelial cell growth.

    Peyton, Kelly J; Liu, Xiao-ming; Durante, William

    2016-01-01

    The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state. PMID:26709656

  16. Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

    Avraham, H; Cowley, S; Chi, S. Y.; Jiang, S.; Groopman, J E

    1993-01-01

    Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor ...

  17. Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

    Caterina Mammi; Donatella Pastore; Lombardo, Marco F; Francesca Ferrelli; Massimiliano Caprio; Claudia Consoli; Manfredi Tesauro; Lucia Gatta; Massimo Fini; Massimo Federici; Paolo Sbraccia; Giulia Donadel; Alfonso Bellia; Giuseppe M Rosano; Andrea Fabbri

    2011-01-01

    BACKGROUND: The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cel...

  18. In Vitro Guidance of Dental Pulp Cells by Nd:YAG Laser-Irradiated Endothelial Cells

    Masuda, Yoshiko Murakami; Yamada, Yoshishige; Kimura, Yuichi

    2012-01-01

    Objective: After endothelial cells were ablated by neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation, we investigated the response of pulp cells by examining the expression of transforming growth factor beta-1 (TGF-β1). Background data: The reaction of stimulated blood vessels is related to the initiation of dentinogenesis. After artificial injury of endothelial cells, pulp cells migrate to the site of the injured endothelial cells. Materials and methods: Rat aortic endothelial cel...

  19. Silencing of directional migration in roundabout4 knockdown endothelial cells

    Roberts David D

    2008-11-01

    Full Text Available Abstract Background Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4, the predominant Robo in endothelial cells using small interfering RNA technology in vitro. Results Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. Conclusion This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.

  20. Radiation-induced apoptosis in microvascular endothelial cells.

    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...

  1. Angiogenic potential of endothelial progenitor cells and embryonic stem cells

    Rae Peter C

    2011-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPCs are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs by direct differentiation using EC-conditioned medium (ECCM. Results The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs. Conclusions We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

  2. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

    Guang-fa WANG; Shao-yu WU; Jin-jun RAO; Lin L(U); Wei XU; Jian-xin PANG; Zhong-qiu LIU; Shu-guang WU; Jia-jie ZHANG

    2009-01-01

    Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin,an aglycon of geniposide, inhibits endothelial exocytosis.Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

  3. Carotid Repair Using Autologous Adipose-Derived Endothelial Cells

    Froehlich, Harald; Gulati, Rajiv; Boilson, Barry; Witt, Tyra; Harbuzariu, Adriana; Kleppe, Laurel; Dietz, Allan B.; Lerman, Amir; Simari, Robert D.

    2009-01-01

    Background and Purpose Adipose tissue is an abundant source of endothelial cells as well as stem and progenitor cells which can develop an endothelial phenotype. It has been demonstrated that these cells have distinct angiogenic properties in vitro and in vivo. However, whether these cells have the capacity to directly improve large vessel form and function following vascular injury remains unknown. To define whether delivery of adipose-derived endothelial cells (ADECs) would improve healing of injured carotid arteries, a rabbit model of acute arterial injury was employed. Methods Autologous rabbit ADECS were generated utilizing defined culture conditions. To test the ability of ADECs to enhance carotid artery repair, cells were delivered intra-arterially following acute balloon injury. Additional delivery studies were performed following functional selection of cells prior to delivery. Results Following rabbit omental fat harvest and digestion, a proliferative, homogenous, and distinctly endothelial population of ADECs was identified. Direct delivery of autologous ADECs resulted in marked re-endothelialization 48 hours following acute vascular injury as compared to saline controls (82.2 ±26.9% vs 4.2±3.0% pADECs that were selected for their ability to take up acetylated LDL significantly improved vasoreactivity and decreased intimal formation following vascular injury. Conclusions Taken together, these data suggest that ADECs represent an autologous source of proliferative endothelial cells which demonstrate the capacity to rapidly improve re-endothelialization, improve vascular reactivity, and decrease intimal formation in a carotid artery injury model. PMID:19286583

  4. Radioprotection of mouse CNS endothelial cells in vivo

    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)

  5. Radioprotection of mouse CNS endothelial cells in vivo

    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 γ-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)

  6. Nipah virus infection and glycoprotein targeting in endothelial cells

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  7. Expression of platelet-endothelial cell adhesion molecule-1 in human umbilical vein endothelial cells by exposure to advanced glycosylation end products and inflammatory mediators

    孟丹; 刘乃丰

    2003-01-01

    Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α+IFN-γ and AGEs-BSA+TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods (P> 0.05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-γ, TNF-α+IFN-γ and AGEs-BSA+TNF-α. The level of PECAM-1 treated with AGEs-BSA+TNF-α was lower than that of TNF-α treated alone (P<0.01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.

  8. Listeria monocytogenes Virulence Factors That Stimulate Endothelial Cells

    Drevets, Douglas A.

    1998-01-01

    Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L. monocytogenes or L. monocytogenes mutants; then surface expression of E-selectin and neutro...

  9. Paclitaxel Induces Thrombomodulin Downregulation in Human Aortic Endothelial Cells

    Wang, Huang-Joe; Lu, Te-Ling; Huang, Haimei; Huang, Huey-Chun

    2011-01-01

    Patients with paclitaxel-eluting stents are at risk of developing stent thrombosis upon premature discontinuation of dual antiplatelet therapy. In this study, we set out to clarify whether paclitaxel can modulate thrombomodulin expression in human aortic endothelial cells. Human aortic endothelial cells were stimulated with paclitaxel. Methoxyphenyl tetrazolium inner salt cell viability assay, Western blot analysis, real-time polymerase chain reaction, and immunohistochemical assay were perfo...

  10. Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10.

    Hebeda, C B; Teixeira, S A; Tamura, E K; Muscará, M N; de Mello, S B V; Markus, R P; Farsky, S H P

    2011-08-01

    We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions. PMID:21564091

  11. Hypertension alters phosphorylation of VASP in brain endothelial cells.

    Arlier, Zulfikar; Basar, Murat; Kocamaz, Erdogan; Kiraz, Kemal; Tanriover, Gamze; Kocer, Gunnur; Arlier, Sefa; Giray, Semih; Nasırcılar, Seher; Gunduz, Filiz; Senturk, Umit K; Demir, Necdet

    2015-04-01

    Hypertension impairs cerebral vascular function. Vasodilator-stimulated phosphoprotein (VASP) mediates active reorganization of the cytoskeleton via membrane ruffling, aggregation and tethering of actin filaments. VASP regulation of endothelial barrier function has been demonstrated by studies using VASP(-/-) animals under conditions associated with tissue hypoxia. We hypothesize that hypertension regulates VASP expression and/or phosphorylation in endothelial cells, thereby contributing to dysfunction in the cerebral vasculature. Because exercise has direct and indirect salutary effects on vascular systems that have been damaged by hypertension, we also investigated the effect of exercise on maintenance of VASP expression and/or phosphorylation. We used immunohistochemistry, Western blotting and immunocytochemistry to examine the effect of hypertension on VASP expression and phosphorylation in brain endothelial cells in normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rats under normal and exercise conditions. In addition, we analyzed VASP regulation in normoxia- and hypoxia-induced endothelial cells. Brain endothelial cells exhibited significantly lower VASP immunoreactivity and phosphorylation at the Ser157 residue in SHR versus WKY rats. Exercise reversed hypertension-induced alterations in VASP phosphorylation. Western blotting and immunocytochemistry indicated reduction in VASP phosphorylation in hypoxic versus normoxic endothelial cells. These results suggest that diminished VASP expression and/or Ser157 phosphorylation mediates endothelial changes associated with hypertension and exercise may normalize these changes, at least in part, by restoring VASP phosphorylation. PMID:24894047

  12. Endothelial cell growth factor and ionophore A23187 stimulation of production of inositol phosphates in porcine aorta endothelial cells.

    Moscat, J; Moreno, F.; Herrero, C.; C. López; García-Barreno, P.

    1988-01-01

    The existence of a bovine brain-derived endothelial cell growth factor has recently been reported, but its mode of action is unknown. We show that the endothelial cell growth factor is a potent stimulant of inositol monophosphate release in porcine aorta endothelial cells. Although the activation of phospholipase C by this factor does not appear to be dependent on Ca2+, the Ca2+ ionophore A23187 stimulates release of inositol phosphates. It is suggested that the inositol 1,4,5-trisphosphate 3...

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

    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.

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

    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. Alk1 controls arterial endothelial cell migration in lumenized vessels.

    Rochon, Elizabeth R; Menon, Prahlad G; Roman, Beth L

    2016-07-15

    Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs. PMID:27287800

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

    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.

  17. Endothelial cell pseudopods and angiogenesis of breast cancer tumors

    Sun LuZhe

    2005-05-01

    Full Text Available Abstract Background A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse. Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than 35 mm3 were stained with PAS, with CD-31 antibody (an endothelial cell maker, or with hypoxia inducible factor 1α antibody (HIF. The extent of blood vessel and endothelial cell pseudopod volume density was measured by ocular grid intercept counting in the PAS stained slides. Results The tumor area within 100–150 μm of the well-vascularized capsule had few blood vessels and only occasional endothelial cell pseudopods, whereas the area greater than 150 μm from the capsule had more blood vessels, capillaries, and a three-fold increase in volume density of pseudopods sprouting from the capillary endothelial cells. This subcortical region, rich in pseudopods, some of which were observed to have vacuoles/lumens, was strongly positive for presence of HIF. In some larger tumors, pseudopods were observed to insinuate for mm distances through hypoxic regions of the tumor. Conclusion The positive correlation between presence of HIF and the increased extent of pseudopods suggests volume density measure of the latter as a quantifiable marker of tumor hypoxia. Apparently, hypoxic regions of the tumor produce HIF leading to production of vascular endothelial growth factors that stimulate sprouting of capillary endothelial cells and formation of endothelial cell pseudopods.

  18. Uptake of gold nanoparticles in primary human endothelial cells

    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....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  19. Endothelial Progenitor Cells Dysfunction and Senescence: Contribution to Oxidative Stress

    Imanishi, Toshio; Tsujioka, Hiroto; Akasaka, Takashi

    2008-01-01

    The identification of endothelial progenitor cells (EPCs) has led to a significant paradigm in the field of vascular biology and opened a door to the development of new therapeutic approaches. Based on the current evidence, it appears that EPCs may make both direct contribution to neovascularization and indirectly promote the angiogenic function of local endothelial cells via secretion of angiogenic factors. This concept of arterial wall repair mediated by bone marrow (BM)-derived EPCs provid...

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

    Donatella Del Bufalo; Daniela Trisciuoglio; Marco Scarsella; Giulia D'Amati; Antonio Candiloro; Angela Iervolino; Carlo Leonetti; Gabriella Zupi

    2004-01-01

    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 secreti...

  1. Mitochondrial function in vascular endothelial cell in diabetes

    Pangare, Meenal; Makino, Ayako

    2012-01-01

    Micro- and macrovascular complications are commonly seen in diabetic patients and endothelial dysfunction contributes to the development and progression of the complications. Abnormal functions in endothelial cells lead to the increase in vascular tension and atherosclerosis, followed by systemic hypertension as well as increased incident of ischemia and stroke in diabetic patients. Mitochondria are organelles serving as a source of energy production and as regulators of cell survival (e.g., ...

  2. Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

    Lu, T T; Yan, L G; Madri, J. A.

    1996-01-01

    Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell sprea...

  3. Type 5 phosphodiesterase expression is a critical determinant of the endothelial cell angiogenic phenotype

    Zhu, Bing; Zhang, Li; Alexeyev, Mikhail; Alvarez, Diego F.; Strada, Samuel J.; Stevens, Troy

    2008-01-01

    Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stabl...

  4. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

  5. Lack of vimentin impairs endothelial differentiation of embryonic stem cells.

    Boraas, Liana C; Ahsan, Tabassum

    2016-01-01

    The cytoskeletal filament vimentin is inherent to the endothelial phenotype and is critical for the proper function of endothelial cells in adult mice. It is unclear, however, if the presence of vimentin is necessary during differentiation to the endothelial phenotype. Here we evaluated gene and protein expression of differentiating wild type embryonic stem cells (WT ESCs) and vimentin knockout embryonic stem cells (VIM -/- ESCs) using embryoid bodies (EBs) formed from both cell types. Over seven days of differentiation VIM -/- EBs had altered morphology compared to WT EBs, with a rippled outer surface and a smaller size due to decreased proliferation. Gene expression of pluripotency markers decreased similarly for EBs of both cell types; however, VIM -/- EBs had impaired differentiation towards the endothelial phenotype. This was quantified with decreased expression of markers along the specification pathway, specifically the early mesodermal marker Brachy-T, the lateral plate mesodermal marker FLK1, and the endothelial-specific markers TIE2, PECAM, and VE-CADHERIN. Taken together, these results indicate that the absence of vimentin impairs spontaneous differentiation of ESCs to the endothelial phenotype in vitro. PMID:27480130

  6. The effects of glucocorticoids on cultured human endothelial cells.

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  7. Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

    Douglas, G; van Kampen, E.; Hale, AB; McNeill, E; Patel, J.; Crabtree, MJ; Ali, Z; Hoerr, RA; Alp, NJ; Channon, KM

    2013-01-01

    Aims Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Methods and results Endothelial cell repopulation was assessed en face in stented arteries in ApoE−/− mice with end...

  8. Glioma-associated endothelial cells show evidence of replicative senescence

    The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated β-galactosidase (SA-β-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-β-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population

  9. MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

    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.

  10. Endothelial progenitor cells in acute ischemic stroke

    Martí-Fàbregas, Joan; Crespo, Javier; Delgado-Mederos, Raquel; Martínez-Ramírez, Sergi; Peña, Esther; Marín, Rebeca; Dinia, Lavinia; Jiménez-Xarrié, Elena; Fernández-Arcos, Ana; Pérez-Pérez, Jesús; Querol, Luis; Suárez-Calvet, Marc; Badimon, Lina

    2013-01-01

    Objectives The levels of circulating endothelial progenitor cells (EPCs) in ischemic stroke have not been studied extensively and reported results are inconsistent. We aimed to investigate the time course, the prognostic relevance, and the variables associated with EPC counts in patients with ischemic stroke at different time points. Material and methods We studied prospectively 146 consecutive patients with ischemic stroke within the first 48 h from the onset of symptoms (baseline). We evaluated demographic data, classical vascular risk factors, treatment with thrombolysis and statins, stroke etiology, National Institute of Health and Stroke Scale score and outcome (favorable when Rankin scale score 0–2). Blood samples were collected at baseline, at day 7 after stroke (n = 121) and at 3 months (n = 92). The EPC were measured by flow cytometry. Results We included 146 patients with a mean age of 70.8 ± 12.2 years. The circulating EPC levels were higher on day 7 than at baseline or at 3 months (P = 0.045). Pretreatment with statins (odds ratio [OR] 3.11, P = 0.008) and stroke etiology (P = 0.032) were predictive of EPC counts in the baseline sample. EPC counts were not associated with stroke severity or functional outcome in all the patients. However, using multivariate analyses, a better functional outcome was found in patients with higher EPC counts in large-artery atherosclerosis and small-vessel disease etiologic subtypes. Conclusions After acute ischemic stroke, circulating EPC counts peaked at day 7. Pretreatment with statins increased the levels of EPC. In patients with large-artery atherosclerosis and small-vessel disease subtypes, higher counts were related to better outcome at 3 months. PMID:24363968

  11. Endothelial Progenitor Cells Promote Directional Three-Dimensional Endothelial Network Formation by Secreting Vascular Endothelial Growth Factor

    Yoshinori Abe; Yoshiyuki Ozaki; Junichi Kasuya; Kimiko Yamamoto; Joji Ando; Ryo Sudo; Mariko Ikeda; Kazuo Tanishita

    2013-01-01

    Endothelial progenitor cell (EPC) transplantation induces the formation of new blood-vessel networks to supply nutrients and oxygen, and is feasible for the treatment of ischemia and cardiovascular diseases. However, the role of EPCs as a source of proangiogenic cytokines and consequent generators of an extracellular growth factor microenvironment in three-dimensional (3D) microvessel formation is not fully understood. We focused on the contribution of EPCs as a source of proangiogenic cytoki...

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

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

    2014-01-01

    We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... 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...... that glycocalyx disruption preceded cerebral manifestations. The contribution of this loss to pathogenesis should be studied further....

  13. Unidirectional transfer of prostaglandin endoperoxides between platelets and endothelial cells.

    Schafer, A I; Crawford, D D; Gimbrone, M. A.

    1984-01-01

    An important determinant of platelet-vessel wall interactions is the local balance of production of endothelial prostacyclin (PGI2) and platelet thromboxane (TX) A2, labile eicosanoids with opposing effects on hemostasis. Disputed evidence suggests that platelet-derived prostaglandin endoperoxide intermediates may be utilized as substrates for vascular PGI2 synthesis. Using several different approaches, we have found that platelets can transfer endoperoxides to cultured endothelial cells for ...

  14. Accelerating Vascularization in Polycaprolactone Scaffolds by Endothelial Progenitor Cells

    Singh, Shivani; Wu, Benjamin M.; Dunn, James C.Y.

    2011-01-01

    Vascularization is a major challenge in tissue engineering. The purpose of this study is to expedite the formation of blood vessels in porous polycaprolactone (PCL) scaffolds by the delivery of endothelial progenitor cells (EPCs). To establish a pro-angiogenic and pro-vasculogenic microenvironment, we employed EPCs seeded in PCL scaffold with surface-immobilized heparin and vascular endothelial growth factor (VEGF). EPCs seeded on scaffolds with VEGF exhibited phosphorylation of the receptor....

  15. Fructose Induces the Inflammatory Molecule ICAM-1 in Endothelial Cells

    Glushakova, Olena; Kosugi, Tomoki; Roncal, Carlos; Mu, Wei; Heinig, Marcelo; Cirillo, Pietro; Sánchez-Lozada, Laura G.; Richard J Johnson; Nakagawa, Takahiko

    2008-01-01

    Epidemiologic studies have linked fructose intake with the metabolic syndrome, and it was recently reported that fructose induces an inflammatory response in the rat kidney. Here, we examined whether fructose directly stimulates endothelial inflammatory processes by upregulating the inflammatory molecule intercellular adhesion molecule-1 (ICAM-1). When human aortic endothelial cells were stimulated with physiologic concentrations of fructose, ICAM-1 mRNA and protein expression increased in a ...

  16. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  17. Berberine protects vascular endothelial cells in hypertensive rats

    Wang, Yang; Ding, Yun

    2015-01-01

    Objective: This study is to investigate the effect and mechanism of berberine on vascular endothelial cell injury. Methods: The isolated aortic endothelial cells were divided into negative control group, spontaneous hypertension group, and berberine group (1.25, 2.5, and 5 μmol/L berberine). CCK-8 assay was performed to detect cell proliferation. Annexin V-FITC flow cytometry and Hochest33342/PI staining were used to measure cell apoptosis. Expression of TLR4, Myd88, and NF-κB was detected wi...

  18. An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells

    Kang, Jinjoo; Yoo, Jaehyuk; Lee, Sunju; Tang, Wanli; Aguilar, Berenice; Ramu, Swapnika; Choi, Inho; Otu, Hasan H.; Shin, Jay W.; Dotto, G. Paolo; Koh, Chester J.; Detmar, Michael

    2010-01-01

    Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may coreside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators. PMID:20351309

  19. Endothelial dysfunction in ankylosing spondylitis associated with reduced endothelial progenitor cell population

    Pawan Krishan

    2015-06-01

    Full Text Available Background: Endothelial dysfunction, and cardiovascular (CV morbidity and mortality have been documented in patients with ankylosing spondylitis (AS. Endothelial progenitor cells (EPCs have reparative potential in overcoming the endothelial dysfunction and reducing cardiovascular risk. Aim: To investigate the relationship between endothelial function and EPCs in patients with AS in a cross-sectional study. Methods: Circulating EPCs (CD34+/CD133+ were isolated and quantified from peripheral blood samples of AS (n23 and healthy controls (n=20 matched for age and sex. Endothelium-depended vascular function, i.e. flow-mediated dilation (FMD, was assessed for all subjects. Disease activity was evaluated using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI. Functional ability was monitored using Bath Ankylosing Spondylitis Functional Index (BASFI. All subjects were free of any other known traditional CV risk factors. Results: AS patients had depleted level of circulating %EPCs (0.028 ± 0.001% versus 0.045 ± 0.011%, P <0.001 and reduced FMD% (6.77 ± 2.15 versus 10.06 ± 0.55, P <0.001 than healthy controls. Circulating EPC population significantly positively correlated with FMD% (r 0.538, P = 0.008. Levels of CD34+/CD133+ putative cells showed a significant inverse correlation with disease duration (P = 0.01, BASDAI (P = 0.04, ESR (P = 0.002 and CRP (P = 0.007. Conclusion: AS patients, free of any other known CV risk factors, demonstrated depleted levels of EPCs and reduced endothelial function. These alterations may cause further deterioration of endothelial function in AS patients. EPC would possibly serve as a novel therapeutic target for preventing cardiovascular risk in AS.

  20. Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

    Clayton, Z E; Sadeghipour, S; Patel, S

    2015-10-15

    Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells. PMID:26123569

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

    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.

  2. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

  3. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    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

  4. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    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.

  5. Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue.

    Ugele, B; Lange, F

    2001-01-01

    Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary. PMID:11573814

  6. New insights in endothelial and smooth muscle cell communication.

    Conejo, Víctor Arana; De Haro, Roberto; Sosa-Melgarejo, Jorge; Méndez, José D

    2007-01-01

    Based on immunohistochemical techniques against connexins and the intercellular flux of staining molecules, it has previously been shown that electrotonic communication occurs among endothelial and vascular smooth muscle cells, this due to the presence of myoendothelial gap junctions. The aim of this study was to evaluate the density of myoendothelial contacts in the left coronary and internal mammary arteries as well as in the left saphenous vein by means of electron microscopy, the distance between both cells participating in an myoendothelial contact with a semi-automatic image analysis system and the presence of homocellular and heterocellular gap junctions between endothelial and smooth muscle cells by using the immunohistochemical technique and confocal microscopy in thoracic aorta were also analyzed. The results are that all blood vessels studied present myoendothelial contacts, while density studies show that they are more abundant in the saphenous vein. The myoendothelial contact distance is constant and in no case the cytoplasmic processes reach the plasma membrane of the partner cell toward which they are advanced. Homocellular gap junctions were found between smooth muscle cells and between endothelial cells. Heterocellular gap junctions were absent, evidencing the possibility that signaling molecules between endothelial and smooth muscle cells may be transferred through plasma membranes as was once thought and not necessarily by electrotonic communication. PMID:17383847

  7. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    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.

  8. When the endothelium scores an own goal: endothelial cells actively augment metastatic extravasation through endothelial-mesenchymal transition.

    Gasparics, Ákos; Rosivall, László; Krizbai, István A; Sebe, Attila

    2016-05-01

    Endothelial-mesenchymal transition (EndMT) is an important mechanism during organ development and in certain pathological conditions. For example, EndMT contributes to myofibroblast formation during organ fibrosis, and it has been identified as an important source of cancer-associated fibroblasts, facilitating tumor progression. Recently, EndMT was proposed to modulate endothelial function during intravasation and extravasation of metastatic tumor cells. Evidence suggests that endothelial cells are not passive actors during transendothelial migration (TEM) of cancer cells, as there are profound changes in endothelial junctional protein expression, signaling, permeability, and contractility. This review describes these alterations in endothelial characteristics during TEM of metastatic tumor cells and discusses them in the context of EndMT. EndMT could play an important role during metastatic intravasation and extravasation, a novel hypothesis that may lead to new therapeutic approaches to tackle metastatic disease. PMID:26993222

  9. Recovery of Corneal Endothelial Cells from Periphery after Injury.

    Sang Ouk Choi

    Full Text Available Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.To investigate the recovery process of corneal endothelial cells (CECs from corneal endothelial injury.Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group. Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.

  10. Cytotoxicity of proparacaine to human corneal endothelial cells in vitro.

    Wen, Qian; Fan, Tingjun; Bai, Suran; Sui, Yunlong

    2015-08-01

    Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic. PMID:26165639

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

    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.

  12. Modulation of human vascular endothelial cell behaviors by nanotopographic cues.

    Liliensiek, Sara J; Wood, Joshua A; Yong, Jiang; Auerbach, Robert; Nealey, Paul F; Murphy, Christopher J

    2010-07-01

    Basement membranes possess a complex three-dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges > or =800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell-types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated. PMID:20400175

  13. Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience

    Thuret, G; Manissolle, C; Acquart, S.; Petit, J-C Le; Maugery, J; Campos-Guyotat, L; Doughty, M J; Gain, P

    2003-01-01

    Aim: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant.

  14. Improved endothelialization of titanium vascular implants by extracellular matrix secreted from endothelial cells.

    Tu, Qiufen; Zhao, Yuancong; Xue, Xiaoqing; Wang, Jin; Huang, Nan

    2010-12-01

    A variety of metals have been widely used in construction of cardiovascular implants (CVIs), such as artificial heart valves, ventricular pumps, and vascular stents. Although great effects have been put into rigorous anticoagulation, late thrombosis still occurred due to inferior blood and cell compatibility. Natural endothelium is popularly regarded as the only substance that has long-term anticoagulant ability. So, establishment of a compact endothelial cell (EC) monolayer on CVIs surface is a guarantee for their long-term potency. In the work described here, titanium (Ti) disks were coated with extracellular matrix (ECM) directly secreted by human umbilical vein endothelial cells (HUVECs), so as to help ECs proliferate and migrate and to improve their endothelialization in vivo. Deposition of ECM on Ti disks was detected by immunofluorescence microscopy, diffuse reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The surface topography and wettability of the Ti disks significantly changed after ECM deposition. Most importantly, it was found that ECM deposition inhibited platelet adhesion, stimulated EC proliferation, increased EC migration speed in vitro, and eventually accelerated the re-cellularization speed of Ti disks in vivo. These important results render it reasonable and feasible to modify CVIs with ECM secreted from ECs for improving their long-term potency. PMID:20666613

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

    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 (PEDV compared to ACh-induced EDV in aortae maintained in normal glucose (NG, 11 mM) Krebs. The reduction of EDV was partially reversed following a 3-h exposure to 50 μM metformin; 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 (P<0.05) reversed the HG-induced reduction in phosphorylation of both eNOS and Akt; however, no changes were detected for phosphorylation of AMPK or the expression of SIRT1. Our data indicate that a 3-h exposure to metformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt. PMID:26467186

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

    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.

  17. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    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+ 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

  18. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    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.

  19. Biomechanical changes in endothelial cells result from an inflammatory response

    Vaitkus, Janina; Stroka, Kimberly; Aranda-Espinoza, Helim

    2012-02-01

    During periods of infection and disease, the immune system induces the release of TNF-α, an inflammatory cytokine, from a variety of cell types, such as macrophages. TNF-α, while circulating in the vasculature, binds to the apical surface of endothelial cells and causes a wide range of biological and mechanical changes to the endothelium. While the biological changes have been widely studied, the biomechanical aspects have been largely unexplored. Here, we investigated the biomechanical changes of the endothelium as a function of TNF-α treatment. First, we studied the traction forces applied by the endothelium, an effect that is much less studied than others. Through the use of traction force microscopy, we found that TNF-α causes an increase in traction forces applied by the endothelial cells as compared to non-treated cells. Then, we investigated cell morphology, cell mechanics, migration, and cytoskeletal dynamics. We found that in addition to increasing applied traction forces, TNF-α causes an increase in cell area and aspect ratio on average, as well as a shift in the organization of F-actin filaments within the cell. Combining these findings together, our results show that an inflammatory response heavily impacts the morphology, cell mechanics, migration, cytoskeletal dynamics, and applied traction forces of endothelial cells.

  20. Intracellular pathways of insulin transport across vascular endothelial cells

    Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C

  1. Functional impairment of endothelial cells by the antimycotic amphotericin B.

    Pelzmann, Brigitte; Di Giuro, Cristiana M L; Zorn-Pauly, Klaus; Rossmann, Christine; Hallström, Seth; Groschner, Klaus; Fameli, Nicola

    2016-03-25

    We set out to determine the membrane potential (Vm) of the endothelial cell line EA.hy926 and its sensitivity to the antimycotic amphotericin B (AmB), a commonly used antifungal component in cell culture media. We measured the endothelial Vm under various experimental conditions by patch clamp technique and found that Vm of AmB-treated cells is (-12.1 ± 9.3) mV, while in AmB-untreated (control) cells it is (-57.1 ± 4.1) mV. In AmB-free extracellular solutions, Vm recovered toward control levels and this gain in Vm rapidly dissipated upon re-addition of AmB, demonstrating a rapid and reversible effect of AmB on endothelial Vm. The consequences of AmB dependent alterations in endothelial transmembrane potential were tested at the levels of Ca(2+) signaling, of nucleotide concentrations, and energy metabolism. In AmB-treated cells we found substantially reduced Ca(2+) entry (to about 60% of that in control cells) in response to histamine induced endoplasmic reticulum (ER) Ca(2+) depletion, and diminished the ATP-to-ADP ratio (by >30%). Our data demonstrate a marked and experimentally relevant dependence of basic functional parameters of cultured endothelial cells on the presence of the ionophoric antimycotic AmB. The profound and reversible effects of the widely used culture media component AmB need careful consideration when interpreting experimental data obtained under respective culture conditions. PMID:26902113

  2. Induction of lymphatic endothelial cell differentiation in embryoid bodies

    Liersch, Ruediger; Nay, Filip; Lu, Lingge; Detmar, Michael

    2006-01-01

    The molecular mechanisms that regulate the formation of the lymphatic vascular system remain poorly characterized. Whereas studies in embryonic stem (ES) cells have provided major new insights into the mechanisms of blood vessel formation, the development of lymphatic endothelium has not been previously observed. We established embryoid bodies (EBs) from murine ES cells in the presence or absence of lymphangiogenic growth factors. We found that lymphatic endothelial cells develop at day 18 af...

  3. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

  4. Apoptosis and calcification of vascular endothelial cell under hyperhomocysteinemia.

    Fang, Kuaifa; Chen, Zhujun; Liu, Meng; Peng, Jian; Wu, Pingsheng

    2015-01-01

    In recent years, it is found that increase in Hcy level in blood can directly or indirectly cause vascular endothelial cell injury and induce vascular calcification. However, the mechanism of vascular endothelial cell injury and vascular calcification has not been studied thoroughly. This paper carried out experiment for research aiming at discussing the effect and action mechanism of Hhcy on endothelial cells and vascular calcification. Firstly, human umbilical vein endothelial cells (HUVECs) were cultured and then intervened by Hcy of different concentrations (0, 0.01, 0.1, 1.0, 3.0, 5.0 mmol/L) and at different action time (3, 6, 12, 24 h). Then apoptosis rate and reactive oxygen were detected by flow cytometry. At the same time, the model for the culture of rat vascular calcification was set up and induced into Hhcy so as to detect the total plasma Hcy level and judge vascular calcification degree. The results showed that with the increase in Hcy concentration and extension of action period, the apoptosis rate and generation of reactive oxygen of HUVECs all significantly increased, and the differences were all statistically significant (P animal calcification model, mass of black particle deposition was seen after Von Kossa staining of rat vessels in calcification group. Compared with the control group, the vascular calcium content, alkaline phosphatase activity and osteocalcin content in calcification group all increased (P benefits on clinical prevention works. PMID:25476479

  5. High glucose augments stress-induced apoptosis in endothelial cells

    Wenwen Zhong; Yang Liu; Hui Tian

    2009-01-01

    Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

  6. Tubulated bodies in teleost (Pimelodus maculatus) endothelial cells.

    Ferri, S; Sesso, A

    1983-01-01

    In the present report tubulated granules are described for the first time in a freshwater teleost (Pimelodus maculatus) endothelial cells. Some ultrastructural characteristics as well as the localization and distribution suggest that tubulated bodies represent the teleost counterpart of the Weibel-Palade bodies described in other animal classes. PMID:6639042

  7. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  8. Virulent Treponema pallidum activates human vascular endothelial cells.

    Riley, B S; Oppenheimer-Marks, N; Hansen, E J; Radolf, J D; Norgard, M V

    1992-03-01

    Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium. PMID:1347056

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    Full Text Available Oth.CDV.10.AllAg.Brachiocephalic_endothelial_cells hg19 TFs and others Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.10.AllAg.Brachiocephalic_endothelial_cells.bed ...

  20. File list: Oth.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Oth.CDV.05.AllAg.Brachiocephalic_endothelial_cells hg19 TFs and others Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.05.AllAg.Brachiocephalic_endothelial_cells.bed ...

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

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

    Full Text Available DNS.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 DNase-seq Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

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

    Full Text Available His.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 Histone Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

  4. File list: Unc.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Unc.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 Unclassified Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

  5. File list: Pol.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Pol.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 RNA polymerase Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

  6. File list: ALL.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.05.AllAg.Brachiocephalic_endothelial_cells hg19 All antigens Cardiovascular Brachioceph...alic endothelial cells DRX014747 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.CDV.05.AllAg.Brachiocephalic_endothelial_cells.bed ...

  7. File list: Unc.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Unc.CDV.10.AllAg.Brachiocephalic_endothelial_cells hg19 Unclassified Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.CDV.10.AllAg.Brachiocephalic_endothelial_cells.bed ...

  8. File list: Oth.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Oth.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 TFs and others Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

  9. File list: Unc.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available Unc.CDV.05.AllAg.Brachiocephalic_endothelial_cells hg19 Unclassified Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.CDV.05.AllAg.Brachiocephalic_endothelial_cells.bed ...

  10. File list: His.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available His.CDV.10.AllAg.Brachiocephalic_endothelial_cells hg19 Histone Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.10.AllAg.Brachiocephalic_endothelial_cells.bed ...

  11. File list: His.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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

  12. File list: His.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available His.CDV.05.AllAg.Brachiocephalic_endothelial_cells hg19 Histone Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.05.AllAg.Brachiocephalic_endothelial_cells.bed ...

  13. File list: Unc.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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

  14. Endothelial cell senescence is associated with disrupted cell-cell junctions and increased monolayer permeability

    Krouwer Vincent J D

    2012-08-01

    Full Text Available Abstract Background Cellular senescence is associated with cellular dysfunction and has been shown to occur in vivo in age-related cardiovascular diseases such as atherosclerosis. Atherogenesis is accompanied by intimal accumulation of LDL and increased extravasation of monocytes towards accumulated and oxidized LDL, suggesting an affected barrier function of vascular endothelial cells. Our objective was to study the effect of cellular senescence on the barrier function of non-senescent endothelial cells. Methods Human umbilical vein endothelial cells were cultured until senescence. Senescent cells were compared with non-senescent cells and with co-cultures of non-senescent and senescent cells. Adherens junctions and tight junctions were studied. To assess the barrier function of various monolayers, assays to measure permeability for Lucifer Yellow (LY and horseradish peroxidase (PO were performed. Results The barrier function of monolayers comprising of senescent cells was compromised and coincided with a change in the distribution of junction proteins and a down-regulation of occludin and claudin-5 expression. Furthermore, a decreased expression of occludin and claudin-5 was observed in co-cultures of non-senescent and senescent cells, not only between senescent cells but also along the entire periphery of non-senescent cells lining a senescent cell. Conclusions Our findings show that the presence of senescent endothelial cells in a non-senescent monolayer disrupts tight junction morphology of surrounding young cells and increases the permeability of the monolayer for LY and PO.

  15. Cilengitide inhibits proliferation and differentiation of human endothelial progenitor cells in vitro

    Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of pre-existing vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133+ progenitor cells in vitro. αvβ3-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of αvβ3- and αvβ5-integrins in our in vitro system. We could show expression of αvβ3-integrin on 60 ± 9% of non-adherent endothelial progenitors and on 91 ± 7% of differentiated endothelial cells. αvβ3-integrin was absent on CD133+ hematopoietic stem cells. Cilengitide inhibited proliferation of CD133+ cells in a dose-dependent manner. The development of adherent endothelial cells from expanded CD133+ cells was reduced even stronger by Cilengitide underlining its effect on integrin mediated cell adhesion. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was decreased by Cilengitide. In summary, Cilengitide inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects

  16. Endothelial cell markers reflecting endothelial cell dysfunction in patients with mixed connective tissue disease

    Soltész Pál (1961-) (belgyógyász, kardiológus); Bereczki Dániel (1960-) (neurológus); Szodoray Péter (1973-) (belgyógyász, orvos); Magyar Mária Tünde (1970-) (neurológus); Dér Henrietta (1977-) (orvos); Csípő István (1953-) (vegyész); Hajas Ágota Helga (1985-) (orvos); Paragh György (1953-) (belgyógyász, kardiológus, endokrinológus, lipidológus, sürgősségi orvostani szakorvos, belgyógyászati angiológiai minősített orvos); Szegedi Gyula (1936-2013) (belgyógyász, immunológus); Bodolay Edit (1950-) (belgyógyász, allergológus és klinikai immunológus)

    2010-01-01

    Introduction The aim of the present study was to investigate the association between cardiovascular risk factors and endothelial dysfunction in patients with mixed connective tissue disease (MCTD) and to determine which biomarkers are associated with atherosclerotic complications, such as cardiovascular disease. Methods Fifty MCTD patients and 38 healthy age-matched and sex-matched controls were enrolled in this study. In order to describe endothelial dysfunction, we assessed flow-mediated di...

  17. Endothelial induced EMT in breast epithelial cells with stem cell properties

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla;

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether...... endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of...... keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D...

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

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

    1992-01-01

    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, O

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

    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.

  20. Endothelial cell pseudopods and angiogenesis of breast cancer tumors

    Sun LuZhe; Short Nicholas; Cameron Ivan L; Hardman W Elaine

    2005-01-01

    Abstract Background A neoplastic tumor cannot grow beyond a millimeter or so in diameter without recruitment of endothelial cells and new blood vessels to supply nutrition and oxygen for tumor cell survival. This study was designed to investigate formation of new blood vessels within a human growing breast cancer tumor model (MDA MB231 in mammary fat pad of nude female mouse). Once the tumor grew to 35 mm3, it developed a well-vascularized capsule. Histological sections of tumors greater than...

  1. Adiponectin promotes endothelial progenitor cell number and function

    Shibata, Rei; Skurk, Carsten; Ouchi, Noriyuki; Galasso, Gennaro; Kondo, Kazuhisa; Ohashi, Taiki; Shimano, Masayuki; Kihara, Shinji; Murohara, Toyoaki; Walsh, Kenneth

    2008-01-01

    Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells ...

  2. Endothelial cells and cathepsins: Biochemical and biomechanical regulation.

    Platt, Manu O; Shockey, W Andrew

    2016-03-01

    Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention. PMID:26458976

  3. The effect of nicotine on aortic endothelial cell turnover

    Endothelial injury and increased mitotic activity are early features in the pathogenesis of intimal thickening in arteries. This study examines the effect of systemic nicotine on mitotic activity in endothelial cells. Nine adult mice were given nicotine in their drinking water for 5 weeks. The dose (5 mg/kg body wt/day) was equivalent to a human smoking 50-100 cigarettes/day. A group of 8 similar mice, not exposed to nicotine, was the control. At the end of the exposure period all mice were injected with (3H)thymidine (1uCi/g body wt) and were killed 24 h later. After perfusion fixation, en-face preparations of aortic endothelium were processed for autoradiography. In nicotine-affected endothelium 0.46.+-0.11% (SEM) of cells were labeled, which was significantly higher (P<0.01) than in controls (0.14+-0.06). However, there was no difference in cell density between the groups. On this evidence it was concluded that the rate of cell loss, or cell turnover, was greater in nicotine-affected endothelium. Because other studies have shown that increased mitotic acitivity and cell loss are established features of endothelial injury, the present findings provide evidence in support of the hypothesis that nicotine contributes to the pathogenesis of arterial disease in smokers. (author)

  4. Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

    Zhu Minmin

    2013-01-01

    Full Text Available Abstract Background Hyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB, up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs. Methods Protein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα, protein kinase Cβ2 (PKCβ2, and phosphorylation of PKCβ2 (Ser660 were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.- accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution. Results High glucose induced the expression of endothelial selectin (E-selectin, intercellular adhesion molecule 1 (ICAM-1, vascular cell adhesion molecule 1 (VCAM-1, and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion. Conclusion Propofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression

  5. Angiotensin-converting enzyme kinetics in an endothelial cell column

    The kinetics of saturable endothelial metabolic functions have been assessed in vivo by transient (indicator-dilution) measurements and in culture by steady-state measurements, but comparisons between the two are difficult. Therefore, we used indicator-dilution methods to assess the kinetics of angiotensin-converting enzyme (ACE) activity in cultured endothelium. Bovine fetal aortic endothelial cells were grown to confluence on microcarrier beads. Cell-covered beads were poured into polypropylene columns and perfused with serum-free culture medium. Six injections, containing [3H]benzol-Phe-Ala-Pro [( 3H]BPAP, an ACE substrate) and varying amounts of unlabeled BPAP, were applied to each column and effluent was collected in serial samples. The apparent kinetics of BPAP metabolism were determined by four models used previously to determine pulmonary endothelial ACE kinetics in vivo, the most useful model incorporating transit time heterogeneity. The Km averaged 5 microM, which is close to values determined previously in vivo and in vitro. The Amax (Vmax.reaction volume) and Amax/Km averaged 6 nmol/min and 1.5 ml/min, respectively, which are lower than estimates in vivo. In conclusion, we have developed a new method for investigating saturable metabolic activity in cultured endothelium, which after further exploration should also enable better comparisons of endothelial metabolic functions in vivo and in culture

  6. XIAP reverses various functional activities of FRNK in endothelial cells

    Highlights: ► FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. ► XIAP binds the FRNK domain of FAK. ► XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. ► XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  7. Cancer stem cell-vascular endothelial cell interactions in glioblastoma.

    Sharma, Aman; Shiras, Anjali

    2016-05-01

    Glioblastoma (GBM), a higher grade glial tumor, is highly aggressive, therapy resistant and often shows poor patient prognosis due to frequent recurrence. These features of GBM are attributed to presence of a significantly smaller proportion of glioma stem cells (GSCs) that are endowed with self-renewal ability, multi-potent nature and show resistance to therapy in patients. GSCs preferably take shelter close to tumor vasculature due to paracrine need of soluble factors secreted by endothelial cells (ECs) of vasculature. The physical proximity of GSCs to ECs creates a localized perivascular niche where mutual GSC-EC interactions regulate GSC stemness, migration, therapy resistance, and cellular kinetics during tumor growth. Together, perivascular niche presents a therapeutically targetable tumor structure for clinical management of GBM. Thus, understanding cellular and non-cellular components in perivascular niche is vital for designing in vitro and in vivo GBM tumor models. Here, we discuss the components and structure of tumor vascular niche and its impact on tumor progression. PMID:26692486

  8. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  9. Nitrative Stress Participates in Endothelial Progenitor Cell Injury in Hyperhomocysteinemia.

    Dong, Yu; Sun, Qi; Liu, Teng; Wang, Huanyuan; Jiao, Kun; Xu, Jiahui; Liu, Xin; Liu, Huirong; Wang, Wen

    2016-01-01

    In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that

  10. Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

    Lu, Xiaoyan; Chen, Dong; Liu, Zhiping; Li, Chaoyang; Liu, Ying; Zhou, Jin; Wan, Pengxia; Mou, Yong-Gao; Wang, Zhichong

    2010-01-01

    Purpose To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Methods Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcr...

  11. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals

    Yoder, Mervin C.; Mead, Laura E.; Prater, Daniel; Krier, Theresa R.; Mroueh, Karim N.; Li, Fang; Krasich, Rachel; Temm, Constance J.; Prchal, Josef T.; Ingram, David A.

    2007-01-01

    The limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies “endothelial cell colony-forming units” (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast...

  12. Wall shear stress effects on endothelial-endothelial and endothelial-smooth muscle cell interactions in tissue engineered models of the vascular wall.

    Dalit Shav

    Full Text Available Vascular functions are affected by wall shear stresses (WSS applied on the endothelial cells (EC, as well as by the interactions of the EC with the adjacent smooth muscle cells (SMC. The present study was designed to investigate the effects of WSS on the endothelial interactions with its surroundings. For this purpose we developed and constructed two co-culture models of EC and SMC, and compared their response to that of a single monolayer of cultured EC. In one co-culture model the EC were cultured on the SMC, whereas in the other model the EC and SMC were cultured on the opposite sides of a membrane. We studied EC-matrix interactions through focal adhesion kinase morphology, EC-EC interactions through VE-Cadherin expression and morphology, and EC-SMC interactions through the expression of Cx43 and Cx37. In the absence of WSS the SMC presence reduced EC-EC connectivity but produced EC-SMC connections using both connexins. The exposure to WSS produced discontinuity in the EC-EC connections, with a weaker effect in the co-culture models. In the EC monolayer, WSS exposure (12 and 4 dyne/cm(2 for 30 min increased the EC-EC interaction using both connexins. WSS exposure of 12 dyne/cm(2 did not affect the EC-SMC interactions, whereas WSS of 4 dyne/cm(2 elevated the amount of Cx43 and reduced the amount of Cx37, with a different magnitude between the models. The reduced endothelium connectivity suggests that the presence of SMC reduces the sealing properties of the endothelium, showing a more inflammatory phenotype while the distance between the two cell types reduced their interactions. These results demonstrate that EC-SMC interactions affect EC phenotype and change the EC response to WSS. Furthermore, the interactions formed between the EC and SMC demonstrate that the 1-side model can simulate better the arterioles, while the 2-side model provides better simulation of larger arteries.

  13. Junctional communication is induced in migrating capillary endothelial cells.

    Pepper, M S; Spray, D C; Chanson, M; Montesano, R; Orci, L; Meda, P

    1989-12-01

    Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration. PMID:2592412

  14. The expression of ADAMTS13 in human microvascular endothelial cells.

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells. PMID:26366828

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

    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

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

    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.

  17. Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene.

    Abrass, C K; Berfield, A K; Ryan, M C; Carter, W G; Hansen, K M

    2006-09-01

    Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus. PMID:16850021

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

    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. PMID:26863518

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

    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.

  20. Enhancement of tumor necrosis factor-induced endothelial cell injury by cycloheximide

    Tumor necrosis factor (TNF), a potent polypeptide mediator released by activated monocytes and macrophages, has a number of proinflammatory effects on endothelial cells. TNF is cytotoxic to tumor cells in vivo and in vitro, but TNF-induced toxicity to endothelial cells is less well established. We now report that cycloheximide (CHX), an inhibitor of protein synthesis, renders endothelial cells highly susceptible to TNF-induced lysis. TNF alone did not change the overall rate of protein synthesis by endothelial cells, whereas the addition of CHX completely abolished protein synthesis. Endothelial cells incubated in TNF alone in high concentrations (up to 1,000 U/ml) showed minimal rounding up and release of 51Cr. Likewise, CHX alone (5 micrograms/ml) had no significant effect on endothelial cell morphology and release of 51Cr. However, incubation of endothelial cells in both CHX and TNF caused injury in a dose-dependent manner. Morphological evidence of cell retraction, rounding, and detachment began within 2 h, but specific 51Cr release did not begin to rise until after 4 h. These changes were not observed when endothelial cells were incubated with TNF/CHX at 4 degrees C. The combination of TNF/CHX was lethal to all endothelial cells tested (bovine pulmonary artery, human umbilical vein, and human aorta), with human aortic cells showing the most pronounced changes. We conclude that healthy endothelial cells are resistant to TNF-induced lysis, but inhibition of their ability to make protein renders them highly susceptible

  1. Lymphatic Endothelial Differentiation in Pulmonary Lymphangioleiomyomatosis Cells

    Davis, Jennifer M.; Hyjek, Elizabeth; Husain, Aliya N.; Shen, Le; Jones, Jennifer; Schuger, Lucia A.

    2013-01-01

    Pulmonary lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm affecting almost exclusively women of childbearing age. LAM belongs to the family of perivascular epithelioid cell tumors, characterized by spindle and epithelioid cells with smooth muscle and melanocytic differentiation. LAM cells infiltrate the lungs, producing multiple, bilateral lesions rich in lymphatic channels and forming cysts, leading to respiratory insufficiency. Here we used antibodies against four lymphatic end...

  2. Volume changes of human endothelial cells induced by photodynamic treatment

    Leunig, Andreas; Staub, Frank; Plesnila, Nick; Peters, Jurgen; Feyh, Jens; Gutmann, Ralph; Goetz, Alwin E.

    1996-01-01

    Photodynamic therapy (PDT) has shown promising results in treatment of malignant tumors. However, the mechanisms leading to tumor destruction during PDT are still not completely understood. In addition to effects on the microcirculation, damage to cellular structures has been observed following exposure of cells to PDT. A phenomenon preceding these events might possibly be cell swelling. We therefore studied the influence of treatment with Photofrin (PF) and laser light on volume changes and cell viability of endothelial cells. Endothelial cells were obtained from human umbilical cord veins (HUVEC) by an adaption of the method of Maruyama (1963). After subcultivation the cells were harvested and transferred as a cell suspension into a specially designed incubation chamber. Cells received either PF in concentrations of 1.5 or 3.0 (mu) g/ml and laser illumination (630 nm; 40 mW/cm2, 4 Joule), PF alone, or laser treatment only. Following start of PF incubation and after phototreatment cell samples were taken for volume measurements using flow cytometry and for studies of cellular morphology using scanning electron microscopy. Simultaneously, cell viability was monitored by the trypan blue exclusion test and colorimetric MTT assay. (abstract truncated)

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

    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...

  4. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Shumei Man

    2008-02-01

    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.

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

    Masamitsu Kanada

    2014-12-01

    Full Text Available 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. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

  6. Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis.

    Castets, Marie; Coissieux, Marie-May; Delloye-Bourgeois, Céline; Bernard, Laure; Delcros, Jean-Guy; Bernet, Agnès; Laudet, Vincent; Mehlen, Patrick

    2009-04-01

    Netrin-1 was recently proposed to play an important role in embryonic and pathological angiogenesis. However, data reported led to the apparently contradictory conclusions that netrin-1 is either a pro- or an antiangiogenic factor. Here, we reconcile these opposing observations by demonstrating that netrin-1 acts as a survival factor for endothelial cells, blocking the proapoptotic effect of the dependence receptor UNC5B and its downstream death signaling effector, the serine/threonine kinase DAPK. The netrin-1 effect on blood vessel development is mimicked by caspase inhibitors in ex vivo assays, and the inhibition of caspase activity, the silencing of the UNC5B receptor, and the silencing of DAPK are each sufficient to rescue the vascular sprouting defects induced by netrin-1 silencing in zebrafish. Thus, the proapoptotic effect of unbound UNC5B and the survival effect of netrin-1 on endothelial cells finely tune the angiogenic process. PMID:19386270

  7. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C; Urich, Eduard; Heckel, Tobias; O'Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H C; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L; Gerszten, Robert E; Graf, Martin; Iacone, Roberto; Cowan, Chad A

    2015-08-01

    The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

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

    Barkhordari, A.; CJP Jones; RW Stoddart; SF McClure; McClure, J; S Rahimi Moghadam

    2014-01-01

    [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 pan...

  9. Endothelial progenitor cells regenerate infracted myocardium with neovascularisation development ☆

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

    2013-01-01

    We achieved possibility of isolation, characterization human umbilical cord blood endothelial progenitor cells (EPCs), examination potency of EPCs to form new blood vessels and differentiation into cardiomyoctes in canines with acute myocardial infarction (AMI). EPCs were separated and cultured from umbilical cord blood. Their phenotypes were confirmed by uptake of double stains dioctadecyl tetramethylindocarbocyanine-labeled acetylated LDL and FITC-labeled Ulex europaeus agglutinin 1 (DILDL-...

  10. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

    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

  11. Norepinephrine Stimulates Mobilization of Endothelial Progenitor Cells after Limb Ischemia

    Jiang, Qijun; Ding, Shifang; Wu, Jianxiang; Liu, Xing; Wu, Zonggui

    2014-01-01

    Objective During several pathological processes such as cancer progression, thermal injury, wound healing and hindlimb ischemia, the mobilization of endothelial progenitor cells (EPCs) mobilization was enhanced with an increase of sympathetic nerve activity and norepinephrine (NE) secretion, yet the cellular and molecular mechanisms involved in the effects of NE on EPCs has less been investigated. Methods and Results EPCs from BMs, peripheral circulation and spleens, the VEGF concentration in...

  12. Modulation of Human Vascular Endothelial Cell Behaviors by Nanotopographic Cues

    Liliensiek, S.J.; Wood, J.A.; Yong, J.; Auerbach, R.; Nealey, P.F.; Murphy, C. J.

    2010-01-01

    Basement membranes possess a complex three dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimeti...

  13. Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells

    Rárová, L.; Zahler, S.; Liebl, J.; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav

    2012-01-01

    Roč. 77, č. 13 (2012), s. 1502-1509. ISSN 0039-128X R&D Projects: GA MŠk(CZ) LC06077 Grant ostatní: GA MŠk(CZ) ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : Angiogenesis * Human umbilical vein endothelial cells * Migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.803, year: 2012

  14. Double suicide genes selectively kill human umbilical vein endothelial cells

    Liu Lunxu; Wang Yanping; Mei Longyong; Jia Weiguo; Che Guowei

    2011-01-01

    Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simplex virus-thymidine kinase (TK) gene were cloned using polymerase chain reaction (PCR). Plasmid pKDR-CDglyTK was constructed wi...

  15. Atorvastatin Affects Several Angiogenic Mediators in Human Endothelial Cells

    Dulak, Jozef; Loboda, Agnieszka; Jazwa, Agnieszka; Zagorska, Anna; Dörler, Jacob; Alber, Hannes; Dichtl, Wolfgang; Weidinger, Franz; Frick, Matthias; Jozkowicz, Alicja

    2005-01-01

    The pleiotropic effects of statins, inhibitors of 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase, have been recently extended to the modulation of angiogenesis. Here, to get more insight into the statins action, the authors have investigated the effect of atorvastatin on the expression of several angiogenic and inflammatory genes in human umbilical endothelial cells (HUVECs). Atorvastatin was proangiogenic at the dose of 10 nM, and antiangiogenic at the concentrations of 1 to 10 μM...

  16. Effect of Intracameral Use of Dexamethasone on Corneal Endothelial Cells

    Objective: To evaluate the effect of intracameral dexamethasone on corneal endothelium. Study Design: Quasi experimental study. Place and Duration of Study: Layton Rehmatulla Benevolent Trust Eye Hospital, Lahore, from May 2011 to January 2012. Methodology: Study subjects were adults of either gender with senile cataract who underwent phacoemulsification. They were divided in two groups, each had 110 patients. Group-A received subconjunctival injection of dexamethasone (2 mg/0.5 ml) at the end of surgery while group-B received intracameral injection of dexamethasone (0.4 mg/0.1 ml) at the end of surgery. Endothelial cell count was performed by specular microscopy pre-operatively and postoperatively at first week, first month and three months. Outcome measures included changes in endothelial cell count. Results were compared using t-test for means. Results: There were 55 (50%) males and 55 (50%) females in group-A and 44 (40%) males and 66 (60%) females in group-B. In group-A, there were 66 (60%) right and 44 (40%) left eyes while group-B had 62 (56.36%) right and 48 (43.63%) left eyes. Mean age in group-A was 55.17 A +- 5.93 years and 54.87 A +- 5.55 years in group-B. Mean phacoemulsification time in group-A was 1.92 A +- 0.63 minutes and 1.82 A +- 0.54 minutes in group-B. After 3 months, in group-A, there was 7.55 A +- 1.19% endothelial cell loss while in group-B, there was 7.63 A +- 1.10% endothelial cell loss. The difference between the two groups was not statistically significant (p=0.614). Conclusion: Use of intracameral dexamethasone at the end of cataract surgery is safe for corneal endothelium. (author)

  17. Ultrasound fails to induce proliferation of human brain and mouse endothelial cell lines

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    Both in vitro and in vivo studies suggest that ultrasound (US) is capable of inducing angiogenesis. There is no information, however, on whether ultrasound can induce proliferation of brain endothelial cells. We therefore explored the angiogenic potential of ultrasound on a novel immortalised human brain endothelial cell line (hCMEC/D3) and on mouse brain microvascular endothelial cells (bEND3). Ultrasound failed to enhance cell proliferation in both cell lines at all acoustic pressures studied. Endothelial cell damage occurred at 0.24 MPa with significantly slower proliferation. Cells growing in Opticell{trade mark, serif} dishes did not show damage or reduced proliferation at these pressures.

  18. Effect of endothelial progenitor cells in neovascularization and their application in tumor therapy

    DONG Fang; HA Xiao-qin

    2010-01-01

    Objective To review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors.Data sources The data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells".Study selection Articles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected.Results Endothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notchsignal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer.Conclusions Endothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

  19. Shear-Induced Nitric Oxide Production by Endothelial Cells.

    Sriram, Krishna; Laughlin, Justin G; Rangamani, Padmini; Tartakovsky, Daniel M

    2016-07-12

    We present a biochemical model of the wall shear stress-induced activation of endothelial nitric oxide synthase (eNOS) in an endothelial cell. The model includes three key mechanotransducers: mechanosensing ion channels, integrins, and G protein-coupled receptors. The reaction cascade consists of two interconnected parts. The first is rapid activation of calcium, which results in formation of calcium-calmodulin complexes, followed by recruitment of eNOS from caveolae. The second is phosphorylation of eNOS by protein kinases PKC and AKT. The model also includes a negative feedback loop due to inhibition of calcium influx into the cell by cyclic guanosine monophosphate (cGMP). In this feedback, increased nitric oxide (NO) levels cause an increase in cGMP levels, so that cGMP inhibition of calcium influx can limit NO production. The model was used to predict the dynamics of NO production by an endothelial cell subjected to a step increase of wall shear stress from zero to a finite physiologically relevant value. Among several experimentally observed features, the model predicts a highly nonlinear, biphasic transient behavior of eNOS activation and NO production: a rapid initial activation due to the very rapid influx of calcium into the cytosol (occurring within 1-5 min) is followed by a sustained period of activation due to protein kinases. PMID:27410748

  20. V-ATPase regulates communication between microvascular endothelial cells and metastatic cells.

    Sennoune, S R; Arutunyan, A; del Rosario, C; Castro-Marin, R; Hussain, F; Martinez-Zaguilan, R

    2014-01-01

    To metastasize distant organs, tumor cells and endothelial cells lining the blood vessels must crosstalk. The nature of this communication that allows metastatic cells to intravasate and travel through the circulation and to extravasate to colonize different organs is poorly understood. In this study, we evaluated one of the first steps in this process—the proximity and physical interaction of endothelial and metastatic cells. To do this, we developed a cell separator chamber that allows endothelial and metastatic cells to grow side by side. We have shown in our previous studies that V-ATPases at the cell surface (pmV-ATPase) are involved in angiogenesis and metastasis. Therefore, we hypothesized that the physical proximity/interaction between endothelial and metastatic cells expressing pmV-ATPase will increase its activity in both cell types, and such activity in turn will increase pmV-ATPase expression on the membranes of both cell types. To determine pmV-ATPase activity we measured the proton fluxes (JH+) across the cell membrane. Our data indicated that interaction between endothelial and metastatic cells elicited a significant increase of JH+ via pmV-ATPase in both cell types. Bafilomycin, a V-ATPase inhibitor, significantly decrease JH+. In contrast, JH+ of the non-metastatic cells were not affected by the endothelial cells and vice-versa. Altogether, our data reveal that one of the early consequences of endothelial and metastatic cell interaction is an increase in pmV-ATPase that helps to acidify the extracellular medium and favors protease activity. These data emphasize the significance of the acidic tumor microenvironment enhancing a metastatic and invasive phenotype. PMID:24606724

  1. Differential adhesion of tumor cells to capillary endothelial cells in vitro.

    Alby, L; Auerbach, R

    1984-01-01

    Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular ...

  2. In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

    Zaniboni, Andrea; Bernardini, Chiara; Bertocchi, Martina; Zannoni, Augusta; Bianchi, Francesca; Avallone, Giancarlo; Mangano, Chiara; Sarli, Giuseppe; Calzà, Laura; Bacci, Maria Laura; Forni, Monica

    2015-09-01

    Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes. PMID:26135800

  3. Small vulvar squamous cell carcinomas and adjacent tissues. A morphologic study

    Poulsen, Hemming; Junge, Jette; Vyberg, Mogens;

    2003-01-01

    Vulvar squamous cell carcinomas are of different subtypes and degrees of differentiation, and may be associated with adjacent lichen sclerosus and/or varying degrees of dysplasia. The aim of this investigation was to study small carcinomas with a diameter of less than 2 cm in order to find a poss...

  4. Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention.

    Uthamaraj, Susheil; Tefft, Brandon J; Hlinomaz, Ota; Sandhu, Gurpreet S; Dragomir-Daescu, Dan

    2015-01-01

    Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing. PMID:26436434

  5. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    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.

  6. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    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

  7. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    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.

  8. Endothelial progenitor cells give rise to pro-angiogenic smooth muscle-like progeny

    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 circ

  9. Endothelial Cell Injury Caused by Candida albicans Is Dependent on Iron

    Fratti, Rutilio A.; Belanger, Paul H.; Ghannoum, Mahmoud A.; Edwards, John E.; Filler, Scott G.

    1998-01-01

    Although it is known that Candida albicans causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in Candida-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated ...

  10. Bile acids increase intracellular Ca2+ concentration and nitric oxide production in vascular endothelial cells

    Nakajima, Toshiaki; Okuda, Yukichi; Chisaki, Keigo; Shin, Wee-Soo; Iwasawa, Kuniaki; Morita, Toshihiro; Matsumoto, Akihiro; Suzuki, Jun-ichi; Suzuki, Seizi; Yamada, Nobuhiro; Toyo-Oka, Teruhiko; Nagai, Ryozo; Omata, Masao

    2000-01-01

    The effects of bile acids on intracellular Ca2+ concentration [Ca2+]i and nitric oxide production were investigated in vascular endothelial cells.Whole-cell patch clamp techniques and fluorescence measurements of [Ca2+]i were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO2−.Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca2+]i concent...

  11. Angiogenic potential modulation of human endothelial progenitor cells by vascular plasmatic biomarkers

    d'Audigier, Clément,

    2013-01-01

    Rationale: The pro-angiogenic capacities of endothelial progenitor cells are now well established, and their involvement in neovascularization events in adults has stimulated the research in the field of angiogenic therapy based on transplant of these cells. Current data converge towards the notion of two cell types with endothelial phenotype, defined at least by their kinetics of appearance in culture: early endothelial progenitor cells (CFU-EC or CAC) and late (ECFC). Our team has shown tha...

  12. Angiostatin binds ATP synthase on the surface of human endothelial cells

    Moser, Tammy L.; Stack, M. Sharon; Asplin, Iain; Enghild, Jan J; Højrup, Peter; Everitt, Lorraine; Hubchak, Susan; Schnaper, H. William; Pizzo, Salvatore V.

    1999-01-01

    Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin...

  13. Disturbance of Copper Homeostasis Is a Mechanism for Homocysteine-Induced Vascular Endothelial Cell Injury

    Dong, Daoyin; Wang, Biao; Yin, Wen; Ding, Xueqing; Yu, Jingjing; Kang, Y James

    2013-01-01

    Elevation of serum homocysteine (Hcy) levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu) metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs) to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell vi...

  14. Induction of procoagulant activity on human endothelial cells by Streptococcus pneumoniae.

    Geelen, S; Bhattacharyya, C; Tuomanen, E

    1992-01-01

    The inflammatory response in infection caused by gram-negative organisms involves induction of procoagulant activity (PCA) on human endothelial cells. Although infections caused by gram-positive organisms are also associated with fibrin formation and thrombosis, the bacterial determinants inducing PCA are unknown. This study shows that intact pneumococci and the pneumococcal cell wall efficiently induce PCA on human endothelial cells. Upon exposure of endothelial cells to pneumococci, PCA was...

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

    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 < 0.05) compared with non-stimulated muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P < 0.05) in the intensely contracted, but not in the moderately contracted muscle cells....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P < 0.05), whereas endothelial cells in culture alone did not cause extracellular accumulation of adenosine. 4. Skeletal muscle cells were...

  16. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    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. PMID:27127549

  17. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    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.

  18. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells.

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    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. PMID:27127549

  19. In vitro differentiation of human adipose-derived mesenchymal stem cells into endothelial-like cells

    GUAN Lidong; SHI Shuangshuang; PEI Xuetao; LI Shaoqing; WANG Yunfang; YUE Huimin; LIU Daqing; HE Lijuan; BAI Cixian; YAN Fang; NAN Xue

    2006-01-01

    The neovascularization of ischemic tissue is a crucial initial step for the functional rehabilitation and wound healing. However, the short of seed cell candidate for the foundation of vascular network is still a big issue. Human adipose tissue derived mesenchymal stem cells (hADSCs), which possess multilineage potential, are capable of adipogenic, osteogenic, and chondrogenic differentiation. We examined whether this kind of stem cells could differentiate into endothelial-like cells and participate in blood vessel formation, and whether they could be used as an ideal cell source for therapeutic angiogenesis in ischemic diseases or vascularization of tissue constructs. The results showed that hADSCs, grown under appropriately induced conditions, displayed characteristics similar to those of vessel endothelium. The differentiated cells expressed endothelial cell markers CD34 and vWF, and had high metabolism of acetylated low-density lipoprotein and prostacyclin. In addition, the induced cells were able to form tube-like structures when cultured on matrigel. Our data indicated that induced hADSCs could exhibit characteristics of endothelial cells. Therefore, these cells, as a source of human endothelial cells, may find many applications in such realms as engineering blood vessels, endothelial cell transplantation for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

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

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

  1. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  2. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

    Allameh Abdolamir; Jazayeri Maryam; Adelipour Maryam

    2016-01-01

    Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs) to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM) gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor...

  3. Chase-and-run between adjacent cell populations promotes directional collective migration

    Theveneau, Eric; Steventon, Benjamin; Scarpa, Elena; Garcia, Simon; Trepat, Xavier; Streit, Andrea; Mayor, Roberto

    2016-01-01

    Collective cell migration in morphogenesis and cancer progression often involves the coordination of multiple cell types. How reciprocal interactions between adjacent cell populations lead to new emergent behaviours remains unknown. Here we studied the interaction between Neural Crest (NC) cells, a highly migratory cell population, and placodal cells, an epithelial tissue that contributes to sensory organs. We found that NC cells “chase” placodal cells by chemotaxis, while placodal cells “run” when contacted by NC. Chemotaxis to Sdf1 underlies the chase, while repulsion involving PCP and N-Cadherin signalling is responsible for the run. This “chase-and-run” requires the generation of asymmetric forces, which depend on local inhibition of focal adhesions. The cell interactions described here are essential for correct NC migration and for segregation of placodes in vivo and are likely to represent a general mechanism of coordinated migration. PMID:23770678

  4. Endothelial progenitor cells: what use for the cardiologist?

    Siddique Aurangzeb

    2010-02-01

    Full Text Available Abstract Endothelial Progenitor Cells (EPC were first described in 1997 and have since been the subject of numerous investigative studies exploring the potential of these cells in the process of cardiovascular damage and repair. Whilst their exact definition and mechanism of action remains unclear, they are directly influenced by different cardiovascular risk factors and have a definite role to play in defining cardiovascular risk. Furthermore, EPCs may have important therapeutic implications and further understanding of their pathophysiology has enabled us to explore new possibilities in the management of cardiovascular disease. This review article aims to provide an overview of the vast literature on EPCs in relation to clinical cardiology.

  5. Flow-induced Expression and Phosphorylation of VASPin Endothelial Cells

    Muller; SYLYAINE; Jean-FranoisSYOLTZ

    2005-01-01

    1 Introduction It is well known that mechanical forces have important influence on endothelial cells, in particular, on cytoskeleton reorganization. VASP (vasodilator stimulated phosphoprotein) is a 46 KD actin associated protein. It is a member of Ena/VASP protein family and composed of EVH1, proline-rich and EVH2 domains. It is considered as an important component of the sub-cellular regions where remodelling of the actin cytoskeleton takes place, such as the front of spreading lamellipodia in motile cell...

  6. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie

    2009-01-01

    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  7. Adjacent-cell Preconditioners for solving optically thick neutron transport problems

    We develop, analyze, and test a new acceleration scheme for neutron transport methods, the Adjacent-cell Preconditioner (AP) that is particularly suited for solving optically thick problems. Our method goes beyond Diffusion Synthetic Acceleration (DSA) methods in that it's spectral radius vanishes with increasing cell thickness. In particular, for the ID case the AP method converges immediately, i.e. in one iteration, to 10-4 pointwise relative criterion in problems with dominant cell size of 10 mfp or thicker. Also the AP has a simple formalism and is cell-centered hence, multidimensional and high order extensions are easier to develop, and more efficient to implement

  8. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

    2014-01-01

    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. PMID:25514584

  9. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    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.

  10. Endothelial cell motility, coordination and pattern formation during vasculogenesis.

    Czirok, Andras

    2013-01-01

    How vascular networks assemble is a fundamental problem of developmental biology that also has medical importance. To explain the organizational principles behind vascular patterning, we must understand how can tissue level structures be controlled through cell behavior patterns like motility and adhesion that, in turn, are determined by biochemical signal transduction processes? We discuss the various ideas that have been proposed as mechanisms for vascular network assembly: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and multicellular sprouting guided by cell-cell contacts. All of these processes yield emergent patterns, thus endothelial cells can form an interconnected structure autonomously, without guidance from an external pre-pattern. PMID:23857825

  11. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  12. Matrix fibronectin disruption and altered endothelial cell adhesion induced by activated leukocytes

    Sequestration of activated leukocytes (PMN) within the lung may contribute to pulmonary vascular injury following trauma, sepsis, or intravascular coagulation. Monolayers of cultured rat endothelial cells were utilized to evaluate the effect of activated PMNs on endothelial cell attachment and the extracellular fibronectin matrix over a 4 hr incubation interval. Rat endothelial cells were identified by immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with 51Cr in order to establish a cell injury assay in which the release of pelletable (cell associated) or non-pelletable activity was measured in the media. PMN activation was verified by chemiluminescence activity. Following phorbol myristate acetate (PMA) the leukocytes aggregated, chemiluminesced, and caused detachment of 51Cr endothelial cells. Endothelial detachment increased as a function of time with a plateau by 3 hrs. Immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin in association with endothelial cell disadhesion. Matrix fibronectin disruption was not seen with PMNs or PMA alone. Thus, disruption of the fibronectin matrix by released proteases may contribute to endothelial cell detachment

  13. Functional and gene expression analysis of hTERT overexpressed endothelial cells

    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

  14. Synthesis of Prostacyclin from Platelet-derived Endoperoxides by Cultured Human Endothelial Cells

    Marcus, Aaron J.; Weksler, Babette B.; Jaffe, Eric A.; Broekman, M Johan

    1980-01-01

    We have previously shown that aspirin-treated endothelial cells synthesize prostacyclin (PGI2) from the purified prostaglandin endoperoxide PGH2 (1978. J. Biol. Chem.253: 7138). To ascertain whether aspirin-treated endothelial cells produce PGI2 from endoperoxides released by stimulated platelets, [3H]arachidonic acid-prelabeled platelets were reacted in aggregometer cuvettes with the calcium ionophore A 23187, thrombin, or collagen in the presence of aspirin-treated endothelial cell suspensi...

  15. Impact of intense pulsed light irradiation on cultured primary fibroblasts and a vascular endothelial cell line

    Wu, Di; Zhou, BingRong; Xu, Yang; Yin, Zhiqiang; Luo, Dan

    2012-01-01

    The aim of this study was to determine the effects of intense pulsed light (IPL) on cell proliferation and the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in human fibroblasts and vascular endothelial cell lines, and to investigate the effects of IPL on the mRNA expression levels of type I and III procollagens in cultured human fibroblasts. Foreskin fibroblasts and a vascular endothelial cell line (ECV034) were cultured and treated with various ...

  16. Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate

    Martínez-Sales, Vicenta; Vila, Virtudes; Ferrando, Marcos; Reganon, Edelmiro

    2010-01-01

    Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and...

  17. Virulent Treponema pallidum promotes adhesion of leukocytes to human vascular endothelial cells.

    Riley, B S; Oppenheimer-Marks, N; Radolf, J D; Norgard, M V

    1994-01-01

    Perivasculitis and endothelial cell abnormalities are characteristic histopathologic features of syphilis, a sexually transmitted disease caused by Treponema pallidum. To extend earlier studies demonstrating that T. pallidum activates endothelial cells, we now show that virulent T. pallidum, but not heat-killed T. pallidum or nonpathogenic Treponema phagedenis, promotes increased adherence of lymphocytes and monocytes to human umbilical vein endothelial cells. Lymphocytes and monocytes are th...

  18. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

    Allameh Abdolamir

    2016-07-01

    Full Text Available Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF, vascular endothelial growth factor (VEGF receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC staining. Results Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis.

  19. Endothelial cell nitric oxide production in acute chest syndrome.

    Hammerman, S I; Klings, E S; Hendra, K P; Upchurch, G R; Rishikof, D C; Loscalzo, J; Farber, H W

    1999-10-01

    Acute chest syndrome (ACS) is the most common form of acute pulmonary disease associated with sickle cell disease. To investigate the possibility that alterations in endothelial cell (EC) production and metabolism of nitric oxide (NO) products might be contributory, we measured NO products from cultured pulmonary EC exposed to red blood cells and/or plasma from sickle cell patients during crisis. Exposure to plasma from patients with ACS caused a 5- to 10-fold increase in S-nitrosothiol (RSNO) and a 7- to 14-fold increase in total nitrogen oxide (NO(x)) production by both pulmonary arterial and microvascular EC. Increases occurred within 2 h of exposure to plasma in a concentration-dependent manner and were associated with increases in endothelial nitric oxide synthase (eNOS) protein and eNOS enzymatic activity, but not with changes in nitric oxide synthase (NOS) III or NOS II transcripts, inducible NOS (iNOS) protein nor iNOS enzymatic activity. RSNO and NO(x) increased whether plasma was obtained from patients with ACS or other forms of vasoocclusive crisis. Furthermore, an oxidative state occurred and oxidative metabolites of NO, particularly peroxynitrite, were produced. These findings suggest that altered NO production and metabolism to damaging oxidative molecules contribute to the pathogenesis of ACS. PMID:10516198

  20. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

    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.

  1. Adhesion and invasion of bovine endothelial cells by Neospora caninum.

    Hemphill, A; Gottstein, B; Kaufmann, H

    1996-02-01

    Neospora caninum is a recently identified coccidian parasite which was, until 1988, misdiagnosed as Toxoplasma gondii. It causes paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. The life-cycle of Neospora has not yet been elucidated. The only two stages identified so far are tissue cysts and intracellularly dividing tachyzoites. Very little is known about the biology of this species. We have set up a fluorescence-based adhesion/invasion assay in order to investigate the interaction of N. caninum tachyzoites with bovine aorta endothelial (BAE) cells in vitro. Treatment of both host cells and parasites with metabolic inhibitors determined the metabolic requirements for adhesion and invasion. Chemical and enzymatic modifications of parasite and endothelial cell surfaces were used in order to obtain information on the nature of cell surface components responsible for the interaction between parasite and host. Electron microscopical investigations defined the ultrastructural characteristics of the adhesion and invasion process, and provided information on the intracellular development of the parasites. PMID:8851858

  2. ECM-Dependence of Endothelial Progenitor Cell Features.

    Siavashi, Vahid; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Vafaei, Rana; Sariri, Reyhaneh

    2016-08-01

    Preserving self-renewal, multipotent capacity, and large-scale expansion of highly functional progenitor cells, including endothelial progenitor cells (EPCs), is a controversial issue. These current limitations, therefore, raise the need of developing promising in vitro conditions for prolonged expansion of EPCs without loss of their stemness feature. In the current study, the possible role of three different natural extracellular substrates, including collagen, gelatin, and fibronectin, on multiple parameters of EPCs such as cell morphology, phenotype, clonogenic, and vasculogenic properties was scrutinized. Next, EPCs from GFP-positive mice were pre-expanded on each of these ECM substrates and then systemically transplanted into sublethaly irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed considerable promise for fibronectin for EPC expansion with maintenance of stemness characteristics, whereas gelatin and collagen matrices directed the cells toward a mature endothelial phenotype. Transplantation of EPCs pre-expanded on fibronectin resulted in widespread distribution and appropriate engraftment to various tissues with habitation in close association with the microvasculature. In addition, fibronectin pre-expanded cells were gradually enriched in the bone marrow after transplantation, resulting in marrow repopulation and hematologic recovery, leading to improved survival of recipient mice whereas gelatin- and collagen-expanded cells failed to reconstitute the bone marrow. This study demonstrated that, cell characteristics of in vitro expanded EPCs are determined by the subjacent matrix. Fibronectin-expanded EPCs are heralded as a source of great promise for bone marrow reconstitution and neo-angiogenesis in therapeutic bone marrow transplantation. J. Cell. Biochem. 117: 1934-1946, 2016. © 2016 Wiley Periodicals, Inc. PMID:26756870

  3. Endothelial monolayers on collagen-coated nanofibrous membranes: cell-cell and cell-ECM interactions.

    Kang, Donggu; Kim, Jeong Hwa; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik; Jin, Songwan

    2016-06-01

    Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems. PMID:27186924

  4. Exogenous endothelial cells as accelerators of hematopoietic reconstitution

    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.

  5. Glycoconjugates and Related Molecules in Human Vascular Endothelial Cells

    Norihiko Sasaki

    2013-01-01

    Full Text Available Vascular endothelial cells (ECs form the inner lining of blood vessels. They are critically involved in many physiological functions, including control of vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and immunity. It is considered that impairment of EC functions leads to the development of vascular diseases. The carbohydrate antigens carried by glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans mainly present on the cell surface serve not only as marker molecules but also as functional molecules. Recent studies have revealed that the carbohydrate composition of the EC surface is critical for these cells to perform their physiological functions. In this paper, we consider the expression and functional roles of endogenous glycoconjugates and related molecules (galectins and glycan-degrading enzymes in human ECs.

  6. Oxidative stress modulates PPAR gamma in vascular endothelial cells.

    Blanquicett, Carmelo; Kang, Bum-Yong; Ritzenthaler, Jeffrey D; Jones, Dean P; Hart, C Michael

    2010-06-15

    The peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in vascular regulation. However, the impact of oxidative stress on PPAR gamma expression and activity has not been clearly defined. Human umbilical vein endothelial cells (HUVECs) were exposed to graded concentrations of H(2)O(2) for 0.5-72h, or bovine aortic endothelial cells (BAECs) were exposed to alterations in extracellular thiol/disulfide redox potential (E(h)) of the cysteine/cystine couple. Within 2h, H(2)O(2) reduced HUVEC PPAR gamma mRNA and activity and reduced the expression of two PPAR gamma-regulated genes without altering PPAR gamma protein levels. After 4h H(2)O(2) exposure, mRNA levels remained reduced, whereas PPAR gamma activity returned to control levels. PPAR gamma mRNA levels remained depressed for up to 72 h after exposure to H(2)O(2), without any change in PPAR gamma activity. Catalase prevented H(2)O(2)-induced reductions in PPAR gamma mRNA and activity. H(2)O(2) (1) reduced luciferase expression in HUVECs transiently transfected with a human PPAR gamma promoter reporter, (2) failed to alter PPAR gamma mRNA half-life, and (3) transiently increased expression and activity of c-Fos and phospho-c-Jun. Treatment with the AP1 inhibitor curcumin prevented H(2)O(2)-mediated reductions in PPAR gamma expression. In addition, medium having an oxidized E(h) reduced BAEC PPAR gamma mRNA and activity. These findings demonstrate that oxidative stress, potentially through activation of inhibitory redox-regulated transcription factors, attenuates PPAR gamma expression and activity in vascular endothelial cells through suppression of PPAR gamma transcription. PMID:20302927

  7. Carnosine facilitates nitric oxide production in endothelial f-2 cells.

    Takahashi, Satoru; Nakashima, Yukiko; Toda, Ken-Ichi

    2009-11-01

    We examined the effect of carnosine (beta-alanyl-histidine) on nitric oxide (NO) production and endothelial NO synthase (eNOS) activation in endothelial F-2 cells. Carnosine enhanced NO production in a dose-dependent manner, and the stimulatory effect of carnosine was observed at concentrations exceeding 5 mM. The carnosine-stimulated NO production was inhibited by N(G)-nitro-L-arginine methyl ester, but not by N(G)-nitro-D-arginine methyl ester. In contrast, beta-alanine, histidine (carnosine components) and anserine (N-methyl carnosine) failed to increase NO production. Carnosine had no effect on NO production for the initial 5 min, but thereafter resulted in a gradual increase in NO production up to 15 min. Carnosine did not induce phosphorylation of eNOS at Ser1177. The carnosine-induced increase in NO production was observed even when extracellular Ca2+ was depleted by ethylene glycol bis(2-aminoethyl ether)-N,N,N'-N'-tetraacetic acid however, the effect was abolished upon depletion of intracellular Ca2+ by BAPTA. After F-2 cells were incubated with carnosine for 4 min, intracellular Ca2+ concentration gradually increased. The carnosine-induced increase in intracellular Ca2+ concentration occurred even in the absence of extracellular Ca2+. These results indicate that carnosine facilitates NO production in endothelial F-2 cells. It is also suggested that eNOS is activated by Ca2+, which might be released from intracellular Ca2+ stores in response to carnosine. PMID:19881293

  8. Small vulvar squamous cell carcinomas and adjacent tissues. A morphologic study

    Poulsen, Hemming; Junge, Jette; Vyberg, Mogens; Horn, Thomas; Lundvall, Finn

    2003-01-01

    each case. Seven patients with keratinizing squamous cell carcinomas (median age 65) had adjacent lichen sclerosus. All carcinomas were completely surrounded by areas of VIN1. VIN2 and VIN3 were not found. Seven patients without lichen sclerosus (median age 58) showed squamous cell carcinomas of the...... keratinizing type (n=2) or the basaloid type (n=5). Five of these cases were incompletely surrounded by varying degrees of dysplasia, mainly VIN2 and VIN3. Two different pathogenetic pathways for the development of vulvar squamous cell carcinoma are likely....

  9. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  10. DNA damage in human endothelial cells after irradiation in anoxia

    Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from 60Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells. (orig.)

  11. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    Patsch, Christoph; Challet-Meylan, Ludivine; Eva C Thoma; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F.; Grainger, Stephanie J.; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of ei...

  12. 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

    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.

  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

    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. Cellular Dewetting: Opening of Macroapertures in Endothelial Cells

    Gonzalez-Rodriguez, David; Maddugoda, Madhavi P.; Stefani, Caroline; Janel, Sebastien; Lafont, Frank; Cuvelier, Damien; Lemichez, Emmanuel; Brochard-Wyart, Françoise

    2012-05-01

    Pathogenic bacteria can cross from blood vessels to host tissues by opening transendothelial cell macroapertures (TEMs). To induce TEM opening, bacteria intoxicate endothelial cells with proteins that disrupt the contractile cytoskeletal network. Cell membrane tension is no longer resisted by contractile fibers, leading to the opening of TEMs. Here we model the opening of TEMs as a new form of dewetting. While liquid dewetting is irreversible, we show that cellular dewetting is transient. Our model predicts the minimum radius for hole nucleation, the maximum TEM size, and the dynamics of TEM opening, in good agreement with experimental data. The physical model is then coupled with biological experimental data to reveal that the protein missing in metastasis (MIM) controls the line tension at the rim of the TEM and opposes its opening.

  15. C-reactive protein increases plasminogen activator inhibitor–1 expression in human endothelial cells

    Chen, Changyi; Nan, Bicheng; Lin, Peter; Yao, Qizhi

    2007-01-01

    C-reactive protein (CRP) is an inflammatory marker which predicts cardiovascular disease. However, it is not fully understood whether CRP has direct effects on endothelial functions and gene expression. The purpose of current study was to determine the effects and molecular mechanisms of CRP on the expression of plasminogen activator inhibitor-1 (PAI-1) in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations for d...

  16. Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

    Sukumar, Piruthivi; Viswambharan, Hema; Imrie, Helen; Cubbon, Richard M.; Yuldasheva, Nadira; Gage, Matthew; Galloway, Stacey; Skromna, Anna; Kandavelu, Parkavi; Santos, Celio X.; Gatenby, V. Kate; Smith, Jessica; Beech, David J; Wheatcroft, Stephen B.; Channon, Keith M.

    2013-01-01

    Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which cou...

  17. Expression and Function of the Homeostatic Molecule Del-1 in Endothelial Cells and the Periodontal Tissue

    Jieun Shin; Hosur, Kavita B.; Kalyani Pyaram; Ravi Jotwani; Shuang Liang; Triantafyllos Chavakis; George Hajishengallis

    2013-01-01

    Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interf...

  18. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  19. Biological behaviour and role of endothelial progenitor cells in vascular diseases

    ZHANG Qiu-hua; SHE Ming-peng

    2007-01-01

    Obiective To review the biological behaviour of endothelial progenitor cells and their role in vascular diseases.Data sources The data used in this review were mainly from Medline and PubMed for relevant English language articles published from 1985 to March 2007.The search term was "endothelial progenitor cells".Study selection Articles about the biological behaviour of endothelial progenitor cells and their roles in the pathogenesis of vascular diseases such as atherogenesis were used.Results Progenitor cells in bone marrow,peripheral blood and adventitia can differentiate into mature endothelial cells (ECs).The progenitor cells,which express certain surface markers including AC133,CD34 and KDR,enable restoration of the microcirculation and ECs when injury or ischaemia occurs.Endothelial progenitor cells used in experimental models and clinical trials for ischaemic syndromes could restore endothelial integrity and inhibit neointima development.Moreover,their number and functional properties are influenced by certain cytokines and atherosclerotic risk factors.Impairment of the progenitor cells might limit the regenerative capacity,even lead to the development of atherosclerosis or other vascular diseases.Conclusions Endothelial progenitor cells have a particular role in prevention and treatment of certain cardiovascular diseases.However,many challenges remain in understanding differentiation of endothelial progenitor cells,their mobilization and revascularization.

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

    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.

  1. Endothelial progenitor cell recruitment in a microfluidic vascular model.

    Lewis, Daniel M; Abaci, Hasan E; Xu, Yu; Gerecht, Sharon

    2015-12-01

    During vessel injury, endothelial progenitors cells (EPCs) are recruited from bone marrow and directed to the hypoxic injury site. The hypoxic conditions in the damaged blood vessel promote TNF-α, which upregulates intercellular adhesion molecule-1 (ICAM-1). EPCs attach to endothelial cell lining using ICAM-1. Here we aimed to examine EPC attachment to ECs in an injured-blood vessel conditions. We first determined ICAM-1 expression in stimulated HUVECs. We stimulated HUVECs with 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and hypoxia with TNF-α-supplemented media and found the highest ECFC attachment on HUVECs stimulated with TNF-α and hypoxia, correlating with the highest ICAM-1 expression. We next designed, fabricated and tested a three-dimensional microbioreactor (3D MBR) system with precise control and monitoring of dissolve oxygen and media flow rate in the cellular environment. We utilized a step-wise seeding approach, producing monolayer of HUVECs on all four walls. When stimulated with both TNF-α and hypoxia, ECFC retention on HUVECs was significantly increased under low shear stress compared to static controls. Overall, the 3D MBR system mimics the pathological oxygen tension and shear stress in the damaged vasculature, providing a platform to model vascular-related disorders. PMID:26693599

  2. Endothelial cell counts after Descemet’s stripping automated endothelial keratoplasty versus penetrating keratoplasty in Asian eyes

    Ang M

    2012-04-01

    Full Text Available Marcus Ang1,2, Jodhbir S Mehta1–4, Arundhati Anshu1,2, Hon Kiat Wong5, Hla M Htoon2, Donald Tan1–31Singapore National Eye Centre, 2Singapore Eye Research Institute, 3Department of Ophthalmology, National University Health Systems, 4Department of Clinical Sciences, Duke-NUS Graduate Medical School, 5Department of Ophthalmology, Tan Tock Seng Hospital, SingaporeBackground: 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 < 0.001. DSAEK-treated eyes had significantly superior uncorrected visual acuity (mean difference = 0.42 ± 0.0059; P < 0.001 and best spectacle-corrected visual acuity (mean difference = 0.14 ± 0.032; P < 0.001 as compared with penetrating 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 < 0.001. Graft survival at 1 year was comparable in both groups, ie, 66/68 (97.0% DSAEK-treated eyes

  3. Influence of pro-angiogenic cytokines on proliferative activity and survival of endothelial cells

    Solyanik G. I.

    2010-04-01

    Full Text Available Aim. Tumor angiogenesis in contrast to physiological one is characterized by high level of malignant cell production of proangiogenic cytokines, which have different influence on functional activity of endothelial cells. The goal of the study – to carry out a comparative analysis of the influence of a vascular endothelial growth factor (VEGF and an epidermal growth factor (EGF on proliferative activity and survival of endothelial cells upon their confluent and exponential growth. Methods. The proliferative activity of endothelial cells was determined by MTT-test and their viability was detected by the trypane blue exclusion test. Results. It was shown that EGF (irrespectively of the level of serum factors in concentrations higher than 10 ng/ml activated the proliferative activity of confluent endotheliocytes in a concentration-dependent manner by 18–36 % (ð < 0.05 as compared to the control, while this cytokine didn’t affect the endothelial cells in the exponential growth phase. VEGF in wide concentration range didn’t display the mitogenic effect on endotheliocytes in both confluent and exponential growth phases. Furthermore, VEGF in concentrations higher than 100 ng/ml inhibited proliferative activity of confluent endothelial cells by 12 % (ð < 0.05. In case of deficiency of nutrients, EGF and VEGF promoted the survival of endothelial cells, considerably decreasing their death. Conclusions. EGF, in contrast to VEGF, stimulates proliferation and survival of the endothelial cells, whereas VEGF has significant influence only on the survival of the cells

  4. SNEV overexpression extends the life span of human endothelial cells

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells

  5. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis. PMID:26321757

  6. Tp17 membrane protein of Treponema pallidum activates endothelial cells in vitro.

    Zhang, Rui-Li; Wang, Qian-Qiu; Zhang, Jing-Ping; Yang, Li-Jia

    2015-04-01

    Tp17, a membrane immunogen of Treponema pallidum subsp. pallidum, was initially recognized as an inflammatory mediator of syphilis. Because the histopathology of syphilis is characterized by endothelial cell abnormalities, we investigated the effects of recombinant Tp17 (rTp17) on endothelial cell activation in vitro. Using real-time reverse transcription-PCR and whole-cell ELISA, we found that rTp17 activated endothelial cells, as demonstrated by the up-regulated expression and increased gene transcription of intercellular adhesion molecule 1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1). rTp17 also enhanced the migration and subsequent adhesion of monocytes to endothelial cells as well as increased transendothelial migration of monocytes. These data suggest that the ability of Tp17 to activate endothelial cells may play an important role in the immunopathogenesis of syphilis. PMID:25744604

  7. Pro-angiogenic Hematopoietic Progenitor Cells and Endothelial Colony Forming Cells in Pathological Angiogenesis of Bronchial and Pulmonary Circulation

    Duong, Heng; Erzurum, Serpil; Asosingh, Kewal

    2011-01-01

    Dysregulation of angiogenesis is a common feature of many disease processes. Vascular remodeling is believed to depend on the participation of endothelial progenitor cells, but the identification of endothelial progenitors in postnatal neovascularization remains elusive. Current understanding posits a role for circulating pro-angiogenic hematopoietic cells, which interact with local endothelial cells to establish an environment that favors angiogenesis in physiologic and pathophysiologic resp...

  8. Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner.50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did.Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  9. Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis

    Xiao-ming Wu; Yan-bin Gao; Fang-qiang Cui; Na Zhang

    2016-01-01

    The interaction between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is an essential aspect of diabetic nephropathy (DN). Therefore, understanding how GECs communicate with GMCs in the diabetic environment is crucial for the development of new targets for the prevention and treatment of DN. Exosomes, nanometer-sized extracellular membrane vesicles secreted by various cell types, play important roles in cell-to-cell communication via the transfer of mRNA, microRNA ...

  10. Double suicide genes selectively kill human umbilical vein endothelial cells

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  11. Caveolin-1 Deficiency Induces Spontaneous Endothelial-to-Mesenchymal Transition in Murine Pulmonary Endothelial Cells in Vitro

    Li, Zhaodong; Wermuth, Peter J.; Benn, Bryan S.; Lisanti, Michael P.; Jimenez, Sergio A.

    2013-01-01

    It was previously demonstrated that transforming growth factor β (TGF-β) induces endothelial-to-mesenchymal transition (EndoMT) in murine lung endothelial cells (ECs) in vitro. Owing to the important role of caveolin-1 (CAV1) in TGF-β receptor internalization and TGF-β signaling, the participation of CAV1 in the induction of EndoMT in murine lung ECs was investigated. Pulmonary ECs were isolated from wild-type and Cav1 knockout mice using immunomagnetic methods with sequential anti-CD31 and a...

  12. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules

    1991-01-01

    Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their ...

  13. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells. Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine

  14. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  15. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. PMID:27208492

  16. Effects of amelogenins on angiogenesis-associated processes of endothelial cells

    Almqvist, S; Kleinman, H K; Werthén, M; Thomsen, P; Ågren, Sven Per Magnus

    2011-01-01

    To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay.......To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay....

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

    Wechsler, S J; Luedke, A. J.

    1991-01-01

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

  18. Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus

    Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian;

    2011-01-01

    To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....

  19. INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINEAORTIC ENDOTHELIAL CELLS

    2000-01-01

    Objective To examine the effects of insulin on cell proliferation, nitric oxide (NO) release and nitric oxide synthase (NOS) gene expression in bovine aortic endothelial cells ( BAEC ) . Methods The mi togenesis was assessed by MTT method; the products of NO in the culture media, by Griess reaction; and the levels of NOS mRNA in BAEC , by RT/PCR tech nique. Results BAEC were not responsive to the growth-promoting effects of insulin. Stimulation with insulin resulted a dose-dependent rise of NO in the culture supernatants 2h later, with a maximum at 12~24h and a decline at 24h. This rise was inhibited by an inhibitor of NOS (L-NAME). NOS mRNA increased slightly in BAEC without statistical significance. Conelu sion The study suggested that the insulin-induced NO release might be caused directly by NOS activation.

  20. Inhibitory Effect of the Punica granatum Fruit Extract on Angiotensin-II Type I Receptor and Thromboxane B2 in Endothelial Cells Induced by Plasma from Preeclamptic Patients

    Widya Kusumawati; Kusnarman Keman; Setyawati Soeharto

    2016-01-01

    This study aims to evaluate whether the Punica granatum fruit extract modulates the Angiotensin-II Type I receptor (AT1-R) and thromboxane B2 level in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluence, endothelial cells were divided into five groups, which included endothelial cells exposed to 2% plasma from normal pregnancy (NP), endothelial cells exposed to 2% plasma from preeclampti...

  1. [The irido-corneo-endothelial syndrome. The loss of the control of corneal endothelial cell cycle. A review].

    Robert, A M; Renard, G; Robert, L; Bourges, J-L

    2013-04-01

    The three major symptoms of the irido-corneo-endothelial syndrome are the alterations of the corneal endothelium and of the iris with a loss of the regulation of the cell cycle, and the progressive obstruction of the irido-corneal angle. This rare pathology attacks mainly young adult women. Most of the symptoms and complications originate from the excessive proliferation of the corneal endothelial cells accompanied by the evolution of their phenotype towards that of the epithelial cells. In normal conditions the corneal endothelial cells do not divide, they are blocked in the G1 stage of the cell cycle, mainly because of the action of the inhibitors of cyclin-dependent kinases. Still these cells retain a good capacity for proliferation, which can be induced by the down-regulation of the expression of the inhibitors of the cyclin-dependent kinases. This proliferative capacity declines with age and is also different according to the localization of the cells: it is more intense with those originating from the central area then in those from the peripheral area of the cornea. The age-related decline of the proliferative capacity is not due to the shortening of the telomers, but to the stress-induced accelerated senescence of the cells. PMID:23123109

  2. Endothelial progenitor cells display clonal restriction in multiple myeloma

    Dai Kezhi

    2006-06-01

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

  3. Circulating endothelial progenitor cells in kidney transplant patients.

    Giovana S Di Marco

    Full Text Available BACKGROUND: Kidney transplantation (RTx leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. METHODS: We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males. RTx patients received a calcineurin inhibitor (CNI-based (65% or a CNI-free therapy (35% and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1 levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+ and CD26+ cells were determined by flow cytometry. RESULTS: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1--a cytokine responsible for EPC mobilization--is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. CONCLUSIONS: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in

  4. Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

    Ye-Ji Jeong

    Full Text Available Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA, an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

  5. Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

    Ulisse Garbin

    2008-01-01

    Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

  6. CD34+ cells represent highly functional endothelial progenitor cells in murine bone marrow.

    Junjie Yang

    Full Text Available BACKGROUND: Endothelial progenitor cells (EPCs were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: CD34(+ cells, c-Kit(+/Sca-1(+/Lin(- (KSL cells, c-Kit(+/Lin(- (KL cells and Sca-1(+/Lin(- (SL cells were isolated from mouse bone marrow mononuclear cells (BMMNCs using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34(+ cells showed the lowest EPC colony forming activity, CD34(+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34(+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34(+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34(+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. CONCLUSION: These findings suggest that mouse CD34(+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

  7. Activation of the canonical Wnt/β-catenin pathway enhances monocyte adhesion to endothelial cells

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/β-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3β or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/β-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/β-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules

  8. Modeling human endothelial cell transformation in vascular neoplasias

    Victoria W. Wen

    2013-09-01

    Full Text Available Endothelial cell (EC-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia.

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

    Jeanine M. Roodhart

    2010-01-01

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

  10. Surface-enhanced Raman spectroscopy of the endothelial cell membrane.

    Simon W Fogarty

    Full Text Available We applied surface-enhanced Raman spectroscopy (SERS to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.

  11. The effect of 193 nm excimer laser radiation on the human corneal endothelial cell density

    The effect of 193 nm excimer laser radiation on human corneal endothelial cell density was examined. Fifty-five eyes from 35 patients underwent photorefractive keratectomy for myopia. Photomicrographs of the endothelium were taken a short time before the operation and on an average of 7 months postoperatively with a specular microscope. The average endothelial cell densities were preoperatively 3375 ± 266 cells/mm2 (means ± SD) and postoperatively 3348 ± 287 cells/mm2, corresponding to a fall of 27 cells/mm2 (N = 55). This fall in endothelial cell density was not statistically significant. A significant correlation between the change in cell density and age of the patient was found, with older patients losing more cells (N = 35, 2p < 0.05). The magnification of the specular microscope was found to change with corneal thickness. The importance of correcting the endothelial cell densities for corneal thickness is discussed. (au) 14 refs

  12. The effect of 193 nm excimer laser radiation on the human corneal endothelial cell density

    Isager, P.; Hjortdal, J.Oe.; Ehlers, N. [Aarhus Univ. Hospital, Dept. of Ophthalmology, Aarhus (Denmark)

    1996-06-01

    The effect of 193 nm excimer laser radiation on human corneal endothelial cell density was examined. Fifty-five eyes from 35 patients underwent photorefractive keratectomy for myopia. Photomicrographs of the endothelium were taken a short time before the operation and on an average of 7 months postoperatively with a specular microscope. The average endothelial cell densities were preoperatively 3375 {+-} 266 cells/mm{sup 2} (means {+-} SD) and postoperatively 3348 {+-} 287 cells/mm{sup 2}, corresponding to a fall of 27 cells/mm{sup 2} (N = 55). This fall in endothelial cell density was not statistically significant. A significant correlation between the change in cell density and age of the patient was found, with older patients losing more cells (N = 35, 2p < 0.05). The magnification of the specular microscope was found to change with corneal thickness. The importance of correcting the endothelial cell densities for corneal thickness is discussed. (au) 14 refs.

  13. Triglyceride-rich lipoprotein lipolysis increases aggregation of endothelial cell membrane microdomains and produces reactive oxygen species

    Wang, Limin; Sapuri-Butti, Annapoorna R.; Aung, Hnin Hnin; Parikh, Atul N.; Rutledge, John C

    2008-01-01

    Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a proinflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis-induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized by confocal microscopy ...

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

    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

    2015-09-01

    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 intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  15. Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

    Introduction: Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. Methods: Endothelial cell proliferation is determined by the amount of [3H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels in the conditioned medium were determined by ELISA. Results: Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. Conclusion: Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors

  16. Rapamycin inhibits proliferation and differentiation of human endothelial progenitor cells in vitro

    Bone-marrow-derived, circulating endothelial precursor cells contribute to neoangiogenesis in various diseases. Rapamycin has recently been shown to have anti-angiogenic effects in an experimental tumor model. Our group has developed a culture system that allows expansion and endothelial differentiation of human CD133+ precursor cells. We could show by PCR analysis that mTOR, the rapamycin-binding protein, was expressed in fresh CD133+ cells, in expanded cells after 28 days, and in differentiated endothelial cells. Rapamycin inhibited proliferation of CD133+ cells dose dependently at similar concentrations as hematopoietic Jurkat or HL-60 cells. Apoptosis was induced by rapamycin after 48 h of treatment, which could be reduced by preincubation with FK 506. Furthermore, the development of adherent endothelial cells from expanded CD133+ cells was dose dependently inhibited. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was reduced by rapamycin. In summary, rapamycin inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects

  17. Hydrogel Surfaces to Promote Attachment and Spreading of Endothelial Progenitor Cells

    Camci-Unal, Gulden; Nichol, Jason William; Bae, Hojae; Tekin, Halil; Bischoff, Joyce; Khademhosseini, Ali

    2012-01-01

    Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA-based hydrogels provides an attractiv...

  18. Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

    Rong Liu; Hua Liu; Yonju Ha; Tilton, Ronald G.; Wenbo Zhang

    2014-01-01

    Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protei...

  19. Protective effects of tanshinone IIA on endothelial progenitor cells injured by tumor necrosis factor-α

    Wang, Xing-xiang; YANG, JIN-XIU; PAN, YAN-YUN; ZHANG, YE-FEI

    2015-01-01

    Tanshinone IIA (Tan IIA) is a Traditional Chinese Medicine commonly used in Asian and Western countries for the prevention and treatment of cardiovascular disorders, such as atherosclerosis. Endothelial dysfunction and associated inflammatory processes have a critical role in the development of atherosclerosis. Endothelial progenitor cells (EPCs) have been demonstrated to be involved in certain aspects of the endothelial repair process. The present study aimed to investigate the putative prot...

  20. Regulation of Thrombomodulin Expression and Release in Human Aortic Endothelial Cells by Cyclic Strain

    Martin, Fiona A.; Alisha McLoughlin; Rochfort, Keith D.; Colin Davenport; Murphy, Ronan P.; Cummins, Philip M.

    2014-01-01

    Background and Objectives Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods This ...

  1. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  2. Role of endothelial cells in bovine mammary gland health and disease.

    Ryman, Valerie E; Packiriswamy, Nandakumar; Sordillo, Lorraine M

    2015-12-01

    The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood-milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium. PMID:26303748

  3. Nanoparticle accumulation and transcytosis in brain endothelial cell layers

    Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A.

    2013-10-01

    The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In order to investigate the capacity of nanoparticles to access and transport across the BBB, several different nanomaterials, including silica, titania and albumin- or transferrin-conjugated gold nanoparticles of different sizes, were exposed to a human in vitro BBB model of endothelial hCMEC/D3 cells. Extensive transmission electron microscopy imaging was applied in order to describe nanoparticle endocytosis and typical intracellular localisation, as well as to look for evidence of eventual transcytosis. Our results show that all of the nanoparticles were internalised, to different extents, by the BBB model and accumulated along the endo-lysosomal pathway. Rare events suggestive of nanoparticle transcytosis were also observed for several of the tested materials.The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight junctions, which limit paracellular permeability. Several reports suggest that nanoparticles have a unique capacity to cross the BBB. However, direct evidence of nanoparticle transcytosis is difficult to obtain, and we found that typical transport studies present several limitations when applied to nanoparticles. In

  4. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51Cr release from radiolabeled monolayers

  5. File list: ALL.CDV.50.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.50.AllAg.Aortic_valve_endothelial_cells hg19 All antigens Cardiovascular Aortic valve end...othelial cells SRX285599,SRX285598 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.CDV.50.AllAg.Aortic_valve_endothelial_cells.bed ...

  6. File list: Unc.CDV.10.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

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  7. File list: Oth.CDV.50.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

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  8. File list: ALL.CDV.20.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.20.AllAg.Aortic_valve_endothelial_cells hg19 All antigens Cardiovascular Aortic valve end...othelial cells SRX285599,SRX285598 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.CDV.20.AllAg.Aortic_valve_endothelial_cells.bed ...

  9. File list: Unc.CDV.20.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

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  10. File list: NoD.CDV.05.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

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  11. File list: InP.CDV.05.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

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  12. File list: ALL.CDV.05.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.05.AllAg.Aortic_valve_endothelial_cells hg19 All antigens Cardiovascular Aortic valve end...othelial cells SRX285599,SRX285598 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.CDV.05.AllAg.Aortic_valve_endothelial_cells.bed ...

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

    Full Text Available Oth.CDV.20.AllAg.Aortic_valve_endothelial_cells hg19 TFs and others Cardiovascular Aortic valve end...othelial cells SRX285599 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.20.AllAg.Aortic_valve_endothelial_cells.bed ...

  14. File list: His.CDV.05.AllAg.Aortic_valve_endothelial_cells [Chip-atlas[Archive

    Full Text Available His.CDV.05.AllAg.Aortic_valve_endothelial_cells hg19 Histone Cardiovascular Aortic valve end...othelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.CDV.05.AllAg.Aortic_valve_endothelial_cells.bed ...

  15. File list: InP.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  16. File list: NoD.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  17. File list: NoD.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  18. File list: InP.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  19. File list: NoD.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  20. File list: InP.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Full Text Available InP.CDV.50.AllAg.Brachiocephalic_endothelial_cells hg19 Input control Cardiovascular Brachioceph...alic endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.CDV.50.AllAg.Brachiocephalic_endothelial_cells.bed ...

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

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

  2. File list: InP.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  3. File list: Pol.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available Pol.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells hg19 RNA polymerase Cardiovascular Primary umbi...lical vein endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells.bed ...

  4. File list: DNS.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available DNS.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells hg19 DNase-seq Cardiovascular Primary umbi...lical vein endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells.bed ...

  5. File list: Pol.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available Pol.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells hg19 RNA polymerase Cardiovascular Primary umbi...lical vein endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells.bed ...

  6. File list: NoD.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  7. File list: InP.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  14. File list: NoD.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  15. Transdifferentiation of human endothelial progenitors into smooth muscle cells.

    Ji, HaYeun; Atchison, Leigh; Chen, Zaozao; Chakraborty, Syandan; Jung, Youngmee; Truskey, George A; Christoforou, Nicolas; Leong, Kam W

    2016-04-01

    Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction. PMID:26874281

  16. INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

    2001-01-01

    Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co-culture or direct contact co-culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

  17. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    Incubation of [35S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [35S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [35S]heparin proteoglycan after 24 hours and the appearance of free [35S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [35S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

  18. IL-1β regulates the mouse Fas ligand expression in corneal endothelial cells

    ZHANG Jie; Yang Ke; TAN DeYong; ZENG JunYing; Alan FINE

    2007-01-01

    Constitutively expressed Fas ligand (FasL) in several distinct epithelial cell types appears to protect tissues by inducing apoptosis of Fas+ immune cells during inflammatory reactions.To study the relationship of FasL and inflammation process in cornea, we examined the effects of inflammatory cytokine IL-1βon the FasL production, expression and cytotoxic function in corneal endothelial cells.In this paper, we demonstrate that IL-1βinhibits the FasL production and expression in corneal endothelial cells.The promoter activities of FasL in these cells are reduced by IL-1βin a dose-dependent manner.Finally, we also find that IL-1βblock the cytotoxic effects of FasL derived from corneal endothelial cells to the Fas+ target cells.These data support the view that FasL derived from corneal endothelial cells modulate inflammation within cornea.

  19. Polylactic Acid Nanoparticles Targeted to Brain Microvascular Endothelial Cells

    WANG Huafang; HU Yu; SUN Wangqiang; XIE Changsheng

    2005-01-01

    In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells (BMECs) targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can be loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 (T-80) can facilitate BMECs targeting of PLA nanoparticles.

  20. Shear Stress Inhibits Apoptosis of Ischemic Brain Microvascular Endothelial Cells

    Xiafeng Shen

    2013-01-01

    Full Text Available As a therapeutic strategy for ischemic stroke, to restore or increase cerebral blood flow (CBF is the most fundamental option. Laminar shear stress (LS, as an important force generated by CBF, mainly acts on brain microvascular endothelial cells (BMECs. In order to study whether LS was a protective factor in stroke, we investigated LS-intervented ischemic apoptosis of rat BMECs (rBMECs through PE Annexin V/7-AAD, JC-1 and Hoechst 33258 staining to observe the membranous, mitochondrial and nuclear dysfunction. Real-time PCR and western blot were also used to test the gene and protein expressions of Tie-2, Bcl-2 and Akt, which were respectively related to maintain membranous, mitochondrial and nuclear norm. The results showed that LS could be a helpful stimulus for ischemic rBMECs survival. Simultaneously, membranous, mitochondrial and nuclear regulation played an important role in this process.

  1. Bovine aortic endothelial cells are susceptible to Hantaan virus infection

    Hantavirus serotype Hantaan (HTN) is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS, lethality up to 10%). The natural host of HTN is Apodemus agrarius. Recent studies have shown that domestic animals like cattle are sporadically seropositive for hantaviruses. In the present study, the susceptibility of bovine aortic endothelial cells (BAEC) expressing αVβ3-integrin to a HTN infection was investigated. Viral nucleocapsid protein and genomic RNA segments were detected in infected BAEC by indirect immunofluorescence assay, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The results of this study strongly support our previous observation on Puumala virus (PUU) that has been propagated efficiently in BAEC. These findings open a new window to contemplate the ecology of hantavirus infection and transmission route from animal to man

  2. Effects of AMPK on high glucose stimulated apoptosis of endothelial cells via regulation of calcium influx

    Ting LU

    2015-11-01

    Full Text Available Objective To investigate the inhibitory effect of adenosine monophosphate (AMP-dependent protein kinase (AMPK on high glucose-stimulated endothelial cell apoptosis and its mechanism. Methods MS-1 endothelial cells were cultured in vitro, and they were treated with AMPK agonist, AMPK inhibitor, 2-APB (a blocker of store operated Ca2+ channel (SOCC and (or high glucose, and a control group without any intervention were set up. TUNEL assay was performed to determine apoptotic cells. Laser scanning confocal microscopy was used to assess the Ca2+ influx into cells, and Western-blotting was performed to determine the expressions of Stim1 and Orai1 of the store operated Ca2+ channel (SOCC proteins. Results Apoptosis of endothelial cells was induced significantly, and the expressions of Stim1 and Orai1 were upregulated in high glucose group compared with that in control group (P<0.05. The rate of apoptosis of high glucose-induced endothelial cell was found to be increased in AMPK inhibitor group and decreased in AMPK agonist group, and the expressions of Stim1 and Orai1 were found to be down-regulated in AMPK agonist group as compared with that in high glucose group (P<0.05. Compared with the control group, high glucose stimulation significantly induced the Ca2+ influx to endothelial cells; compared with high glucose group, 2-APB significantly inhibited high glucose-induced Ca2+ influx to endothelial cells, and blocked the inducing effect of high-glucose on endothelial cell apoptosis. Compared with high glucose group, AMPK agonist significantly inhibited high glucose-induced cell Ca2+ influx. Conclusion By reducing the expressions of Stim1 and Orai1, AMPK may inhibit SOCC-mediated Ca2+ influx, and block the high glucose-stimulated endothelial cell apoptosis, thus play an important protective role in sustaining endothelial cell function. DOI: 10.11855/j.issn.0577-7402.2015.10.01

  3. Gene expression programs of mouse endothelial cells in kidney development and disease.

    Brunskill, Eric W; Potter, S Steven

    2010-01-01

    Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease. PMID:20706631

  4. Gene expression programs of mouse endothelial cells in kidney development and disease.

    Eric W Brunskill

    Full Text Available Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

  5. Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature

    Witkiewicz Halina

    2011-03-01

    Full Text Available Abstract Background Endothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale. Results We performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface. Conclusion Our unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial

  6. Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

    Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

    1996-08-01

    Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

  7. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  8. Acrolein Oxidizes the Cytosolic and Mitochondrial Thioredoxins in Human Endothelial Cells

    Szadkowski, Adam; Myers, Charles R.

    2007-01-01

    Acrolein is a reactive aldehyde that is a widespread environmental pollutant and can be generated endogenously from lipid peroxidation. The thioredoxin (Trx) system in endothelial cells plays a major role in the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, cells maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. In human microvascular endothelial cells, Trx1 was more sensitive than Trx2 to oxidation by...

  9. Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway*

    Qin, Li; Zhang, Ming

    2010-01-01

    Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the ac...

  10. Ultrastructural study of the endothelial cells in teleost liver sinusoids under normal and experimental conditions.

    Ferri, S; Sesso, A

    1981-01-01

    The ultrastructure of the endothelial cells of liver sinusoids was studied in the teleost, Pimelodus maculatus. These cells have the ability to form pinocytotic vacuoles, starting with the formation of marginal folds. The latter occur in many cells after stimulation by India ink injections and ink particles are ingested by pinocytosis and by micropinocytosis. Desmosomes, structures rarely described between liver sinusoidal endothelial cells, are present in this species. PMID:7273119

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

    Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine; Grant, Christina L.; Vogel, Lotte K.; Rodriguez-Pinto, Daniel; Holmes, Kathryn V.; Ortega, Enrique; Shapiro, Linda H.

    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 myelo...

  12. Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

    Mackay, Laura S; Dodd, Sara; Dougall, Iain G; Tomlinson, Wendy; Lordan, James; Fisher, Andrew J.; Corris, Paul A

    2013-01-01

    Background Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding...

  13. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra

    2008-10-15

    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  14. Impact of diabetic serum on endothelial cells: An in-vitro-analysis of endothelial dysfunction in diabetes mellitus type 2

    Diabetic endothelial dysfunction was characterized by altered levels of adhesion molecules and cytokines. Aim of our study was to evaluate the effects of diabetic serum on cell-growth and proinflammatory markers in human saphenous vein endothelial cells (HSVEC) from diabetic and non-diabetic patients. Diabetic serum showed (1) complementary proliferative activity for non-diabetic and diabetic HSVEC, (2) unchanged surface expression of adhesion molecules, and (3) elevated levels of sICAM-1 in HSVEC of all donors. The concentration of sVCAM-1 was increased only in diabetic cells. The proinflammatory state of diabetic HSVEC characterized by increased levels of cytokines was compensated. We concluded that even under normoglycemic conditions the serum itself contains critical factors leading to abnormal regulation of inflammation in diabetics. We introduced an in vitro model of diabetes representing the endothelial situation at the beginning of diabetes (non-diabetic cells/diabetic serum) as well as the diabetic chronic state (diabetic cells/diabetic serum)

  15. Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation.

    Cary, Lynnette H; Noutai, Daniel; Salber, Rudolph E; Williams, Margaret S; Ngudiankama, Barbara F; Whitnall, Mark H

    2014-06-01

    Detonation of an improvised nuclear device near a population center would cause significant casualties from the acute radiation syndrome (ARS) due to exposure to mixed neutron/gamma fields (MF). The pathophysiology of ARS involves inflammation, microvascular damage and alterations in immune function. Interactions between endothelial cells (EC) and hematopoietic cells are important not only for regulating immune cell traffic and function, but also for providing the microenvironment that controls survival, differentiation and migration of hematopoietic stem and progenitor cells in blood-forming tissues. Endothelial cells/leukocyte interactions also influence tumor progression and the results of anticancer therapies. In this study, we hypothesized that irradiation of endothelial cells would modulate their effects on hematopoietic cells and vice versa. Human umbilical vein endothelial cells (HUVEC) and immortalized T lymphocytes (Jurkat cells) were cultured individually and in co-culture after exposure to mixed fields. Effects of nonirradiated cells were compared to effects of irradiated cells and alterations in signaling pathways were determined. Mitogen-activated protein kinases (MAPKs) p38 and p44/42 (ERK1/2) in HUVEC exhibited higher levels of phosphorylated protein after exposure to mixed field radiation. IL-6, IL-8, G-CSF, platelet derived growth factor (PDGF) and angiopoietin 2 (ANG2) protein expression were upregulated in HUVEC by exposure to mixed field radiation. PCR arrays using HUVEC mRNA revealed alterations in gene expression after exposure to mixed fields and/or co-culture with Jurkat cells. The presence of HUVEC also influenced the function of Jurkat cells. Nonirradiated Jurkat cells showed an increase in proliferation when co-cultured with nonirradiated HUVEC, and a decrease in proliferation when co-cultured with irradiated HUVEC. Additionally, nonirradiated Jurkat cells incubated in media from irradiated HUVEC exhibited upregulation of activated

  16. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood

  17. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    Joo, Hyung Joon; Seo, Ha-Rim [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Jeong, Hyo Eun [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Chung, Seok [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Lim, Do-Sun, E-mail: dslmd@kumc.or.kr [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  18. Systemic endothelial activation occurs in both mild and severe malaria. Correlating dermal microvascular endothelial cell phenotype and soluble cell adhesion molecules with disease severity.

    Turner, G D; Ly, V. C.; Nguyen, T.H.; Nguyen, H.P.; Bethell, D.; Wyllie, S.; Louwrier, K.; Fox, S B; Gatter, K C; Day, N P; Tran, T. H.; White, N J; Berendt, A R

    1998-01-01

    Fatal Plasmodium falciparum malaria is accompanied by systemic endothelial activation. To study endothelial activation directly during malaria and sepsis in vivo, the expression of cell adhesion molecules on dermal microvascular endothelium was examined in skin biopsies and correlated with plasma levels of soluble (circulating) ICAM-1, E-selectin, and VCAM-1 and the cytokine tumor necrosis factor (TNF)-alpha. Skin biopsies were obtained from 61 cases of severe malaria, 42 cases of uncomplicat...

  19. Derived vascular endothelial cells induced by mucoepidermoid carcinoma cells: 3-dimensional collagen matrix model*

    Yang, Sen; Guo, Li-Juan; Gao, Qing-hong; Xuan, Ming; Tan, Ke; Zhang, Qiang; Wen, Yu-ming; Wang, Chang-mei; Tang, Xiu-fa; Wang, Xiao-yi

    2010-01-01

    Mucoepidermoid carcinoma undergoes uniquely vigorous angiogenic and neovascularization processes, possibly due to proliferation of vascular endothelial cells (ECs) induced by mucoepidermoid carcinoma cells (MCCs) in their three-dimensional (3D) microenvironment. To date, no studies have dealt with tumor cells and vascular ECs from the same origin of mucoepidermoid carcinoma using the in vitro 3D microenvironment model. In this context, the current research aims to observe neovascularization w...

  20. Red cell adhesion molecules, foetal haemoglobin and endothelial factors in sickle cell disorders

    Mundee, Y.

    2001-01-01

    Sickle cell anaemia (SS) is a haemoglobinopathy involving production of sickle haemoglobin (HbS, β⁶Glu-->Val), which is able to polymerise leading to vaso-occlusion. Hydroxyurea (HU) treatment increases foetal haemoglobin (HbF) levels but decreases vaso-occlusion and red cell adhesion molecule (AM) expression, and therefore improves clinical symptoms. In this thesis, the contribution of AMs, HbF and endothelial factors to the severity of sickle cell disease has been studied....

  1. The Histone Demethylase PHF8 Is Essential for Endothelial Cell Migration

    Gu, Lunda; Hitzel, Juliane; Moll, Franziska; Kruse, Christoph; Malik, Randa Abdel; Preussner, Jens; Looso, Mario; Leisegang, Matthias S.; Steinhilber, Dieter; Brandes, Ralf P.; Fork, Christian

    2016-01-01

    Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function. PMID:26751588

  2. Low immunogenicity of endothelial derivatives from rat embryonic stem cell-like cells

    Juliane Ladhoff; Michael Bader; Sabine Br(o)sel; Elke Effenberger; Dirk Westermann; Hans-Dieter Volk; Martina Seifert

    2009-01-01

    Embryonic stem cells (ESC) are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells (RESC) under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells (ECs). Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, immune reactions in vivo were assessed by allo-antibody production and determination of interferon-γ (IFNγ)-secreting allo-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNγ MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished susceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these cells do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.

  3. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.

    Yoder, Mervin C; Mead, Laura E; Prater, Daniel; Krier, Theresa R; Mroueh, Karim N; Li, Fang; Krasich, Rachel; Temm, Constance J; Prchal, Josef T; Ingram, David A

    2007-03-01

    The limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies "endothelial cell colony-forming units" (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast, other EPCs with blood vessel-forming ability, termed endothelial colony-forming cells (ECFCs), have been isolated from human peripheral blood. We compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system using progenitor assays, and analysis of donor cells from polycythemia vera patients harboring a Janus kinase 2 V617F mutation in hematopoietic stem cell clones. Further, CFU-ECs possess myeloid progenitor cell activity, differentiate into phagocytic macrophages, and fail to form perfused vessels in vivo. In contrast, ECFCs are clonally distinct from CFU-ECs, display robust proliferative potential, and form perfused vessels in vivo. Thus, these studies establish that CFU-ECs are not EPCs and the role of these cells in angiogenesis must be re-examined prior to further clinical trials, whereas ECFCs may serve as a potential therapy for vascular regeneration. PMID:17053059

  4. Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling

    Baljinnyam, Erdene; Umemura, Masanari; Chuang, Christine;

    2014-01-01

    Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Ther...

  5. Endothelial cell death and intimal foam cell accumulation in the coronary artery of infected hypercholesterolemic minipigs

    Birck, Malene Muusfeldt; Saraste, Antti; Hyttel, Poul;

    2013-01-01

    Apoptosis of endothelial cells (ECs) has been suggested to play a role in atherosclerosis. We studied the synergism of hypercholesterolemia with Chlamydia pneumoniae and influenza virus infections on EC morphology and intimal changes in a minipig model. The coronary artery was excised at euthanasia...

  6. Cheiradone: a vascular endothelial cell growth factor receptor antagonist

    Ahmed Nessar

    2008-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing and pathological conditions (tumour development. Vascular endothelial growth factor (VEGF, fibroblast growth factor-2 (FGF-2 and epidermal growth factor (EGF are the major angiogenic regulators. We have identified a natural product (cheiradone isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation and in vivo (the chick chorioallantoic membrane models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50 was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

  7. Matrix stiffness regulates endothelial cell proliferation through septin 9.

    Yi-Ting Yeh

    Full Text Available Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa in comparison to those with low stiffness (LSG, 1.72 kPa. ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9, the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin α(vβ(3 caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.

  8. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells.

    Tseng, Shuei-Kuen; Chang, Mei-Chi; Su, Cheng-Yao; Chi, Lin-Yang; Chang, Jenny Zwei-Ching; Tseng, Wan-Yu; Yeung, Sin-Yuet; Hsu, Ming-Lun; Jeng, Jiiang-Huei

    2012-08-01

    Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases. PMID:21847594

  9. Estrogen Stimulates Homing of Endothelial Progenitor Cells to Endometriotic Lesions.

    Rudzitis-Auth, Jeannette; Nenicu, Anca; Nickels, Ruth M; Menger, Michael D; Laschke, Matthias W

    2016-08-01

    The incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 μg/kg β-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein(+)/Tie2(+) EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis. PMID:27315780

  10. Dibutyryl cyclic AMP reduces the radiosensitivity of cultured endothelial cells

    The purpose of this study was to determine whether dibutyryl cyclic AMP modifies the radiosensitivity of confluent monolayers of bovine aortic endothelial cells (BAEC). Three indices of BAEC function were monitored from 4-24 hrs after exposure to 1-10 Gy of 60Co gamma rays: the release of 51Cr from prelabeled cells, and release of lactate dehydrogenase (LDH) and plasminogen activator (PLA) into the culture medium. There was a time- and radiation dose-dependent increase in 51Cr, LDH and PLA release from the BAEC, detectable within 12 hrs after 5 Gy or higher, and by 24 hrs after 1 Gy or higher. This increased release was accompanied by a radiation dose-dependent decrease in 51Cr and LDH, and an increase in PLA activity in the lysate of cells adherent to the monolayer at 24 hrs. The continuous presence of cAMP from 1 hr before to 24 hrs after irradiation reduced all of these radiation reactions, although mM concentrations of cAMP were required for significant sparing. The presence of cAMP from 1 hr before to 10 min after irradiation had no effect on BAEC sensitivity, whereas cAMP added 10 min after irradiation was fully as effective as continuously administered drug. Thus, cultured BAEC exhibit membrane dysfunction within 24 hrs after clinically relevant radiation doses, and this dysfunction is ameliorated by cAMP present after irradiation

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

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

    2015-03-01

    We achieved possibility of isolation, characterization human umbilical cord blood endothelial progenitor cells (EPCs), examination potency of EPCs to form new blood vessels and differentiation into cardiomyoctes in canines with acute myocardial infarction (AMI). EPCs were separated and cultured from umbilical cord blood. Their phenotypes were confirmed by uptake of double stains dioctadecyl tetramethylindocarbocyanine-labeled acetylated LDL and FITC-labeled Ulex europaeus agglutinin 1 (DILDL-UEA-1). EPCs of cord blood were counted. Human VEGFR-2 and eNOS from the cultured EPCs were assessed by qPCR. Human EPCs was transplanted intramyocardially in canines with AMI. ECG and cardiac enzymes (CK-MB and Troponin I) were measured to assess severity of cellular damage. Histopathology was done to assess neovascularisation. Immunostaining was done to detect EPCs transdifferentiation into cardiomyocytes in peri-infarct cardiac tissue. qPCR for human genes (hVEGFR-2, and eNOS) was done to assess homing and angiogenic function of transplanted EPCs. Cultured human cord blood exhibited an increased number of EPCs and significant high expression of hVEGFR-2 and eNOS genes in the culture cells. Histopathology showed increased neovascularization and immunostaining showed presence of EPCs newly differentiated into cardiomyocyte-like cells. Our findings suggested that hEPCs can mediate angiogenesis and differentiate into cardiomyoctes in canines with AMI. PMID:25750747

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

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

    2008-01-01

    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 is......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...

  13. Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

    Cicmil, Milenko; Stevens, Jo; Leduc, Mireille; Bon, Cassian; Gibbins, Jonathan M.

    2002-01-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the r...

  14. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells.

    Anagnostou, A; Lee, E. S.; Kessimian, N; Levinson, R.; Steiner, M.

    1990-01-01

    Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. We have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (approximately 27,000) of an apparent single class of rec...

  15. Effect of Zinc and Nitric Oxide on Monocyte Adhesion to Endothelial Cells under Shear Stress

    Lee, Sungmun; Eskin, Suzanne G.; Shah, Ankit K.; Schildmeyer, Lisa A.; McIntire, Larry V.

    2011-01-01

    This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm2 or 1 dyne/cm2) or reversing shear stress (time average 1 dyne/cm2) for 24 hours. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike o...

  16. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

    Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay; SINGHAL, SHARAD S.

    2014-01-01

    Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Ce...

  17. OVERLAPPING ROLES FOR ENDOTHELIAL SELECTINS IN MURINE HEMATOPOIETIC STEM/PROGENITOR CELL HOMING TO BONE MARROW

    Nabors, L. Karina; Wang, Leo D.; Wagers, Amy J.; Kansas, Geoffrey S.

    2013-01-01

    Selectins are carbohydrate-binding adhesion molecules critically involved in leukocyte recognition of endothelium. The endothelial selectins have been implicated in homing of hematopoietic stem/progenitor cell(s) (HSPC) to the bone marrow (BM) during bone marrow transplant (BMT), but the precise role(s) of individual selectins in this process have never been defined. BMT of lethally irradiated mice lacking both endothelial selectins (E/P KO) with limiting numbers of wild-type BM cells rescued...

  18. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    Lotkov, Aleksandr I., E-mail: lotkov@ispms.tsc.ru; Kashin, Oleg A., E-mail: okashin@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); Kudryavtseva, Yuliya A., E-mail: yulia-k1970@mail.ru; Antonova, Larisa V., E-mail: antonova.la@mail.ru; Matveeva, Vera G., E-mail: matveeva-vg@mail.ru; Sergeeva, Evgeniya A., E-mail: sergeewa.ew@yandex.ru [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation); Kudryashov, Andrey N., E-mail: kudryashov@angioline.ru [Angioline Interventional Device Ltd, Novosibirsk, 630090 (Russian Federation)

    2015-10-27

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture.

  19. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    Lotkov, Aleksandr I.; Kashin, Oleg A.; Kudryavtseva, Yuliya A.; Antonova, Larisa V.; Kudryashov, Andrey N.; Matveeva, Vera G.; Sergeeva, Evgeniya A.

    2015-10-01

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture.

  20. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture

  1. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells

    1982-01-01

    Immunofluorescence staining of cultured human umbilical vein endothelial cells has shown the presence of von Willebrand protein in the perinuclear region, in small rodlike structures through the cytoplasm, and on filaments of the extracellular matrix. Nonendothelial cells showed no staining with anti-von Willebrand protein antiserum. At the light microscope level, immunoperoxidase treatment of endothelial cells revealed the same pattern and antibody specificity as the fluorescence staining. T...

  2. Natural antioxidant dihydroxybenzyl alcohol blocks ritonavir-induced endothelial dysfunction in porcine pulmonary arteries and human endothelial cells

    Weakley, Sarah M.; Jiang, Jun; Lü, Jianming; Wang, Xinwen; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

    2011-01-01

    Summary Background Patients with HIV have an increased incidence of pulmonary artery hypertension. This study was designed to determine if the naturally occurring antioxidant dihydroxybenzyl alcohol (DHBA) could counteract the deleterious effects of ritonavir (RTV), an HIV-protease inhibitor known to impair endothelial function and increase oxidative stress. Material/Methods Antioxidant assays were performed on DHBA in a cell free system. Glutathione (GSH) levels were measured in human pulmon...

  3. The effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells

    Papežíková, Ivana; Pekarová, Michaela; Lojek, Antonín; Kubala, Lukáš

    2009-01-01

    Roč. 30, č. 1 (2009), s. 112-115. ISSN 0172-780X R&D Projects: GA ČR(CZ) GP204/07/P539 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : uric acid * homocysteine * endothelial dysfunction Subject RIV: BO - Biophysics Impact factor: 1.047, year: 2009

  4. Effect of uric acid on homocysteine - induced endothelial dysfunction in bovine aortic endothelial cells

    Papežíková, Ivana; Kubala, Lukáš; Lojek, Antonín

    Brno, 2008. s. 1. [III. European Workshop on the Analysis of Phagocyte Functions. 22.05.2008-24.05.2008, Brno] R&D Projects: GA ČR GP204/07/P539 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : uric acid * endothelial dysfunction * homocysteine Subject RIV: BO - Biophysics

  5. Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells

    Rains, Justin L.; Jain, Sushil K.

    2011-01-01

    Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0–10 mM) or β-hydroxybutyrate (BHB) (0–10 mM) for 24 h prior to evaluating adhesion and a...

  6. Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms

    Anna Malashicheva

    2016-01-01

    Full Text Available Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin and smooth muscle (SMA, SM22α, calponin, and vimentin cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis.

  7. Tolerogenic properties of lymphatic endothelial cells are controlled by the lymph node microenvironment.

    Jarish N Cohen

    Full Text Available Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1. Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.

  8. In vitro neutron irradiation of glioma and endothelial cultured cells

    Menichetti, L. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy)], E-mail: luca.menichetti@ifc.cnr.it; Gaetano, L. [University Scuola Superiore Sant' Anna, Pisa (Italy); Zampolli, A.; Del Turco, S. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Ferrari, C. [University of Pavia, Department of Surgery, Laboratory of experimental Surgery, Pavia (Italy); Bortolussi, S.; Stella, S.; Altieri, S. [University of Pavia, Department of Nuclear Physics, Pavia (Italy); National Institute for Nuclear Physics (INFN), Section of Pavia (Italy); Salvadori, P.A. [Department of PET and Radiopharmaceutical Chemistry, C.N.R. Institute of Clinical Physiology, Pisa (Italy); Cionini, L. [Unit of Radiotherapy, AOUP-University Hospital, Pisa (Italy)

    2009-07-15

    To fully develop its potential boron neutron capture therapy (BNCT) requires the combination of a suitable thermal/epithermal neutron flux together with a selective intake of {sup 10}B-boron nuclei in the target tissue. The latter condition is the most critical to be realized as none of the boron carriers used for experimental or clinical purposes proved at the moment an optimal selectivity for cancer cells compared to normal cells. In addition to complex physical factors, the assessment of the intracellular concentration of boron represent a crucial parameter to predict the dose delivered to the cancer cells during the treatment. Nowadays the dosimetry calculation and then the prediction of the treatment effectiveness are made using Monte Carlo simulations, but some of the model assumption are still uncertain: the radiobiological dose efficacy and the probability of tumour cell survival are crucial parameters that needs a more reliable experimental approach. The aim of this work was to evaluate the differential ability of two cell lines to selectively concentrate the boron-10 administered as di-hydroxyboryl-phenylalanine (BPA)-fructose adduct, and the effect of the differential boron intake on the damage produced by the irradiation with thermal neutrons; the two cell lines were selected to be representative one of normal tissues involved in the active/passive transport of boron carriers, and one of the tumour. Recent in vitro studies demonstrated how BPA is taken by proliferating cells, however the mechanism of BPA uptake and the parameters driving the kinetics of influx and the elimination of BPA are still not clarified. In these preliminary studies we analysed the survival of F98 and human umbilical vein endothelial cells (HUVEC) cells line after irradiation, using different thermal fluencies at the same level of density population and boron concentration in the growing medium prior the irradiation. This is first study performed on endothelium model obtained by

  9. Isolation and expansion of human and mouse brain microvascular endothelial cells.

    Navone, Stefania E; Marfia, Giovanni; Invernici, Gloria; Cristini, Silvia; Nava, Sara; Balbi, Sergio; Sangiorgi, Simone; Ciusani, Emilio; Bosutti, Alessandra; Alessandri, Giulio; Slevin, Mark; Parati, Eugenio A

    2013-09-01

    Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d. PMID:23928501

  10. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells

    Anagnostou, A.; Kessimian, N.; Steiner, M. (Memorial Hospital of Rhode Island, Pawtucket (USA) Brown Univ. Program in Medicine, Providence, RH (USA)); Lee, Eun Sun (Memorial Hospital of Rhode Island, Pawtucket (USA)); Levinson, R. (Brown Univ. Program in Medicine, Providence, RI (USA))

    1990-08-01

    Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. The authors have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number ({approx}27,000) of an apparent single class of receptors with an affinity in the 10{sup {minus}9} M range. Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed them to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration of endothelial cells.

  11. Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells

    Erythropoietin is known to be a hematopoietic growth factor with a singularly specific action on the proliferation and differentiation of erythroid progenitor cells. The authors have observed a dose-dependent proliferative action of human recombinant erythropoietin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (∼27,000) of an apparent single class of receptors with an affinity in the 10-9 M range. Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed them to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration of endothelial cells

  12. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with [3H]-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, α-methyldopa, L-DOPA, α-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB

  13. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    Audus, K.L.; Borchardt, R.T.

    1986-03-01

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.

  14. Somatostatin regulates intracellular signaling in human carotid endothelial cells

    Somatostatin (somatotropin release inhibitory factor; SRIF) is an endogenous peptide produced at sites of inflammation, making the SRIF a candidate in regulating vascular inflammation. We have used primary human coronary artery endothelial cells (hCAEC) as a model to study SRIF's vascular actions. RT-PCR analysis of hCAEC total mRNA demonstrated the presence of the sst4 receptor subtype, providing a target for SRIF intracellular signaling. Western blotting with phospho-specific ERK1/2 antibodies showed that SRIF-14 acutely inhibited basal phosphorylation of the extracellular regulated kinases (ERK1/2) by 80%. In addition, SRIF-14 treated hCAEC cell lysates showed a 2.6-fold increase in phosphatase activity, which was inhibited by sodium vanadate. Furthermore, SRIF-14 appeared to be anti-inflammatory in hCAEC as IL-1β-induced adhesion molecule expression was reduced by 50%. Together, these results show that the coronary artery endothelium is a direct target of SRIF action

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

    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

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

    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

    2016-03-01

    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

  17. Endothelial derived factors inhibit anoikis of head and neck cancer stem cells

    Campos, Marcia S.; Neiva, Kathleen G.; Meyers, Kristy A.; Krishnamurthy, Sudha; Nör, Jacques E.

    2011-01-01

    Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells. Objective The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC). Materials and methods HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH−CD44−) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells. Results ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B. Conclusion This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer. PMID:22014666

  18. Investigation of Threshold Voltage Disturbance Caused by Programmed Adjacent Cell in Virtual Source/Drain NAND Flash Memory

    Kim, Wandong; Kwon, Dae Woong; Ji, Jung Hwan; Lee, Jung Hoon; Lee, Jong-Ho; Shin, Hyungcheol; Park, Byung-Gook

    2011-04-01

    In this paper, we investigate the threshold voltage disturbance caused by programmed adjacent cells in virtual source/drain (VSD) NAND flash memory device. The fringing field induced by charge in an adjacent memory node inhibits the inversion of virtual source/drain region. So, it increases the threshold voltage of the read cell. This is a drawback for the multi-level cell (MLC) operation. The device simulation and measurement data of fabricated devices show that the disturbance increases as the cell gate length and VSD length decreases. It can be minimized by the electric field concentration induced by the arch shape structure.

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

    Dilyana Todorova

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

  20. Acceleration of Multidimensional Discrete Ordinates Methods Via Adjacent-Cell Preconditioners

    The adjacent-cell preconditioner (AP) formalism originally derived in slab geometry is extended to multidimensional Cartesian geometry for generic fixed-weight, weighted diamond difference neutron transport methods. This is accomplished for the thick-cell regime (KAP) and thin-cell regime (NAP). A spectral analysis of the resulting acceleration schemes demonstrates their excellent spectral properties for model problem configurations, characterized by a uniform mesh of infinite extent and homogeneous material composition, each in its own cell-size regime. Thus, the spectral radius of KAP vanishes as the computational cell size approaches infinity, but it exceeds unity for very thin cells, thereby implying instability. In contrast, NAP is stable and robust for all cell sizes, but its spectral radius vanishes more slowly as the cell size increases. For this reason, and to avoid potential complication in the case of cells that are thin in one dimension and thick in another, NAP is adopted in the remainder of this work. The most important feature of AP for practical implementation in production level codes is that it is cell centered, reducing the size of the algebraic system comprising the acceleration stage compared to face-centered schemes. Boundary conditions for finite extent problems and a mixing formula across material and cell-size discontinuity are derived and used to implement NAP in a test code, AHOT, and a production code, TORT. Numerical testing for algebraically linear iterative schemes for the cases embodied in Burre's Suite of Test Problems demonstrates the high efficiency of the new method in reducing the number of iterations required to achieve convergence, especially for optically thick cells where acceleration is most needed. Also, for algebraically nonlinear (adaptive) methods, AP generally performs better than the partial current rebalance method in TORT and the diffusion synthetic acceleration method in TWODANT. Finally, application of the AP

  1. Effect of Nateglinide and Glibenclamide on Endothelial Cells and Smooth Muscle Cells from Human Coronary Arteries

    Seeger H

    2004-01-01

    Full Text Available In the present work the effect of nateglinide and glibenclamide, two different substances used for therapy of diabetes mellitus type 2, were investigated on the synthesis of markers of endothelial function and on the proliferation of smooth muscle cells in vitro. As cell models endothelial and smooth muscle cells from human coronary arteries were used. Both substances were tested at concentrations of 0.1, 1 and 10 mmol/l. As markers of endothelial function prostacyclin, endothelin and plasminogen-activator-inhibitor-1 (PAI-1 were tested. Nateglinide and glibenclamide were similarly able to inhibit endothelial endothelin and PAI-1 synthesis, but only at the highest concentration tested. Endothelial prostacyclin synthesis and proliferation of smooth muscle cells were not significantly changed by both substances. These results indicate that both nateglinide and glibenclamide may have potential in reducing negative long-term effects of diabetes such as atherogenesis. Kurzfassung: Effekt von Nateglinid und Glibenclamid auf Endothel- und Muskelzellen humaner Koronararterien. In der vorliegenden Arbeit wurde die Wirkung von Nateglinid und Glibenclamid, zweier unterschiedlicher Substanzen zur Behandlung des Diabetes mellitus Typ 2, auf die Synthese von Markern der Endothelfunktion und auf die Proliferation glatter Muskelzellen untersucht. Als Zellmodell dienten Endothelzellen und glatte Muskelzellen menschlicher Koronararterien. Beide Substanzen wurden in den Konzentrationen 0,1, 1 und 10 mmol/l getestet. Als Marker der Endothelfunktion dienten Prostazyklin, Endothelin und Plasminogen-Aktivator-Inhibitor-1 (PAI-1. Sowohl Nateglinid als auch Glibenclamid konnten die endotheliale Endothelin- und PAI-1-Produktion in ähnlichem Ausmaß senken, allerdings nur in der höchsten Konzentration. Die Prostazyklinsynthese und die Muskelzellproliferation wurden nicht signifikant beeinflußt. Diese Ergebnisse deuten daraufhin, daß sowohl Nateglinid als auch

  2. An evolving new paradigm: endothelial cells – conditional innate immune cells

    Mai, Jietang; Virtue, Anthony; Shen, Jerry; Wang, Hong; Yang, Xiao-Feng

    2013-01-01

    Endothelial cells (ECs) are a heterogeneous population that fulfills many physiological processes. ECs also actively participate in both innate and adaptive immune responses. ECs are one of the first cell types to detect foreign pathogens and endogenous metabolite-related danger signals in the bloodstream, in which ECs function as danger signal sensors. Treatment with lipopolysaccharide activates ECs, causing the production of pro-inflammatory cytokines and chemokines, which amplify the immun...

  3. Endothelial Cells from Embryonic Stem Cells in a Chemically Defined Medium

    Blancas, Alicia A.; Albert J. Shih; Lauer, Nicholas E.; McCloskey, Kara E.

    2011-01-01

    Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy, cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants. Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies, monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cultures of proliferating ESC-derived ECs without genetic m...

  4. Alert cell strategy in SIRS-induced vasculitis: sepsis and endothelial cells

    Matsuda, Naoyuki

    2016-01-01

    Sepsis refers to systemic inflammatory response syndrome and organ failure resulting from infection. Inflammatory receptors (e.g., Toll-like receptors and nucleotide oligomerization domain) recognize bacterial components as inflammatory ligands. These are expressed not only in leukocytes but also in major organs and vascular endothelial cells. “Alert cell” is defined as the cell that expresses the inflammatory receptor and intracellular signaling system to produce inflammatory mediators such ...

  5. Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

    Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures

  6. Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration.

    Condorelli, G; Borello, U; De Angelis, L; Latronico, M; Sirabella, D; Coletta, M; Galli, R; Balconi, G; Follenzi, A; Frati, G; Cusella De Angelis, M G; Gioglio, L; Amuchastegui, S; Adorini, L; Naldini, L; Vescovi, A; Dejana, E; Cossu, G

    2001-09-11

    The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies. PMID:11535818

  7. Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

    Xiaoying Zhou; Barsky, Lora W.; Adams, Gregor B

    2013-01-01

    Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow suppor...

  8. A 220-kilodalton glycoprotein in yeast extract inhibits Staphylococcus aureus adherence to human endothelial cells.

    Elliott, D.A.; Hatcher, V B; Lowy, F D

    1991-01-01

    A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.

  9. Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

    Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

    2016-07-01

    Cover: The cover image, by Angelino Calderone et al., is based on the Original Research Article Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression, DOI: 10.1002/jcp.25257. PMID:26995059

  10. SNS-032 Prevents Tumor Cell-Induced Angiogenesis By Inhibiting Vascular Endothelial Growth Factor

    M. Aktar Ali

    2007-05-01

    Full Text Available Cell proliferation, migration, and capillary network formation of endothelial cells are the fundamental steps for angiogenesis, which involves the formation of new blood vessels. The purpose of this study is to investigate the effect of a novel aminothiazole SNS-032 on these critical steps for in vitro angiogenesis using a coculture system consisting of human umbilical vein endothelial cells (HUVECs and human glioblastoma cells (U87MG. SNS-032 is a potent selective inhibitor of cyclin-dependent kinases 2, 7, and 9, and inhibits both transcription and cell cycle. In this study, we examined the proliferation and viability of HUVECs and U87MG cells in the presence of SNS-032 and observed a dose-dependent inhibition of cellular proliferation in both cell lines. SNS-032 inhibited threedimensional capillary network formations of endothelial cells. In a coculture study, SNS-032 completely prevented U87MG cell-mediated capillary formation of HUVECs. This inhibitor also prevented the migration of HUVECs when cultured alone or cocultured with U87MG cells. In addition, SNS-032 significantly prevented the production of vascular endothelial growth factor (VEGF in both cell lines, whereas SNS-032 was less effective in preventing capillary network formation and migration of endothelial cells when an active recombinant VEGF was added to the medium. In conclusion, SNS-032 prevents in vitro angiogenesis, and this action is attributable to blocking of VEGF.

  11. Phosphate-Buffered Saline-Based Nucleofection of Primary Endothelial Cells

    Kang, Jinjoo; Ramu, Swapnika; Lee, Sunju; Aguilar, Berenice; Ganesan, Sathish Kumar; Yoo, Jaehyuk; Kalra, Vijay K.; Koh, Chester J.; Hong, Young-Kwon

    2009-01-01

    Although various non-viral transfection methods are available, cell-toxicity, low transfection efficiency and high-cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that utilizes a combination of novel electroporation conditions and specific buffer components that stabilize the cells in the electrical field. Despite its superior transfection efficiency and cell viability, high cost of the technology has discouraged the cardiovascular researchers to liberally adopt this new technology. Here, we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular and lymphatic endothelial cells were performed by using PBS. Compared to the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and non-primary cells. Moreover, PBS-mediated nucleofection of siRNA showed more than 90% knockdown of the expression of target genes in primary endothelial cells. Together, we demonstrate that PBS can be an unprecedented economical alternative for the high-cost buffers for nucleofection of various primary and non-primary cells. PMID:19150324

  12. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  13. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    Nakashima, Yukiko; Morimoto, Mayuka [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Toda, Ken-ichi [Department of Dermatology, Kitano Hospital, The Tazuke Kofukai Nedical Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480 (Japan); Shinya, Tomohiro; Sato, Keizo [Department of Clinical Biochemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Miyazaki 882-8508 (Japan); Takahashi, Satoru, E-mail: imwalrus@mukogawa-u.ac.jp [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Institute for Biosciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan)

    2015-07-03

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.

  14. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells

  15. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

    Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid

  16. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

    Ioana Alesutan

    2013-09-01

    Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

  17. Low-level laser irradiation effect on endothelial cells under conditions of hyperglycemia.

    Góralczyk, Krzysztof; Szymańska, Justyna; Szot, Katarzyna; Fisz, Jacek; Rość, Danuta

    2016-07-01

    Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT-low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture-HUVEC line (human umbilical vein endothelial cell)-under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted. PMID:26861982

  18. Lonidamine Causes Inhibition of Angiogenesis-Related Endothelial Cell Functions1

    Del Bufalo, Donatella; Trisciuoglio, Daniela; Scarsella, Marco; D'Amati, Giulia; Candiloro, Antonio; Iervolino, Angela; Leonetti, Carlo; Zupi, Gabriella

    2004-01-01

    Abstract 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. PMID:15548359

  19. Effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells

    Papežíková, Ivana; Kubala, Lukáš; Pekarová, Michaela; Lojek, Antonín

    Elsevier. Roč. 45, č. 1 (2008), s. 310. ISSN 0891-5849. [SFRBM's Annual Meeting /15./. 19.11.2008-23.11.2008, Indianapolis] R&D Projects: GA ČR(CZ) GP204/07/P539 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : uric acid * endothelial dysfunction * homocysteine Subject RIV: BO - Biophysics

  20. Vascular endothelial growth factor attachment to hydroxyapatite via self-assembled monolayers promotes angiogenic activity of endothelial cells

    Currently, tissue engineered constructs for critical sized bone defects are non-vascularized. There are many strategies used in order to promote vascularization, including delivery of growth factors such as vascular endothelial growth factor (VEGF). In this study, hydroxyapatite (HA) was coated with self-assembled monolayers (SAMs). The SAMs were in turn used to covalently bind VEGF to the surface of HA. The different SAM chain length ratios (phosphonoundecanoic acid (11-PUDA):16-phosphonohexadecanoic acid (16-PHDA) utilized in this study were 0:100, 25:75, 50:50, 75:25, and 100:0. Surfaces were characterized by contact angle (CA) and atomic force microscopy, and an in vitro VEGF release study was performed. It was observed that CA and root-mean-squared roughness were not significantly affected by the addition of SAMs, but that CA was significantly lowered with the addition of VEGF. VEGF release profiles of bound VEGF groups all demonstrated less initial burst release than adsorbed control, indicating that VEGF was retained on the HA surface when bound by SAMs. An in vitro study using human aortic endothelial cells (HAECs) demonstrated that bound VEGF increased metabolic activity and caused sustained production of angiopoietin-2, an angiogenic marker, over 28 days. In conclusion, SAMs provide a feasible option for growth factor delivery from HA surfaces, enhancing angiogenic activity of HAECs in vitro. - Highlights: • Vascular endothelial growth factor (VEGF) is attached to hydroxyapatite (HA). • Self-assembled monolayers (SAMs) delay the release of VEGF from hydroxyapatite. • SAM chain length ratio affects the total mass of VEGF released. • VEGF on HA up-regulates proliferation and angiogenic activity of endothelial cells

  1. Vascular endothelial growth factor attachment to hydroxyapatite via self-assembled monolayers promotes angiogenic activity of endothelial cells

    Solomon, Kimberly D., E-mail: solomonk@livemail.uthscsa.edu [Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX (United States); UTSA-UTHSCSA Joint Graduate Program in Biomedical Engineering, San Antonio, TX (United States); Ong, Joo L., E-mail: anson.ong@utsa.edu [Department of Biomedical Engineering, University of Texas at San Antonio, San Antonio, TX (United States); UTSA-UTHSCSA Joint Graduate Program in Biomedical Engineering, San Antonio, TX (United States)

    2013-06-30

    Currently, tissue engineered constructs for critical sized bone defects are non-vascularized. There are many strategies used in order to promote vascularization, including delivery of growth factors such as vascular endothelial growth factor (VEGF). In this study, hydroxyapatite (HA) was coated with self-assembled monolayers (SAMs). The SAMs were in turn used to covalently bind VEGF to the surface of HA. The different SAM chain length ratios (phosphonoundecanoic acid (11-PUDA):16-phosphonohexadecanoic acid (16-PHDA) utilized in this study were 0:100, 25:75, 50:50, 75:25, and 100:0. Surfaces were characterized by contact angle (CA) and atomic force microscopy, and an in vitro VEGF release study was performed. It was observed that CA and root-mean-squared roughness were not significantly affected by the addition of SAMs, but that CA was significantly lowered with the addition of VEGF. VEGF release profiles of bound VEGF groups all demonstrated less initial burst release than adsorbed control, indicating that VEGF was retained on the HA surface when bound by SAMs. An in vitro study using human aortic endothelial cells (HAECs) demonstrated that bound VEGF increased metabolic activity and caused sustained production of angiopoietin-2, an angiogenic marker, over 28 days. In conclusion, SAMs provide a feasible option for growth factor delivery from HA surfaces, enhancing angiogenic activity of HAECs in vitro. - Highlights: • Vascular endothelial growth factor (VEGF) is attached to hydroxyapatite (HA). • Self-assembled monolayers (SAMs) delay the release of VEGF from hydroxyapatite. • SAM chain length ratio affects the total mass of VEGF released. • VEGF on HA up-regulates proliferation and angiogenic activity of endothelial cells.

  2. Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

    Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

    2009-05-26

    Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

  3. Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

    Ellen Ackerstaff

    2007-12-01

    Full Text Available Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MRcompatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs. Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

  4. Angiotensin II Inhibits Insulin Binding to Endothelial Cells

    Su-Jin Oh

    2011-06-01

    Full Text Available BackgroundInsulin-mediated glucose uptake in insulin target tissues is correlated with interstitial insulin concentration, rather than plasma insulin concentration. Therefore, insulin delivery to the interstitium of target tissues is very important, and the endothelium may also play an important role in the development of insulin resistance.MethodsAfter treating bovine aortic endothelial cells with angiotensin II (ATII, we observed the changes in insulin binding capacity and the amounts of insulin receptor (IR on the cell membranes and in the cytosol.ResultsAfter treatment of 10-7M ATII, insulin binding was decreased progressively, up to 60% at 60 minutes (P<0.05. ATII receptor blocker (eprosartan dose dependently improved the insulin binding capacity which was reduced by ATII (P<0.05. At 200 µM, eprosartan fully restored insulin binding capacity, althogh it resulted in only a 20% to 30% restoration at the therapeutic concentration. ATII did not affect the total amount of IR, but it did reduce the amount of IR on the plasma membrane and increased that in the cytosol.ConclusionATII decreased the insulin binding capacity of the tested cells. ATII did not affect the total amount of IR but did decrease the amount of IR on the plasma membrane. Our data indicate that ATII decreases insulin binding by translocating IR from the plasma membrane to the cytosol. The binding of insulin to IR is important for insulin-induced vasodilation and transendothelial insulin transport. Therefore, ATII may cause insulin resistance through this endothelium-based mechanism.

  5. In vivo imaging of tumor vascular endothelial cells

    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.

  6. Differential effects of hydrogen peroxide on indices of endothelial cell function

    1984-01-01

    The responses of pig aortic endothelial cells to sublethal doses of potentially toxic stimuli were investigated by monitoring K+ efflux, prostaglandin production, and the release of cytoplasmic purines. Xanthine plus xanthine oxidase reversibly stimulated these three parameters of endothelial cell function at doses that were not cytotoxic, as measured by chromium release, adenine uptake, and vital dye exclusion. The effects of xanthine plus xanthine oxidase were inhibited by catalase but not ...

  7. PECAM-1 (CD31) regulates a hydrogen peroxide–activated nonselective cation channel in endothelial cells

    Ji, Guangju; O'Brien, Christopher D.; Feldman, Morris; Manevich, Yefim; Lim, Poay; Sun, Jing; Albelda, Steven M.; Kotlikoff, Michael I.

    2002-01-01

    Hydrogen peroxide (H2O2) released by neutrophils is an important mediator of endothelial cell (EC) injury and vascular inflammation via its effect on EC-free Ca2+, [Ca2+]i. Although the underlying mechanisms are not well understood, platelet endothelial cell adhesion molecule (PECAM)-1/CD-31 is a critical modulator of neutrophil–EC transmigration. PECAM-1 is also known to regulate EC calcium signals and to undergo selective tyrosine phosphorylation. Here, we report that PECAM-1 molecules tran...

  8. Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells.

    Meier, C.; Oelschlaeger, T A; Merkert, H; Korhonen, T K; Hacker, J

    1996-01-01

    Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epit...

  9. Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: Elucidation of a novel glycosphingolipid-signaling pathway

    Dasgupta, Somsankar; Wang, Guanghu; Robert K Yu

    2011-01-01

    Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid (GSL) of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the HNK-1 sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-H...

  10. Matrix-Bound VEGF Mimetic Peptides: Design and Endothelial Cell Activation in Collagen Scaffolds

    Chan, Tania R.; Stahl, Patrick J.; Yu, S. Michael

    2011-01-01

    Long term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial cell (EC) differentiation and vasculature formation are dependent on spatio-temporal cues in the extracellular matrix that dynamically interact with cells, a process difficult to reproduce in artificial systems. Here we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF) and can be used to encode spatially controlled angiogenic signa...

  11. Toxic Effects of Xylazine on Endothelial Cells in Combination with Cocaine and 6-monoacetylmorphine

    Silva-Torres, L.A.; C. Vélez; Alvarez, J. Lyvia; Ortiz, J.G.; Zayas, B.

    2014-01-01

    The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its c...

  12. Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

    Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

    1995-01-01

    Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present ...

  13. Enhanced phosphorylation of caveolar PKC-α limits peptide internalization in lung endothelial cells

    Hutchinson, Tarun E.; Zhang, Jianliang; Xia, Shen-Ling; Kuchibhotla, Sudeep; Block, Edward R.; Patel, Jawaharlal M.

    2011-01-01

    We previously reported that the vasoactive peptide 1 (P1, “SSWRRKRKESS”) modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were...

  14. Involvement of Heme Oxygenase-1 in Orexin-A-induced Angiogenesis in Vascular Endothelial Cells

    Kim, Mi-Kyoung; Park, Hyun-Joo; Kim, Su-Ryun; Choi, Yoon Kyung; Bae, Soo-Kyung; Bae, Moon-Kyoung

    2015-01-01

    The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin...

  15. Lonidamine Causes Inhibition of Angiogenesis-Related Endothelial Cell Functions1

    Del Bufalo, Donatella; Trisciuoglio, Daniela; Scarsella, Marco; d'Amati, Giulia; Candiloro, Antonio; Iervolino, Angela; Leonetti, Carlo; Zupi, Gabriella

    2004-01-01

    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 secreti...

  16. Characterization of novel NADPH oxidases in endothelial cells under basal and stress conditions

    Petry, Andreas

    2009-01-01

    Increased levels of reactive oxygen species (ROS) contribute to vascular diseases like pulmonary hypertension and atherosclerosis. Although a NOX2-containing NADPH oxidase similar to the neutrophil one has been described to be active in endothelial cells, the contribution of newly discovered NOX homologues (NOX1-NOX5) was still unclear. Therefore, the overall aim of this study was to better characterize the expression, regulation and function of NOX homologues in different endothelial cell mo...

  17. Intravascularly Administered RGD-Displaying Measles Viruses Bind to and Infect Neovessel Endothelial Cells In Vivo

    Ong, Hooi Tin; Trejo, Theodore R; Pham, Linh D.; Oberg, Ann L.; Russell, Stephen J.; Peng, Kah-Whye

    2009-01-01

    Systemically administered vectors must cross the endothelial lining of tumor blood vessels to access cancer cells. Vectors that interact with markers on the lumenal surface of these endothelial cells might have enhanced tumor localization. Here, we generated oncolytic measles viruses (MVs) displaying αvβ3 integrin-binding peptides, cyclic arginine-glycine-aspartate (RGD) or echistatin, on the measles hemagglutinin protein. Both viruses had expanded tropisms, and efficiently entered target cel...

  18. Stiffness memory of EA.hy926 endothelial cells in response to chronic hyperglycemia

    Targosz-Korecka, Marta; Brzezinka, Grzegorz D; Malek, Katarzyna E; Stȩpień, Ewa; Szymonski, Marek

    2013-01-01

    Background Glycemic memory of endothelial cells is an effect of long-lasting hyperglycemia and is a cause of various diabetics complications, that arises despite of the treatment targeted towards returning low glucose level in blood system. On the other hand, endothelial dysfunction, which is believed to be a main cause of cardiovascular complications, is exhibited in the changes of mechanical properties of cells. Although formation of the glycemic memory was widely investigated, its impact o...

  19. Analysis and performance of adjacent-cell preconditioners for accelerating multidimensional transport calculations

    The formal development of the Adjacent-cell Preconditioner (AP) and its implementation in the TORT code are briefly reviewed. Based on earlier experience with diffusion type acceleration, and excellent results in slab geometry the reciprocal averaging formula is used to mix the preconditioner elements across material and mesh discontinuities. Numerical testing of the method employing the Burre Suite of Test Problems (BSTeP), a collection of 144 cases covering a wide range in parameter space, using AP, Partial Current Rebalance (PCR), and TWODANT's Diffusion Synthetic Acceleration (DSA) is presented. While AP outperforms the other two methods for the majority of the cases included in BSTeP it consumes many more iterations than can be explained by spectral analysis of the homogeneous model problem in cases with sharp material discontinuity. In order to verify this undesirable behavior and explore potential remedies a model problem, the Periodic Horizontal Interface (PHI), is developed that permits discontinuity of nuclear properties and cell height across the interface. Fourier mode decomposition is applied to AP with the reciprocal averaging mixing formula for the PHI configuration and shown to possess a spectral radius that approaches unity as the material discontinuity gets larger. The question of whether an unconditionally stable AP exists for PHI is tackled and preliminary indications are negative. Novel preconditioners that have nontraditional cell-coupling schemes that remain stable in these regimes may have to be sought

  20. Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro.

    Morioka, Hideo; Morii, Takeshi; Vogel, Tikva; Hornicek, Francis J; Weissbach, Lawrence

    2003-07-01

    Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes. PMID:12799192

  1. Endothelial cell-initiated signaling promotes the survival and self-renewal of cancer stem cells

    Krishnamurthy, Sudha; Dong, Zhihong; Vodopyanov, Dmitry; Imai, Atsushi; Helman, Joseph I.; Prince, Mark E.; Wicha, Max S.; Nör, Jacques E.

    2010-01-01

    Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used Aldehyde Dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin−) led to tumors in 13 (out of 15) mice, while 10,000 non-cancer stem cells (NCSC; ALDH−CD44−Lin−) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a sub-population of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin− cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-µm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared to controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck cancer stem cells. PMID:21098716

  2. Real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow.

    Kroon, Jeffrey; Daniel, Anna E; Hoogenboezem, Mark; van Buul, Jaap D

    2014-01-01

    During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium. PMID:25146919

  3. Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells

    FANG Ning-tao; XIE Shang-zhe; WANG Song-mei; GAO Hong-yang; WU Chun-gen; PAN Luan-feng

    2007-01-01

    Background Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.Methods Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 × 106 cells/cm2 and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).Results EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin,Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the

  4. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  5. Does VEGF concentration in pre-eclamptic serum induce sVCAM-1 production in endothelial cell culture?

    Sri B. Subakir

    2005-03-01

    Full Text Available Serum concentrations of VEGF (Vascular Endothelial Growth Factor are elevated in preeclampsia. In addition to inducing mitosis and increase permeability of endothelial cells, VEGF was reported to activate endothelial cells to produce cell adhesion molecules. Cell adhesion molecules play an important role in the inflammation process by inducing adherence of leukocytes in blood stream to the endothelial cells. The aim of this study is to investigate the effect of VEGF in serum from preeclamptic patients on sVCAM-1 (soluble vascular adhesion molecules-1 production in endothelial cell culture. Twelve sera from women with preeclampsia and 11 from women with normal pregnancy (controls in 20% concentration were added to human umbilical vein endothelial cell culture (HUVEC and incubated for 24 hours. All subjects have agreed to participate in this study and signed the informed consent form. sVCAM-1 concentration in the supernatant was measured by ELISA. VEGF concentration tends to be higher in preeclamptic serum than control, but the difference is not stastitically significant. The production of sVCAM-1 by endothelial cells exposed to preeclamptic serum was significantly higher than the production by endothelial cells exposed to serum from control (p<0.05. No correlation was found between the difference in VEGF concentrations in preeclamptic and control sera, and sVCAM-1 production by endothelial cell culture. (Med J Indones 2005; 14: 3-6Keywords: endothelial cell, preeclampsia, VCAM, VEGF

  6. Engineering anastomosis between living capillary networks and endothelial cell-lined microfluidic channels.

    Wang, Xiaolin; Phan, Duc T T; Sobrino, Agua; George, Steven C; Hughes, Christopher C W; Lee, Abraham P

    2016-01-21

    This paper reports a method for generating an intact and perfusable microvascular network that connects to microfluidic channels without appreciable leakage. This platform incorporates different stages of vascular development including vasculogenesis, endothelial cell (EC) lining, sprouting angiogenesis, and anastomosis in sequential order. After formation of a capillary network inside the tissue chamber via vasculogenesis, the adjacent microfluidic channels are lined with a monolayer of ECs, which then serve as the high-pressure input ("artery") and low pressure output ("vein") conduits. To promote a tight interconnection between the artery/vein and the capillary network, sprouting angiogenesis is induced, which promotes anastomosis of the vasculature inside the tissue chamber with the EC lining along the microfluidic channels. Flow of fluorescent microparticles confirms the perfusability of the lumenized microvascular network, and minimal leakage of 70 kDa FITC-dextran confirms physiologic tightness of the EC junctions and completeness of the interconnections between artery/vein and the capillary network. This versatile device design and its robust construction methodology establish a physiological transport model of interconnected perfused vessels from artery to vascularized tissue to vein. The system has utility in a wide range of organ-on-a-chip applications as it enables the physiological vascular interconnection of multiple on-chip tissue constructs that can serve as disease models for drug screening. PMID:26616908

  7. Research of the Effect of the Shear Stress on Endothelial Cells

    2005-01-01

    1 IntroductionCellular mechanism is one of the foundations of regenerating medicine and tissue engineering, which is also an advanced subject in cell mechanism in recent years~([1]). The form and function of a cell, and the growing, reproducing and death, even canceration are related to the characteristics of cell mechanism. While the research of the shear stress on endothelial cells is an important field in cell mechanism. The main bio-functions of endothelial cells are as follows: anti-cruor, regulating t...

  8. Studies on angiogenesis and endothelial cell apoptosis in radiation-combined wound healing in Wistar rats

    Objective: To study the changes and significance of angiogenesis and endothelial cell apoptosis in radiation-combined wound healing. Methods: Wistar rats were wounded and then local irradiation was performed immediately with a dose of 25 Gy 60Co γ-rays in the irradiation wound group. Tissue specimens were obtained at 2, 5, 10, 15, 21 and 28 days after injury and angiogenesis and apoptosis of endothelial cells were investigated by means of LM, EM and in situ terminal labelling. Results: In the non-irradiation wound group, angiogenesis began from day 2 and the capillary number reached its peak on day 10 and decreased on day 15 after injury when the endothelial cells occurred apoptosis and scarcely necrosis. In the irradiation wound group angiogenesis began on day 5 when endothelial cells occurred apoptosis. The capillary number reached its peak on day 15 and decreased on day 21 after injury when endothelial cells occurred both apoptosis and necrosis. Conclusions: Radiation may delay the angiogenesis in wound healing, induce apoptosis of endothelial cells earlier and delay the peak of the capillary number. That radiation may delay the angiogenesis is one of reasons in delay of wound healing by radiation

  9. Interleukin 1 is an autocrine regulator of human endothelial cell growth

    Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, the authors demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth the suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells

  10. Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells.

    Camci-Unal, Gulden; Nichol, Jason William; Bae, Hojae; Tekin, Halil; Bischoff, Joyce; Khademhosseini, Ali

    2013-05-01

    Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin-based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA-based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA-heparin hydrogels, using standard EDC/NHS amine-coupling strategies. We found that EPC adhesion and spreading on CD34 Ab-immobilized HA-heparin hydrogels was significantly higher than their non-modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non-modified and CD34 Ab-modified HA-heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA-heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non-thrombogenic surface. PMID:22223475

  11. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng

    2009-01-01

    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  12. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    Askari, Ara A. [Barts and the London, Queen Mary University, London (United Kingdom); Thomson, Scott [Comparative Biomedical Sciences, Royal Veterinary College, London (United Kingdom); Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C. [Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (United States); Bishop-Bailey, David, E-mail: dbishopbailey@rvc.ac.uk [Comparative Biomedical Sciences, Royal Veterinary College, London (United Kingdom)

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  13. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity

  14. Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

    Oh, Myung-Jin; Zhang, Chongxu; LeMaster, Elizabeth; Adamos, Crystal; Berdyshev, Evgeny; Bogachkov, Yedida; Kohler, Erin E; Baruah, Jugajyoti; Fang, Yun; Schraufnagel, Dean E; Wary, Kishore K; Levitan, Irena

    2016-05-01

    Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity. PMID:26989083

  15. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays

    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 rec...

  16. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    Bosche, Bert, E-mail: bert.bosche@uk-essen.de [Department of Neurology, University of Duisburg-Essen (Germany); Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Schäfer, Matthias, E-mail: matthias.schaefer@sanofi.com [Institute of Physiology, Justus-Liebig-University Giessen (Germany); Graf, Rudolf, E-mail: rudolf.graf@nf.mpg.de [Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Härtel, Frauke V., E-mail: frauke.haertel@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany); Schäfer, Ute, E-mail: ute.schaefer@medunigraz.at [Research Unit for Experimental Neurotraumatology, Medical University of Graz (Austria); Noll, Thomas, E-mail: thomas.noll@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany)

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  17. Tomato Aqueous Extract Modulates the Inflammatory Profile of Immune Cells and Endothelial Cells

    Joseph Schwager

    2016-01-01

    Full Text Available Nutrients transiently or chronically modulate functional and biochemical characteristics of cells and tissues both in vivo and in vitro. The influence of tomato aqueous extract (TAE on the in vitro inflammatory response of activated human peripheral blood leukocytes (PBLs and macrophages was investigated. Its effect on endothelial dysfunction (ED was analyzed in human umbilical vein endothelial cells (HUVECs. Murine macrophages (RAW264.7 cells, PBLs and HUVECs were incubated with TAE. They were activated with LPS or TNF-α in order to induce inflammatory processes and ED, respectively. Inflammatory mediators and adhesion molecules were measured by immune assay-based multiplex analysis. Gene expression was quantified by RT-PCR. TAE altered the production of interleukins (IL-1β, IL-6, IL-10, IL-12 and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10 in PBLs. TAE reduced ED-associated expression of adhesion molecules (ICAM-1, VCAM-1 in endothelial cell. In macrophages, the production of nitric oxide, PGE2, cytokines and ILs (TNF-α, IL-1β, IL-6, IL-12, which reflects chronic inflammatory processes, was reduced. Adenosine was identified as the main bioactive of TAE. Thus, TAE had cell-specific and context-dependent effects. We infer from these in vitro data, that during acute inflammation TAE enhances cellular alertness and therefore the sensing of disturbed immune homeostasis in the vascular-endothelial compartment. Conversely, it blunts inflammatory mediators in macrophages during chronic inflammation. A novel concept of immune regulation by this extract is proposed.

  18. Tomato Aqueous Extract Modulates the Inflammatory Profile of Immune Cells and Endothelial Cells.

    Schwager, Joseph; Richard, Nathalie; Mussler, Bernd; Raederstorff, Daniel

    2016-01-01

    Nutrients transiently or chronically modulate functional and biochemical characteristics of cells and tissues both in vivo and in vitro. The influence of tomato aqueous extract (TAE) on the in vitro inflammatory response of activated human peripheral blood leukocytes (PBLs) and macrophages was investigated. Its effect on endothelial dysfunction (ED) was analyzed in human umbilical vein endothelial cells (HUVECs). Murine macrophages (RAW264.7 cells), PBLs and HUVECs were incubated with TAE. They were activated with LPS or TNF-α in order to induce inflammatory processes and ED, respectively. Inflammatory mediators and adhesion molecules were measured by immune assay-based multiplex analysis. Gene expression was quantified by RT-PCR. TAE altered the production of interleukins (IL-1β, IL-6, IL-10, IL-12) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10) in PBLs. TAE reduced ED-associated expression of adhesion molecules (ICAM-1, VCAM-1) in endothelial cell. In macrophages, the production of nitric oxide, PGE2, cytokines and ILs (TNF-α, IL-1β, IL-6, IL-12), which reflects chronic inflammatory processes, was reduced. Adenosine was identified as the main bioactive of TAE. Thus, TAE had cell-specific and context-dependent effects. We infer from these in vitro data, that during acute inflammation TAE enhances cellular alertness and therefore the sensing of disturbed immune homeostasis in the vascular-endothelial compartment. Conversely, it blunts inflammatory mediators in macrophages during chronic inflammation. A novel concept of immune regulation by this extract is proposed. PMID:26840280

  19. Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells.

    Atienzar-Aroca, Sandra; Flores-Bellver, Miguel; Serrano-Heras, Gemma; Martinez-Gil, Natalia; Barcia, Jorge M; Aparicio, Silvia; Perez-Cremades, Daniel; Garcia-Verdugo, Jose M; Diaz-Llopis, Manuel; Romero, Francisco J; Sancho-Pelluz, Javier

    2016-08-01

    The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell-to-cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT-PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes. PMID:26999719

  20. Endothelial cell cytotoxicity of cotton bracts tannin and aqueous cotton bracts extract

    Using an in vitro cytotoxicity assay based on the release of 51Cr from cultured porcine thoracic aortic and pulmonary arterial endothelial cells, we have demonstrated that cotton bracts tannin is a potent endothelial cell cytotoxin. It produces dose-dependent lethal injury to both types of endothelial cells with the aortic cells, being somewhat more sensitive to tannin-mediated injury than the pulmonary arterial cells. Cytotoxic injury to the cells was biphasic. During the first 3 hr of exposure to tannin, no lethal injury was detected. However, during this period, profound changes in morphology were observed suggesting sublethal injury to the cells preceded the ultimate toxic damage. Comparison of the cytotoxicity dose curves for aqueous bracts extracts with those for tannin demonstrated that tannin was major cytotoxin present in bracts

  1. GABAB Receptors Expressed in Human Aortic Endothelial Cells Mediate Intracellular Calcium Concentration Regulation and Endothelial Nitric Oxide Synthase Translocation

    Xu-Ping Wang

    2014-01-01

    Full Text Available GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i in a number of cells (e.g., retina, airway epithelium and smooth muscle, but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA, in cultured human aortic endothelial cells (HAECs, and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

  2. The natural antioxidants, pomegranate extract and soy isoflavones, favourably modulate canine endothelial cell function.

    Baumgartner-Parzer, Sabina M; Waldenberger, Ferdinand Rudolf; Freudenthaler, Angelika; Ginouvès-Guerdoux, Amandine; McGahie, David; Gatto, Hugues

    2012-01-01

    Cardiovascular disease, preceded by vascular endothelial dysfunction, is a prominent cause of death in dogs. L-carnitine and taurine, well known for their antioxidative capacity, beneficially affect cardiovascular disease as well as certain dog cardiomyopathies. It is well established that vascular endothelial dysfunction precedes cardiovascular disease and that "vasoprotective factors" (NO and antioxidants) prevent apoptosis, whereas "risk factors" such as oxidized LDL, hyperglycemia, and free fatty acids trigger it in cultured human vascular endothelial cells. Whereas human vascular cell in vitro models are widely established and used for the characterisation of potential vasoprotective substances, such models are not available for canine endothelial cells. In the present study we therefore developed an in vitro model, which allows the testing of the effects of different substances on proliferation and apoptosis in canine aortic endothelial cells. This model was used to test L-carnitine, taurine, pomegranate extract, and Soy Isoflavones in comparison to reference substances (glutathione and pioglitazone) previously shown to modulate human endothelial cell function. L-carnitine and taurine neither exhibited antiproliferative nor antiapoptotic activities in the context of this study. However extracts from pomegranate and soy isoflavones dramatically reduced proliferation and apoptosis in a dose dependent fashion, being in line with a vasoprotective activity in dogs. PMID:23762588

  3. Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

    Rong Liu

    2014-01-01

    Full Text Available Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated retinoblastoma (Rb protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy.

  4. AAMP Regulates Endothelial Cell Migration and Angiogenesis Through RhoA/Rho Kinase Signaling.

    Hu, Jianjun; Qiu, Juhui; Zheng, Yiming; Zhang, Tao; Yin, Tieying; Xie, Xiang; Wang, Guixue

    2016-05-01

    Angiogenesis is a complicated process including endothelial cell proliferation, migration and tube formation. AAMP plays a role in regulating cell migration of multiple cell types. The purpose of this study was to investigate whether AAMP regulates angiogenesis, and to clarify the role of AAMP in the VEGF-induced angiogenesis. We found that AAMP expressed in multiple cell types and mainly localized in cytoplasm and membrane in vascular endothelial cells. Using tube formation assay in vitro and aortic ring assay, siRNA-mediated knockdown and antibody blockade of AAMP impaired VEGF-induced endothelial cell tube formation and aortic ring angiogenic sprouting. Mechanistic studies showed that AAMP expression was significantly upregulated by VEGF in a concentration and time-dependent manner. Moreover, VEGF recruited AAMP to the cell membrane protrusions. AAMP regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. AAMP knock-down reduced VEGF-induced actin stress fibers and collagen gel contraction. Furthermore, we identified RhoA/Rho kinase signaling as an important factor that contributes to the action of AAMP in regulating endothelial cell migration and angiogenesis. Altogether, these data demonstrated the critical role of AAMP in angiogenesis and suggested blocking AAMP could serve as a potential therapeutic strategy for angiogenesis-related diseases. PMID:26350504

  5. Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

    Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation

  6. QHREDGS enhances tube formation, metabolism and survival of endothelial cells in collagen-chitosan hydrogels.

    Jason W Miklas

    Full Text Available Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvβ3 or α5β1 were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction.

  7. QHREDGS enhances tube formation, metabolism and survival of endothelial cells in collagen-chitosan hydrogels.

    Miklas, Jason W; Dallabrida, Susan M; Reis, Lewis A; Ismail, Nesreen; Rupnick, Maria; Radisic, Milica

    2013-01-01

    Cell survival in complex, vascularized tissues, has been implicated as a major bottleneck in advancement of therapies based on cardiac tissue engineering. This limitation motivates the search for small, inexpensive molecules that would simultaneously be cardio-protective and vasculogenic. Here, we present peptide sequence QHREDGS, based upon the fibrinogen-like domain of angiopoietin-1, as a prime candidate molecule. We demonstrated previously that QHREDGS improved cardiomyocyte metabolism and mitigated serum starved apoptosis. In this paper we further demonstrate the potency of QHREDGS in its ability to enhance endothelial cell survival, metabolism and tube formation. When endothelial cells were exposed to the soluble form of QHREDGS, improvements in endothelial cell barrier functionality, nitric oxide production and cell metabolism (ATP levels) in serum starved conditions were found. The functionality of the peptide was then examined when conjugated to collagen-chitosan hydrogel, a potential carrier for in vivo application. The presence of the peptide in the hydrogel mitigated paclitaxel induced apoptosis of endothelial cells in a dose dependent manner. Furthermore, the peptide modified hydrogels stimulated tube-like structure formation of encapsulated endothelial cells. When integrin αvβ3 or α5β1 were antibody blocked during cell encapsulation in peptide modified hydrogels, tube formation was abolished. Therefore, the dual protective nature of the novel peptide QHREDGS may position this peptide as an appealing augmentation for collagen-chitosan hydrogels that could be used for biomaterial delivered cell therapies in the settings of myocardial infarction. PMID:24013716

  8. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  9. Regular Exercise Training Increases the Number of Endothelial Progenitor Cells and Decreases Homocysteine Levels in Healthy Peripheral Blood

    Choi, Jeong Kyu; Moon, Ki Myung; Jung, Seok Yun; Kim, Ji Yong; Choi, Sung Hyun; Kim, Da Yeon; Kang, Songhwa; Chu, Chong Woo; Kwon, Sang Mo

    2014-01-01

    Endothelial progenitor cells (EPCs) are known to play an important role in the repair of damaged blood vessels. We used an endothelial progenitor cell colony-forming assay (EPC-CFA) to determine whether EPC numbers could be increased in healthy individuals through regular exercise training. The number of functional EPCs obtained from human peripheral blood-derived AC133 stem cells was measured after a 28-day regular exercise training program. The number of total endothelial progenitor cell co...

  10. Tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in mechanically stimulated vascular endothelial cells.

    Osawa, M; Masuda, M; Harada, N; Lopes, R B; Fujiwara, K

    1997-03-01

    Fluid flow triggers signal transducing events, modulates gene expression, and remodels cytoskeletal structures in vascular endothelial cells (ECs). However, the primary steps of mechanoreception are still unknown. We have recently reported that a glycoprotein is rapidly tyrosine-phosphorylated in bovine ECs exposed to fluid flow or osmotic shock. Here were cloned a 3.4 kb cDNA encoding this protein and found that this was bovine PECAM-1. The tyrosine-phosphorylation level of PECAM-1 immunoprecipitated from mechanically stimulated bovine or human ECs increased. The PECAM-1 phosphorylation was not induced by reagents that triggered Ca2+ mobilization in ECs. An autophosphorylatable band comigrating with c-Src was co-immunoprecipitated with anti-PECAM-1, and c-Src phosphorylated and bound to a GST fusion protein containing the PECAM-1 cytoplasmic domain. A spliced mRNA form lacking amino acid residues 703-721 in the cytoplasmic domain was also expressed in bovine ECs, c-Src neither phosphorylated nor bound to the fusion protein containing the spliced PECAM-1 cytoplasmic domain which lacked one (Tyr 713) of the six tyrosine residues in the PECAM-1 cytoplasmic domain. These results suggest that the YSEI motif containing Tyr 713 is the Src phosphorylation/binding site. Our study is the first demonstration of inducible tyrosine phosphorylation of PECAM-1 and suggests involvement of PECAM-1 and Src family kinases in the sensing/signal transduction of mechanical stimuli in ECs. PMID:9084985

  11. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

    Wen-Shih Huang

    2015-12-01

    Full Text Available A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC. Hence, resistin may play a role in CRC development. Fulvic acid (FA, a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative and SW-48 (p53-positive CRC cells and human umbilical vein endothelial cells (HUVECs were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1. Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin.

  12. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Song, Kai [Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Shandong Province (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Song, Yong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Department of Stomatology, Liu Zhou People' s Hospital, Guangxi (China); Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Liu, Ke, E-mail: liuke.1999@aliyun.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China)

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  13. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo

  14. Effects of radiation on capillary endothelial cells derived from Mongolian gerbil brain

    Mori, Shuichi; Tanaka, Ryuichi; Minakawa, Takashi; Onda, Kiyoshi (Niigata Univ. (Japan). Brain Research Inst.)

    1990-09-01

    Confluent monolayers of capillary endothelial cells derived from Mongolian gerbil brain were irradiated with a single exposure of x-rays, and their radiosensitivity and sequential changes in morphology, staining intensity for factor VIII-related antigen (F VIII RAg), and capacity to produce prostacyclin (PGI{sub 2}) were examined. The radiobiologic parameters that characterized the dose-response survival curve for these cells were found to be n=1.9, D{sub q}=140 rad, and D{sub 0}=190 rad. Morphologically, nuclear and cytoplasmic swelling, vacuolation of cytoplasm, and giant cell formation occurred in a dose dependent manner after 24 hours from irradiation. Decreased staining intensity for F VIII RAg was observed in morphologically affected cells. The capacity to synthesize PGI{sub 2} was significantly enhanced at 24 hours, but less significant at 72 hours after irradiation. The present data suggest that the radiosensitivity of brain capillary endothelial cells may be somewhat lower than that of endothelial cells originated from larger vessels, and that radiation induced morphological and functional changes in the brain capillary endothelial cells may be quantitatively similar to the changes in endothelial cells of larger vessels. (author).

  15. Sickle Cell Disease Activates Peripheral Blood Mononuclear Cells to Induce Cathepsins K and V Activity in Endothelial Cells

    Platt, Manu O.; Sindhuja Surapaneni; Keegan, Philip M.

    2012-01-01

    Sickle cell disease is a genetic disease that increases systemic inflammation as well as the risk of pediatric strokes, but links between sickle-induced inflammation and arterial remodeling are not clear. Cathepsins are powerful elastases and collagenases secreted by endothelial cells and monocyte-derived macrophages in atherosclerosis, but their involvement in sickle cell disease has not been studied. Here, we investigated how tumor necrosis alpha (TNFα) and circulating mononuclear cell adhe...

  16. Tpl2 Inhibitors Thwart Endothelial Cell Function in Angiogenesis and Peritoneal Dissemination

    Wen-Jane Lee

    2013-09-01

    Full Text Available Angiogenesis is critical in the development of cancer, which involves several angiogenic factors in its peritoneal dissemination. The role of protein tumor progression locus 2 (Tpl2 in angiogenic factor-related endothelial cell angiogenesis is still unclear. To understand the precise mechanism(s of Tpl2 inhibition in endothelial cells, this study investigated the role of Tpl2 in mediating angiogenic signals using in vitro, in vivo, and ex vivo models. Results showed that inhibition of Tpl2 inhibitor significantly reduced peritoneal dissemination in a mouse model by positron emission tomography/computed tomography imaging. Simultaneously, inhibiting Tpl2 blocked angiogenesis in tumor nodules and prevented angiogenic factor-induced proliferating cell nuclear antigen (PCNA in endothelial cells. Vascular endothelial growth factor (VEGF or chemokine (C-X-C motif ligand 1 (CXCL1 increased Tpl2 kinase activity and phosphorylation in a dose- and time-dependent manner. Furthermore, Tpl2 inhibition or ablation by siRNA prevented the angiogenic signal-induced tube formation in Matrigel plug assay or aortic ring assay. Inhibiting Tpl2 also prevented the angiogenic factor-induced chemotactic motility and migration of endothelial cells. Tpl2 inhibition by CXCL1 or epidermal growth factor in endothelial cells was associated with inactivation of CCAAT/enhancer binding protein β, nuclear factor κ light-chain enhancer of activated B cells, and activating protein 1 and suppression of VEGF expression. Thus, Tpl2 inhibitors thwart Tpl2-regulated VEGF by inactivating transcription factors involved in angiogenic factor-triggered endothelial cell angiogenesis. These results suggest that the therapeutic inhibition of Tpl2 may extend beyond cancer and include the treatment of other diseases involving pathologic angiogenesis.

  17. Effects of bone marrow-derived endothelial progenitor cell transplantation on vein microenvironment in a rat model of chronic thrombosis

    LI Xiao-qiang; MENG Qing-you; WU Hao-rong

    2007-01-01

    Background Endothelial progenitor cells(EPCs) have been used in both experimental studies and clinical treatments of limb ischemia,as well as in the construction of engineered vascular tissue.The objective of this study was to investigate the effects of transplanted bone marrow-derived EPCs on the vein microenvironment in a rat model of chronic vein thrombosis.Methods Mononuclear cells were isolated from the bone marrow of immature rats by density gradient centrifugation,cultured,and then transplanted into experimentally induced thrombi into inferior vena cava through the femoral vein.Vascular endothelial growth factor(VEGF),angiopoietin-1(ANG-1) and monocyte chemotactic protein-1(MCP-1) mRNA and protein expression levels were measured by real-time quantitative polymerase chain reaction and Western blotting of thrombi and adjacent caval walls 28 days post-transplantation.Results Levels of VEGF,ANG-1,and MCP-1 mRNA in EPC-transplanted thrombi were 100%,230.7%,and 212.5% of levels detected in the sham-operated group(P<0.01),and 99.9%,215.4%,and 177.8% of levels detected in the experimental control group(P<0.01).VEGF,ANG-1 and MCP-1 protein levels exhibited a similar trend.Conclusions Transplanted bone marrow-derived EPCs appear to alter the vein microenvironment in experimentally induced chronic vein thrombosis by upregulating cytokines associated with thrombic organization and recanalization.

  18. Bach1 Induces Endothelial Cell Apoptosis and Cell-Cycle Arrest through ROS Generation

    Wang, Xinhong; Liu, Junxu; Jiang, Li; Wei, Xiangxiang; Niu, Cong; Wang, Rui; Zhang, Jianyi; Yao, Kang

    2016-01-01

    The transcription factor BTB and CNC homology 1 (Bach1) regulates genes involved in the oxidative stress response and cell-cycle progression. We have recently shown that Bach1 impairs cell proliferation and promotes apoptosis in cultured endothelial cells (ECs), but the underlying mechanisms are largely uncharacterized. Here we demonstrate that Bach1 upregulation impaired the blood flow recovery from hindlimb ischemia and this effect was accompanied both by increases in reactive oxygen species (ROS) and cleaved caspase 3 levels and by declines in the expression of cyclin D1 in the injured tissues. We found that Bach1 overexpression induced mitochondrial ROS production and caspase 3-dependent apoptosis and its depletion attenuated H2O2-induced apoptosis in cultured human microvascular endothelial cells (HMVECs). Bach1-induced apoptosis was largely abolished when the cells were cultured with N-acetyl-l-cysteine (NAC), a ROS scavenger. Exogenous expression of Bach1 inhibited the cell proliferation and the expression of cyclin D1, induced an S-phase arrest, and increased the expression of cyclin E2, which were partially blocked by NAC. Taken together, our results suggest that Bach1 suppresses cell proliferation and induces cell-cycle arrest and apoptosis by increasing mitochondrial ROS production, suggesting that Bach1 may be a promising treatment target for the treatment of vascular diseases. PMID:27057283

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

    Qijun Jiang

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

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

    Kaemisa Srisen

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

  1. The effect of bisphosphonates on the endothelial differentiation of mesenchymal stem cells.

    Sharma, Dileep; Hamlet, Stephen Mark; Petcu, Eugen Bogdan; Ivanovski, Saso

    2016-01-01

    The contribution of the local stem cell niche to providing an adequate vascular framework during healing cannot be overemphasized. Bisphosphonates (BPs) are known to have a direct effect on the local vasculature, but their effect on progenitor cell differentiation is unknown. This in vitro study evaluated the effect(s) of various BPs on the differentiation of human placental mesenchymal stem cells (pMSCs) along the endothelial lineage and their subsequent functional and morphogenic capabilities. pMSC multipotency was confirmed by successful differentiation into cells of both the osteogenic and endothelial lineages, as demonstrated by positive Alizarin Red S staining and Ac-LDL uptake. pMSC differentiation in the presence of non-cytotoxic BP concentrations showed that nitrogen containing BPs had a significant inhibitory effect on cell migration and endothelial marker gene expression, as well as compromised endothelial differentiation as demonstrated using von Willebrand factor immunofluorescence staining and tube formation assay. This in vitro study demonstrated that at non-cytotoxic levels, nitrogen-containing BPs inhibit differentiation of pMSCs into cells of an endothelial lineage and affect the downstream functional capability of these cells supporting a multi-modal effect of BPs on angiogenesis as pathogenic mechanism contributing to bone healing disorders such as bisphosphonate related osteonecrosis of the jaws (BRONJ). PMID:26857282

  2. Study of apoptosis induced by cytostatics and vegetal extracts on human endothelial cell line.

    Bârzu, Simona Natalia; Bădulescu, Maria Mihaela; Lupu, Andreea Roxana; Cremer, Lidia; Szegli, G; Kerek, F; Călugăru, Ana

    2008-01-01

    Angiogenesis, the biological process by which new capillaries are formed from pre-existing vessels, is a tightly controlled and complex process involving several factors with both stimulating and inhibiting steps. In solid tumor growth, a specific clinical turning point is the transition to the vascular phase. Once it develops an intrinsic vascular network, a tumor grows indefinitely. Tumor angiogenesis depends mainly on the release by neoplasic cells of growth factors specific for endothelial cells (ECs), able to stimulate growth of the host blood vessels. The aim of this study was to analyze the apoptotic effect of some cytostatics, Vinblastine, Rapamycin and Doxorubicin, and vegetal extracts (called VOB) isolated and purified from Vitis sp., on human EA.hy926 endothelial cell line. In a proliferation assay using Crystal Violet, we demonstrated that Vinblastine and Rapamycin cytostatics have synergistic effect on endothelial cell line EA.hy926 growth inhibition. The inhibitory effects of Vinblastine and Doxorubicin were enhanced by VOB vegetal extracts. A combined treatment of cytostatics and VOB vegetal extracts resulted in a stronger antiproliferative effect of EA.hy926 endothelial cells. Results obtained regarding the apoptosis induced on EA.hy926 endothelial cells showed that each compound alone was able to induce a significant percent of apoptotic cells in a dose-dependent manner. PMID:19284159

  3. Internalization of targeted quantum dots by brain capillary endothelial cells in vivo.

    Paris-Robidas, Sarah; Brouard, Danny; Emond, Vincent; Parent, Martin; Calon, Frédéric

    2016-04-01

    Receptors located on brain capillary endothelial cells forming the blood-brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery. PMID:26661181

  4. Influence of curvature on the morphology of brain microvascular endothelial cells

    Ye, Mao; Yang, Zhen; Wong, Andrew; Searson, Peter; Searson Group Team

    2013-03-01

    There are hundreds or thousands of endothelial cells around the perimeter of a single artery or vein, and hence an individual cell experiences little curvature. In contrast, a single endothelial cell may wrap around itself to form the lumen of a brain capillary. Curvature plays a key role in many biological, chemical and physical processes, however, its role in dictating the morphology and polarization of brain capillary endothelial cells has not been investigated. We hypothesize that curvature and shear flow play a key role in determining the structure and function of the blood-brain barrier (BBB). We have developed the ``rod'' assay to study the influence of curvature on the morphology of confluent monolayers of endothelial cells. In this assay cells are plated onto glass rods pulled down to the desired diameter in the range from 5 - 500 μm and coated with collagen. We show that curvature has a significant influence on the morphology of endothelial cells and may have an important role in blood-brain barrier function.

  5. Tirofiban counteracts endothelial cell apoptosis through the VEGF/VEGFR2/pAkt axis.

    Giordano, Arturo; Romano, Simona; D'Angelillo, Anna; Corcione, Nicola; Messina, Stefano; Avellino, Raffaella; Biondi-Zoccai, Giuseppe; Ferraro, Paolo; Romano, Maria Fiammetta

    2016-05-01

    Tirofiban is used in the treatment of patients with acute coronary syndrome submitted to percutaneous coronary intervention (PCI). We have, previously, shown that tirofiban stimulates VEGF expression and promotes proliferation of endothelial cells. VEGF is a well known inhibitor of endothelial cell apoptosis. TNF-α is a pro-apoptotic cytokine released in the site of a vascular injury, including balloon angioplasty. We thought to investigate whether tirofiban was able to protect endothelial cells from cell death induced by TNF-α. For this study, we used human umbilical vein endothelial cells (HUVEC). Analysis of apoptosis was performed by propidium iodide incorporation, annexin V staining and measure of active caspase 3 levels. Western blot served for a semiquantitative measure of Akt activation, VEGF, and the pro-apoptotic Bim and Bak. Our results show that TNF-α was unable to activate caspase 3 and produce cell death in the presence of tirofiban. Activation of apoptosis was preceded by upregulation of Bim and Bak that resulted decreased after addition of tirofiban. The anti-apoptosis effect of tirofiban was reproduced by VEGF and counteracted by VEGFR2 blockade and the cation chelating agent ethylene glycol tetraacetic acid (EGTA). The use of p-Akt inhibitor, BEZ235,and Akt knockdown, suggested that pAkt mediated the prosurvival effect of tirofiban. In conclusion, tirofiban protects endothelial cells from apoptosis stimulated by TNF-α, due to its ability to stimulate VEGF production. PMID:26699078

  6. CD73 represses pro-inflammatory responses in human endothelial cells

    Ridley Anne J

    2010-02-01

    Full Text Available Abstract Background CD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been reported to regulate expression of pro-inflammatory molecules in mouse endothelium. Our aim is to determine the function of CD73 in human endothelial cells. Methods We used RNAi to deplete CD73 levels in human umbilical cord endothelial cells (HUVECs. Results CD73 depletion resulted in a strong reduction in adenosine production, indicating that CD73 is the major source of extracellular adenosine in HUVECs. We find that CD73 depletion induces a similar response to pro-inflammatory stimuli such as the cytokine TNF-α. In CD73-depleted cells, surface levels of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin increase. This correlates with increased translocation of the transcription factor NF-kB to the nucleus, which is known to regulate ICAM-1, VCAM-1 and E-selectin expression in response to TNF-α. Adhesion of monocytic cells to endothelial cells is enhanced. In addition, CD73-depleted cells become elongated, have higher levels of stress fibres and increased endothelial permeability, resembling known responses to TNF-α. Conclusions These results indicate that CD73 normally suppresses pro-inflammatory responses in human endothelial cells.

  7. Disruption of Nrf2 Signaling Impairs Angiogenic Capacity of Endothelial Cells: Implications for Microvascular Aging

    Valcarcel-Ares, M. Noa; Gautam, Tripti; Warrington, Junie P.; Bailey-Downs, Lora; Sosnowska, Danuta; de Cabo, Rafael; Losonczy, Gyorgy; Sonntag, William E; Ungvari, Zoltan; Csiszar, Anna

    2012-01-01

    The redox-sensitive transcription factor NF-E2–related factor 2 (Nrf2) plays a key role in preserving a healthy endothelial phenotype and maintaining the functional integrity of the vasculature. Previous studies demonstrated that aging is associated with Nrf2 dysfunction in endothelial cells, which alters redox signaling and likely promotes the development of large vessel disease. Much less is known about the consequences of Nrf2 dysfunction at the level of the microcirculation...

  8. Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice

    Segal, Mark S.; Sautina, Laura; Li, Shiyu; Diao, YanPeng; Agoulnik, Alexander I.; Kielczewski, Jennifer; McGuane, Jonathan T.; Grant, Maria B.; Conrad, Kirk P.

    2012-01-01

    The ovarian peptide hormone, relaxin, circulates during pregnancy, contributing to profound maternal vasodilation through endothelial and nitric oxide (NO)–dependent mechanisms. Circulating numbers of bone marrow–derived endothelial cells (BMDECs), which facilitate angiogenesis and contribute to repair of vascular endothelium, increase during pregnancy. Thus, we hypothesized that relaxin enhances BMDEC NO production, circulating numbers, and function. Recombinant human relaxin-2 (rhRLX) stimu...

  9. Biophysiochemical properties of endothelial cells cultured on bio-inspired collagen films

    Seo, Eunseok; Seo, Kyung Won; Gil, Jung-Eun; Ha, Young-Ran; Yeom, Eunseop; Lee, Seungchul; Lee, Sang Joon

    2014-01-01

    Background In this study, we investigated the effect of the extracellular matrix on endothelial dysfunction by careful observation of human umbilical vein endothelial cells (HUVECs) cultured on denatured collagen film. Results HUVECs on denatured collagen film showed relatively high surface roughness compared with normal HUVECs. The expression levels of MMP-1, MMP-2 and CD146 increased in the ECs on denatured collagen film. In addition, we examined the accumulation of fluorescent beads on HUV...

  10. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh

    Meng Deng

    2015-01-01

    Full Text Available Adipose-derived stem cell (ADSC is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34− when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs.

  11. Chronic Hypoxia Promotes Pulmonary Artery Endothelial Cell Proliferation through H2O2-Induced 5-Lipoxygenase

    Porter, Kristi M.; Bum-Yong Kang; Adesina, Sherry E.; Murphy, Tamara C.; C Michael Hart; Sutliff, Roy L.

    2014-01-01

    Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimula...

  12. Hyperglycemia affects Matrix Metalloproteinase Secretion in Human Umbilical Vein Endothelial Cells in vitro

    2011-01-01

    Goal: The aim of the current thesis was to study the effect of hyperglycemia (HG) on MMP secretion from human primary endothelial cells. Background: Diabetes Mellitus is characterized by abnormal high levels of glucose in the blood. Endothelial dysfunction is a serious complication of sustained hyperglycemia. Diabetic complications are main reasons for mortality in diabetes. The matrix metalloproteinases (MMPs) and their corresponding inhibitors are the main regulators of the turnover of ...

  13. The Role of Fatty Acids and Caveolin-1 in TNF-α-Induced Endothelial Cell Activation

    Wang, Lei; Lim, Eun-Jin; Toborek, Michal; Hennig, Bernhard

    2008-01-01

    Hypertriglyceridemia and associated high circulating free fatty acids are important risk factors of atherosclerosis. In contrast to omega-3 fatty acids, linoleic acid, the major omega-6 unsaturated fatty acid in the American diet, may be atherogenic by amplifying an endothelial inflammatory response. We hypothesize that omega-6 and omega-3 fatty acids can differentially modulate TNF-α-induced endothelial cell activation and that functional plasma membrane microdomains called caveolae are requ...

  14. Rescue of Brain Function Using Tunneling Nanotubes Between Neural Stem Cells and Brain Microvascular Endothelial Cells.

    Wang, Xiaoqing; Yu, Xiaowen; Xie, Chong; Tan, Zijian; Tian, Qi; Zhu, Desheng; Liu, Mingyuan; Guan, Yangtai

    2016-05-01

    Evidence indicates that neural stem cells (NSCs) can ameliorate cerebral ischemia in animal models. In this study, we investigated the mechanism underlying one of the neuroprotective effects of NSCs: tunneling nanotube (TNT) formation. We addressed whether the control of cell-to-cell communication processes between NSCs and brain microvascular endothelial cells (BMECs) and, particularly, the control of TNT formation could influence the rescue function of stem cells. In an attempt to mimic the cellular microenvironment in vitro, a co-culture system consisting of terminally differentiated BMECs from mice in a distressed state and NSCs was constructed. Additionally, engraftment experiments with infarcted mouse brains revealed that control of TNT formation influenced the effects of stem cell transplantation in vivo. In conclusion, our findings provide the first evidence that TNTs exist between NSCs and BMECs and that regulation of TNT formation alters cell function. PMID:26041660

  15. Investigation of the cytotoxicity of CCVD carbon nanotubes towards human umbilical vein endothelial cells

    Flahaut, Emmanuel; Durrieu, Marie-Christine; Remy-Zolghadri, Murielle; Bareille, Reine; Baquey, Charles

    2006-01-01

    The cytotoxicity of different samples of carbon nanotubes synthesised by catalytic chemical vapour deposition was investigated towards human umbilical vein endothelial cells, using two cytotoxicity standard assays (neutral red assay for the cell viability and MTT assay—tetrazolinium salt—for the cell metabolic activity). No cytotoxicity was found for any sample.

  16. Reinforcing endothelial junctions prevents microvessel permeability increase and tumor cell adhesion in microvessels in vivo

    Fu, Bingmei M.; Yang, Jinlin; Cai, Bin; Fan, Jie; Zhang, Lin; Zeng, Min

    2015-10-01

    Tumor cell adhesion to the microvessel wall is a critical step during tumor metastasis. Vascular endothelial growth factor (VEGF), a secretion of tumor cells, can increase microvessel permeability and tumor cell adhesion in the microvessel. To test the hypothesis that inhibiting permeability increase can reduce tumor cell adhesion, we used in vivo fluorescence microscopy to measure both microvessel permeability and adhesion rates of human mammary carcinoma MDA-MB-231 cells in post-capillary venules of rat mesentery under the treatment of VEGF and a cAMP analog, 8-bromo-cAMP, which can decrease microvessel permeability. By immunostaining adherens junction proteins between endothelial cells forming the microvessel wall, we further investigated the structural mechanism by which cAMP abolishes VEGF-induced increase in microvessel permeability and tumor cell adhesion. Our results demonstrate that 1) Pretreatment of microvessels with cAMP can abolish VEGF-enhanced microvessel permeability and tumor cell adhesion; 2) Tumor cells prefer to adhere to the endothelial cell junctions instead of cell bodies; 3) VEGF increases microvessel permeability and tumor cell adhesion by compromising endothelial junctions while cAMP abolishes these effects of VEGF by reinforcing the junctions. These results suggest that strengthening the microvessel wall integrity can be a potential approach to inhibiting hematogenous tumor metastasis.

  17. Cytotoxicity of Metal Ions Released from Nitinol Alloys on Endothelial Cells

    Haider, W.; Munroe, N.; Tek, V.; Gill, P. K. S.; Tang, Y.; McGoron, A. J.

    2011-07-01

    Most implantable medical devices are expected to function in the body over an extended period of time. Therefore, immersion tests under simulated conditions can be useful for assessing the amount of metal ions released in situ. In this investigation, dissolved ions from as-received binary and ternary Nitinol alloys in cell culture media were periodically measured under static and dynamic conditions. Endothelial cells were grown in aliquots of culture media obtained and the effect of dissolved ions on cell proliferation and viability of endothelial cells (HUVEC) was studied by cytotoxicity assays. The concentration of metal ions in the media was measured by inductively coupled plasma mass spectrometry.

  18. Role of TRPM2 in H(2O(2-induced cell apoptosis in endothelial cells.

    Lei Sun

    Full Text Available Melastatin-like transient receptor potential channel 2 (TRPM2 is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2O(2-induced cytosolic Ca(2+ ([Ca(2+](i elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3, which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+](i rise and whole-cell current change in response to H(2O(2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA had similar inhibitory effect. H(2O(2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2O(2-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+](i and whole-cell currents to H(2O(2. TRPM2 overexpression also aggravated the H(2O(2-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+ overload in response to H(2O(2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.

  19. On endocytosis of foreign ferritin and occurrence of phagolysosomes in fish heart endothelial cells.

    Leknes, Ingvar Leiv

    2016-04-01

    In the present study the ultrastructure and function of the endothelial cells enveloping the muscle trabeculae in heart in two teleosts, platyfish and firemouth cichlid, are described and discussed. These cells displayed a structure making them able to take up large amounts of foreign ferritin particles from the blood stream. The ferritin particles were assembled into huge phagolysosomes. Large amounts of Prussian blue were precipitated throughout these lysosomes when treated with acid ferrohexacyanide solution. The occurrence of Prussian blue precipitations in the control heart endothelial cells after Schmorl's solution, suggests that these cells normally contain undigestible material, a finding which strengthens the view that this tissue is involved in blood clearance in the present species. In conclusion, these heart endothelial cells seem able to perform a very efficient blood clearance of scavenger and foreign macromolecules and particles in the present species. PMID:26852295

  20. Urokinase mediates endothelial cell survival via induction of the X-linked inhibitor of apoptosis protein

    Prager, Gerald W; Mihaly, Judit; Brunner, Patrick M;

    2008-01-01

    factor kappaB (NF-kappaB) p52 activation. Indeed, blocking NF-kappaB activation by using specific NF-kappaB inhibitors abolished uPA-induced cell survival as it blocked uPA-induced XIAP up-regulation. Furthermore, down-regulating XIAP expression by small interfering RNA (siRNA) significantly reduced u......Urokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and......PA-dependent endothelial cell survival. This mechanism is also important for VEGF-induced antiapoptosis because VEGF-dependent up-regulation of XIAP was found defective in uPA(-/-) endothelial cells. This led us to conclude that uPA is part of a novel NF-kappaB-dependent cell survival pathway....