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      Establishment of Immortalized Human Glomerular Endothelial Cell Lines and Their Application

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          Background: The experimental use of cultured endothelial cells derived from the microvasculature such as glomerular endothelial cells possesses many problems, including limited growth rates, heterogeneity and loss of specific cell properties dependent on culture passage. In this study, we attempted to establish immortalized, human glomerular endothelial cell (HGEC) lines. Methods: HGECs of up to 5 passages were transformed by infection with simian virus (SV)-40. After 4–6 weeks the surviving, foci-forming cells were harvested and cloned. Each cell line obtained was examined by immunofluorescence with antibodies to antigens specific for vascular endothelial cells. The expression of adhesion molecules on cells incubated with or without TNF-α was also examined by cellular ELISA. Results: Three of twelve cell lines obtained expressed SV40 large T-antigen and von Willebrand’s factor, as well as endothelial cell adhesion molecules including ICAM-1 (CD54), PECAM-1 (CD31) and E-selectin (CD62E). In these cells, ICAM-1 and E-selectin expression was up-regulated by TNF-α, as in native cultured HGEC. Conclusions: These cell lines maintain the morphologic and functional characteristics of HGEC even after 60 passages. Immortalized HGEC will be useful for research on glomerular cell biology and provide a standardized substrate for anti-endothelial cell antibody detection.

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          Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

          We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.
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            Angiogenesis in vitro.

            Cloned capillary endothelial cells, cultured in tumour-conditioned medium, form capillary tubes. By light and electron microscopy these tubes resemble capillaries in vivo. This first demonstration of angiogenesis in vitro: (1) shows that all the information necessary to develop an entire capillary network in vitro is expressed by one cell type; (2) suggests a mechanism for lumen formation; and (3) offers a possibility of distinguishing between direct and indirect angiogenesis factors.
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              HMEC-1: Establishment of an Immortalized Human Microvascular Endothelial Cell Line


                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                February 2005
                14 January 2005
                : 99
                : 2
                : e38-e45
                aDepartment of Cellular Physiology, Institute of Nephrology and bDivision of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; cDepartment of Immunology, Institute of Medical Microbiology and Hygiene, Freiburg, Germany
                83096 Nephron Exp Nephrol 2005;99:e38–e45
                © 2005 S. Karger AG, Basel

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                Figures: 4, Tables: 1, References: 31, Pages: 1
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