A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92-100% of the cells were positive for beta 2-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the gamma-interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.
We investigated the effects of recombinant human tumor necrosis factor-alpha (TNF) on cell proliferation and on expression of MHC class II antigens and intercellular adhesion molecule ICAM-1 in human dermal microvascular endothelial cells (HDMEC) derived from human foreskin. Second-passage HDMEC were treated with 0.1-10,000 U/ml TNF for up to 6 d, and cell growth was assessed by cell counts and a recently developed fluorogenic assay using 4-methylumbelliferyl heptanoate as a substrate. APAAP immunocytochemistry was performed using monoclonal antibodies against HLA-DR, HLA-DQ, and ICAM-1. TNF did not markedly inhibit the growth of HDMEC but induced expression of HLA-DR (1,000 U/ml and more) and of ICAM-1 (1 U/ml and more). Combination with interferon-gamma led to synergistic ICAM-1 induction. These results demonstrate a profound effect of TNF on the activation of dermal microvascular endothelial cells and suggest a major role of TNF in the mediation of leucocyte adhesion to endothelial cells of the skin microvasculature with possible implications for the initiation and maintenance of inflammatory skin processes.
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