IMMORTALIZED HUMAN SKIN ENDOTHELIAL CELLS

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Endothelial cells are critically important in a variety of physiologic and pathologic processes which occur primarily at the level of the microvasculature. It has been clearly demonstrated that microvascular endothelial cells differ distinctly from large vessel endothelial cells, but extensive study of microvascular endothelial cells has been severely limited by inherent difficulties in isolation and propagation of these cells. Although it is possible to isolate and culture microvascular endothelial cells from human skin, only relatively small numbers of cells with a very limited lifespan can be obtained. This limits the scope of the scientific questions that can be addressed. The study of human microvascular endothelial cells would be greatly facilitated by the development of an immortalized human microvascular endothelial cell line(s) which retains the phenotype and functional properties characteristic of human microvascular endothelial cells. Such a cell line would have widespread utility in the study of a number of physiologic and pathologic processes ranging from gene regulation and cell-cell communication to endothelial cell tumor biology. It would also eliminate the difficulties engendered by the use of mixtures of microvascular endothelial cells from multiple donors. Unfortunately, no human microvascular endothelial cell lines exist that retain an endothelial cell phenotype. In this proposal, we plan to 1) create immortalized microvascular endothelial cells by transforming pure cultures of human dermal microvascular endothelial cells (HDMEC) using simian virus 40 large T antigen. We will also generate transformed HDMEC using temperature sensitive mutants of SV40 large T which will proliferate like other immortalized cells, but revert to a non- immortalized phenotype at the non-permissive temperature; 2) characterize the immortalized cell lines for their retention of essential HDMEC phenotypic and functional characteristics including cell adhesion molecule expression and regulation; 3) establish stable transfectants using these immortalized HDMEC and portions of the transcriptional regulatory region of the human intercellular adhesion molecule 1 (ICAM-1) gene to characterize the role of cellular context in the regulation of ICAM-1 expression; and 4) characterize the signals and molecular mechanisms regulating the skin specific leukocyte homing protein E- selectin. These studies will produce important novel reagents and new insights into the role of microvascular endothelial cells and adherence molecules in cutaneous inflammation.