E-selectin is a cytokine-induced endothelial cell (EC) adhesion molecule that mediates the recruitment of circulating leukocytes into sites of inflammation. Persistent expression of E-selectin in the post-capillary venules of chronic active inflamed skin may favor skin-specific homing of a subset of memory T cells. I previously compared the kinetics of E-selectin expression on cultured human dermal microvascular endothelial cells (HDMEC) to the expression on human umbilical vein endothelial cells (HUVEC), and showed that HDMEC sustain higher levels of expression at 24 hours by slower internalization. My FIRST HYPOTHESIS for the proposed research is that regulation of E-selectin internalization, which controls persistence of expression, is governed by specific interactions between the E-selectin cytoplasmic domain and other EC proteins. My SECOND HYPOTHESIS is that differences in these interactions will account for differences in E-selectin persistence between cultured HDMECs and HUVECs. My THIRD HYPOTHESIS is that recruitment of memory T lymphocytes into diseased skin can be blocked by agents that alter function of EC proteins which determine E-selectin persistence through functional interactions with the cytoplasmic domain of E-selectin.
The Specific Aims of this proposal are 1) to identify key E-selectin cytoplasmic residues mediating internalization in HUVEC, 2) to compare E-selectin internalization signals in HDMEC and HUVEC, and 3) to isolate and characterize HDMEC proteins that recognize and functionally interact with the internalization signal(s) of E-selectin identified in Aims 1 and 2. The third hypothesis, which is the rationale for performing these experiments, will form the basis of future work, allowing me to extend my findings to develop novel therapeutics for inflammatory and malignant skin diseases.