Induction of endothelial-leukocyte adhesion molecules such as E-selectin, ICAM-1 and VCAM-1 on endothelial cells (EC) lining post-capillary venules is a hallmark of TNF-mediated inflammation. To test the hypothesis that TNF-induced gene expression is regulated by the subcellular distribution and covalent modifications of signaling molecules, the investigators will analyze the intracellular trafficking and shedding of TNF receptor I (TNFR1) as well as the ligand-induced assembly, trafficking and modification of the TNFRI -associated signaling complex in human EC in cell and skin organ culture. To do so they will use state-of-the-art optical methods (confocal fluorescence, fluorescence resonance energy transfer and two photon microscopy) complemented by biochemical, immunochemical and genetic approaches. Second, to test the hypothesis that kinetic and anatomic differences in adhesion molecule expression is regulated by differences in NF-KB and AP-1 activation and composition, they will compare transcriptional control of E-selectin to that of ICAM-1 and VCAM-1, focusing on differences revealed by the actions of reductants or of a NEMO binding domain peptide. They will also analyze the basis of differential regulation of the E-selectin gene in arteriolar vs capillary vs. venular EC in a novel model which induces vascular differentiation of human umbilical vein EC (HUVEC) implanted into immunodeficient mice. To do so they will use immunobIotting, EMSA and supershift assays, promoter-reporter gene transduction and chromatin immunoprecipitation. Third, to test the hypothesis that sustained E-selectin expression on dermal microvessels arises from differences in the process of E-selectin internalization, they will analyze internalization pathways in human dermal microvascular EC (HDMEC) and other EC types, identifying the motif in the E-selectin C-terminus and EC proteins that mediate internalization. To do so they will transduce wild type and mutant E-selectin molecules into HDMEC and HUVEC and use biochemical, immunochemical and genetic techniques. They will also use single particle tracking microscopy to analyze the lateral movements and entry into coated pits of individual E-selectin molecules on the surface of HDMEC vs. HUVEC. Insights from these experiments may lead to more nuanced strategies to control inflammation.
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