Microvascular endothelial cells play a central """"""""gatekeeper"""""""" role in inflammation, through their ability to undergo activation, express cell adhesion molecules and recruit circulating immune cells into tissues and foci of inflammation. Nitric oxide (NO) generated by the activated vascular endothelium play a key regulatory role in inflammation by turning off vascular activation and enhanced leukocyte recruitment, key steps in the initiation, maintenance and resolution of an inflammatory response. The acquisition of fundamental knowledge regarding the role of vascular endothelium in human mucosal immunity and inflammatory bowel disease (IBD) has been significantly advanced as a direct result of our novel methodology to isolate, culture and study human intestinal microvascular endothelial microvascular endothelial cells (HIMEC) from normal and IBD- involved surgically resected intestine. Preliminary investigation has revealed that HIMEC isolated from IBD patient tissue have a heightened capacity to bind leukocytes following selective activation with pro-inflammatory cytokines and LPS, compared with endothelial cells isolated from normal intestinal tissue. In control HIMEC, generation of nitric oxide (NO) via the high output, inducible nitric oxide synthase (iNOS) pathway following activation, plays a key role in down-regulating leukocyte adhesion. Therefore, the central hypothesis of this proposal is: Alterations in the ability of IBD HIMEC to produce NO following activation contribute to the heightened leukocyte binding capacity. This hypothesis will be tested with the following, two specific aims: 1) Characterize production of NO in IBD HIMEC following activation at the gene, protein and functional levels; 2) Define the status of endothelial derived NO on IBD HIMEC activation by examining intracellular oxidant stress and its effect on cell adhesion molecular at the gene, protein and function levels. The purpose of this National Institutes of Diabetes, Digestive and Kidney Diseases R03 grant will be to extend the original aims outlined by the Principal Investigator's K award (DK02417) to include investigation of the nitric oxide biology of IBD HIMEC. The information generated by this research will define fundamental alterations in endothelial-leukocyte interaction in IBD, and the mechanisms of nitric oxide modulation of intestinal endothelial activation, and potentially point to novel therapeutic strategies targeting endothelial-leukocyte interaction in IBD patient treatment.
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