T-type Calcium Channels and von Willebrand Factor Release

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The acute chest syndrome is initiated by lung inflammation that induces increased adhesion of sickled red blood cells to pulmonary microvascular endothelium. Emerging evidence indicates the interaction between red blood cells and endothelium is dynamic. While in the non-inflamed state perfusion is facilitated by antiadhesive proteins expressed on the endothelial surface, in the inflamed state vaso-occlusion is partly caused by upregulation of adhesive proteins such as P-selectin and release of von Willebrand factor (vWf) from microvascular endothelium. The secretory organelle in endothelium is the endothelial cell specific organelle called Weibel-Palade body. In the inflamed circulation thrombin and other Gq-linked neurohumoral inflammatory mediators increase endothelial cell cytosolic calcium, and this rise in cytosolic calcium is sufficient to cause rapid translocation of Weibel-Palade bodies to the plasmalemma for vWf secretion and P-selectin up-regulation. Specific calcium entry pathways that stimulate vWf secretion and P-selectin upregulation remain incompletely understood, particularly in microvascular endothelial cells obtained from the prominent site of vaso-occlusion. Preliminary studies suggest that lung microvascular endothelial cells expess T-type, voltage-activated calcium channels which promote a pro-coagulant endothelial phenotype during inflammation. In this proposal, we will test the overall HYPOTHESIS that calcium entry through T-type calcium channels is an important amplification step in release of vWf and up-regulation of P-selectin from lung microvascular endothelium that promotes the retention of sickled red blood cells. The hypothesis will be explored using lung microvascular endothelial cells in culture and an isolated rat lung model, in which the role of the T channel to red blood cell retention can be assessed under flow conditions. The SPECIFIC AIMS test the hypotheses that: [1] Lung microvascular endothelial cells express a T-type calcium channel that is activated by Gq-linked neurohumoral inflammatory mediators, and [2] Activation of T-type calcium channels promotes the release of vWf and up-regulation of P-selectin from lung microvascular endothelial cells important for vaso-occlusion. It is hoped completion of these studies will improve our understanding of mechanisms that regulate erythrocyte-endothelial adherence so that effective therapies can be developed for treatment of sickle cell anemia, as well as other vascular thrombosis disorders.