Ceramide Signaling in Coronary Endothelial Dysfunction

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DESCRIPTION (provided by applicant): Work done over the last funding period has demonstrated that ceramide production and clustering of membrane rafts (MR) (formerly lipid rafts) drive aggregation of NADPH oxidase (Nox) subunits together with other regulatory molecules in the membrane of endothelial cells (ECs) forming a MR redox signaling platform. This signaling platform produces superoxide to regulate coronary endothelial function. This competitive renewal now seeks to elucidate the molecular mechanisms mediating the formation and function of such MR signaling platforms. The hypothesis being tested is that a rapid fusion of membrane proximal lysosomes to plasma membrane via the SNARE-centered exocytic machinery mediates translocation of ASMase, local ceramide production, and consequent formation of MR redox signaling platforms in coronary arterial endothelial cells. This lysosome-MR redox signalosome contributes to endothelial dysfunction in response to activation of death receptors and other danger signals. Specific Aim 1 will determine whether the formation of MR redox signaling platforms are mediated by a rapid fusion of membrane proximal lysosomes to plasma membrane via a SNARE- centered exocytic machinery in coronary ECs. Specific Aim 2 will identify the topographic characteristics of lysosome-MR signalosomes in coronary ECs using scanning and immunogold electron microscopy and determine possible interactions of different molecules. Specific Aim 3 will determine the functionality of the lysosome MR signalosomes in coronary ECs with a focus on interactions of ASMase activity and Nox activation. Specific Aim 4 will determine whether the formation of such lysosome MR signalosomes via the SNARE-centered exocytic machinery contributes to endothelial dysfunction in the intact coronary arterial endothelium. These proposed studies will elucidate the molecular mechanisms mediating the trafficking of intracellular organelles and molecules to cell plasma membrane for transmembrane signaling. The results will reveal a novel lysosomal function serving as a signaling organelle in ECs, which may importantly contribute to the formation of MR redox signaling platform and thereby to the regulation of endothelial function under physiological and pathological conditions.