Ceramide Signaling in Coronary Endothelial Dysfunction

描述(由申请人提供)：在上一个资助期间所做的工作表明，神经酰胺的产生和聚集的膜筏(MR)(以前的脂筏)推动NADPH氧化酶(NOx)亚单位与其他调节分子在内皮细胞(ECs)膜上聚集，形成MR氧化还原信号平台。这个信号平台产生超氧化物来调节冠状动脉内皮功能。这种竞争性的更新现在试图阐明调节这种MR信号平台的形成和功能的分子机制。正在测试的假设是，通过圈套为中心的胞外机制将膜近端的溶酶体快速融合到质膜上，介导ASMase的移位，局部神经酰胺的产生，从而在冠状动脉内皮细胞中形成MR氧化还原信号平台。这个溶酶体-MR氧化还原信号小体有助于内皮功能障碍，以响应死亡受体和其他危险信号的激活。具体目的1将确定冠状动脉内皮细胞中MR氧化还原信号平台的形成是否由膜近端溶酶体通过圈套为中心的胞吐机制与质膜的快速融合所介导。具体目标2将使用扫描和免疫金电子显微镜确定冠状动脉内皮细胞中溶酶体-MR信号小体的形态特征，并确定不同分子之间可能的相互作用。具体目标3将确定冠状动脉内皮细胞中溶酶体MR信号小体的功能，重点是ASMase活性和NOx激活的相互作用。具体目标4将确定这种溶酶体MR信号小体的形成是否通过圈套为中心的胞外机制导致完整的冠状动脉内皮细胞的内皮功能障碍。这些研究将阐明细胞内细胞器和分子转运到细胞膜以进行跨膜信号传递的分子机制。这些结果将揭示一种新的溶酶体功能，它作为内皮细胞中的信号细胞器，可能有助于MR氧化还原信号平台的形成，从而在生理和病理条件下调节内皮功能。

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.