Membrane Raft Platforms in Inflammasome Activation and Endothelial Dysfunction

 描述（由申请人提供）：内皮细胞（EC）中的膜筏（MR）（以前称为脂筏）形成溶酶体-MR信号平台或信号体，以调节内皮功能并参与内皮功能障碍、血管损伤和动脉粥样硬化形成。在持续的资助期间，我们已经表明，MR信号平台与溶酶体酸性鞘磷脂酶（Asm）的易位和局部神经酰胺的产生有关。本提案计划将研究结果从细胞和分子方法扩展到动物疾病模型，以通过使用基因工程动物来解决该内皮MR信号平台的生理和病理相关性。主要的焦点将是它的触发作用，在激活核苷酸寡聚化结构域样受体蛋白与pryin结构域包含3（Nlrp 3）和随之而来的内皮损伤和动脉粥样硬化病变。正在测试的中心假设是Asm-神经酰胺信号传导平台在高胆固醇血症的早期阶段介导EC中Nlrp 3炎性体的活化，从而产生内皮损伤作为触发机制，导致随后的颈动脉壁上的动脉粥样硬化病变与局部炎症反应一致。为了验证这一假设，提出了三个具体目标。具体目标1将使用Asm-/-小鼠、内皮特异性Asm转基因小鼠（EC-Asmtrg）和它们的野生型（WT）同窝出生仔来确定与增强的神经酰胺产生相关的内皮Nlrp 3炎性小体活化是否有助于颈动脉中的高胆固醇血症早期阶段和晚期动脉粥样硬化病变的颈动脉内皮功能障碍或损伤。具体目标2将探索神经酰胺产生增加激活Nlrp 3炎性体的分子机制，主要关注MR氧化还原信号体的形成、溶酶体功能障碍和ras激酶抑制因子（KSR）作为来自Asm-/-、EC-Asmtrg和WT小鼠的分离颈动脉EC中的支架。在特定目标3中，我们试图通过研究活化的caspase-1及其产物在内皮依赖性血管舒张受损（EDVD）、焦亡、粘附和连接蛋白表达改变以及适应性内皮祖细胞（EPCs）着陆或分化中的作用，确定神经酰胺介导的Nlrp 3炎性小体活化如何导致内皮功能障碍或损伤。据我们所知，这些拟议的研究将是第一个调查MR信号平台对细胞凋亡的贡献的研究。 EC中Nlrp 3炎性体的活化，从而导致内皮功能障碍和随后的动脉壁中的动脉粥样硬化病变。Nlrp 3炎性小体激活产生经典炎症反应和非经典血管损伤的发现可能会改变我们如何理解炎症在动脉粥样硬化和心血管疾病中的作用的范式。

 DESCRIPTION (provided by applicant): Membrane raft (MR) (formerly lipid raft) in endothelial cell (EC) forms lysosomal-MR signaling platforms or signalosomes to regulate endothelial function and to be involved in endothelial dysfunction, vascular injury and atherogenesis. Over the lasting funding period, we have shown that the MR signaling platforms are associated with translocation of lysosomal acid sphingomyelinase (Asm) and local ceramide production. The present proposal has planned to extend the findings from cell and molecular approaches to animal disease models to address the physiological and pathological relevance of this endothelial MR signaling platform by using genetically-engineered animals. The major focus will be on its triggering role in the activation of nucleotide oligomerization domain-like receptor protein with pryin domain containing 3 (Nlrp3) and consequent endothelial damage and atherosclerotic lesions. The central hypothesis being tested is that Asm-ceramide signaling platforms mediate the activation of Nlrp3 inflammasomes in ECs at the early stage of hypercholesterolemia and thereby produces endothelial injury as a triggering mechanism to result in subsequent atherosclerotic lesions on the carotid arterial wall in concert with local inflammatory responses. To test this hypothesis, three Specific Aims are proposed. Specific aim 1 will determine whether endothelial Nlrp3 inflammasome activation associated with enhanced ceramide production contributes to carotid endothelial dysfunction or injury at the early stage of hypercholesterolemia and late atherosclerotic lesions in the carotid arteries using Asm-/- mice, endothelium-specific Asm transgenic mice (EC-Asmtrg), and their wild type (WT) littermates. Specific Aim 2 will explore the molecular mechanisms by which increased ceramide production activates Nlrp3 inflammasomes with a main focus on the formation of MR redox signalosomes, lysosome dysfunction and kinase suppressor of ras (KSR) as a scaffold in isolated carotid ECs from Asm-/-, EC-Asmtrg and WT mice. In Specific Aim 3, we attempt to determine how ceramide-mediated Nlrp3 inflammasome activation leads to endothelial dysfunction or injury by studying the role of activated caspase-1 and its products in impaired endothelium-dependent vasodilation (EDVD), pyroptosis, altered expression of adhesion and junction proteins, and adaptive endothelial progenitor cells (EPCs) landing or differentiation. To our knowledge, these proposed studies will be the first to investigate the contribution of MR signaling platforms to the activation of Nlrp3 inflammasomes in EC, thereby leading to endothelial dysfunction and consequent atherosclerotic lesion in the arterial wall. The findings of Nlrp3 inflammasome activation to produce both classical inflammatory response and uncanonical vascular injury may shift the paradigm in how we understand the role of inflammation in atherogenesis and cardiovascular diseases.