Membrane Raft Platforms in Inflammasome Activation and Endothelial Dysfunction

 描述(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，调节内皮功能，参与内皮功能障碍、血管损伤和动脉粥样硬化的形成。在持续的资金支持期间，我们已经证明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。目前的提案计划将细胞和分子方法的发现扩展到动物疾病模型，通过使用基因工程动物来解决这个内皮MR信号平台的生理和病理相关性。主要的焦点将集中在它在核苷酸低聚结构域样受体蛋白3(Nlrp3)的激活中的触发作用，以及随后的内皮损伤和动脉粥样硬化病变。目前正在测试的中心假说是，ASM-神经酰胺信号平台在高胆固醇血症早期介导内皮细胞中Nlrp3炎性小体的激活，从而产生内皮损伤作为触发机制，导致随后的颈动脉壁动脉粥样硬化病变与局部炎症反应相一致。为了验证这一假设，本文提出了三个具体目标。具体目标1将利用ASM-/-小鼠、内皮特异性ASM转基因小鼠(EC-Asmtrg)及其野生型(WT)仔鼠，确定与神经酰胺产生增加相关的内皮Nlrp3炎症小体激活是否有助于高胆固醇血症早期颈动脉内皮细胞功能障碍或损伤以及颈动脉晚期动脉粥样硬化病变。具体目标2将探索神经酰胺产量增加激活Nlrp3炎症体的分子机制，主要集中在在ASM-/-、EC-Asmtrg和WT小鼠分离的颈动脉内皮细胞中形成MR氧化还原信号小体、溶酶体功能障碍和ras激酶抑制因子(KSR)作为支架。在具体目标3中，我们试图通过研究激活的caspase-1及其产物在受损的内皮依赖性血管扩张(EDVD)、下垂、黏附和连接蛋白表达改变以及适应性内皮祖细胞(EPC)着陆或分化中的作用，来确定神经酰胺介导的Nlrp3炎症小体激活是如何导致内皮功能障碍或损伤的。据我们所知，这些拟议的研究将是第一次调查MR信号平台对 Nlrp3在EC中的炎症小体被激活，从而导致内皮功能障碍，从而导致动脉壁的动脉粥样硬化病变。Nlrp3炎性小体激活同时产生经典炎症反应和非典型性血管损伤的发现可能改变我们理解炎症在动脉粥样硬化和心血管疾病中作用的范式。

 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.