Ceramide Signaling in Coronary Endothelial Dysfunction

描述（由申请人提供）：最近的研究表明神经酰胺，一种神经鞘脂有助于不同因素如tnf - α， Fas L和内皮抑素产生内皮功能障碍。然而，内皮细胞中神经酰胺的产生和作用的机制仍然知之甚少。最近，一种涉及膜脂筏及其聚集的新机制被报道参与神经酰胺介导的跨膜和细胞内信号传导。在本研究中，我们假设神经酰胺的产生和脂质筏聚集了不同的信号分子，如NADPH氧化酶亚基，形成一个膜信号放大平台，从而有助于降低NO的生物利用度和不同损伤因素引起的内皮功能障碍。我们将首先展示脂筏平台的形成和相关的受体聚集响应Fas L或tnf - α在牛冠状动脉内皮细胞中使用荧光和共聚焦显微镜和洗涤剂抗性膜的浮选。我们还将确定神经酰胺与脂筏聚集的关联，并通过选择性抑制相关酶或使用siRNA沉默编码这些酶（如鞘磷脂酶）的基因来探索神经酰胺产生的机制。然后，我们将通过共聚焦显微镜、酶动力学分析和亚基易位检测来确定脂筏聚类是否共同聚集并激活内皮细胞NADPH氧化酶。最后，我们将通过对牛冠状动脉完整内皮中NO或02-的荧光成像分析，确定脂质筏聚集是否刺激02-的产生，从而导致与NO相关的内皮功能障碍。这些研究结果将阐明脂质筏聚集和神经酰胺产生在介导Fas L和tnf - α作用中的作用，并为缺血/再灌注、动脉粥样硬化和高血压等不同病理条件下内皮功能障碍的机制提供新的见解。

DESCRIPTION (provided by applicant): Recent studies have indicated that ceramide, a sphingolipid contributes to the detrimental effects of different factors such as TNF-alpha, Fas L and endostatin to produce endothelial dysfunction. However, the mechanisms responsible for the production and actions of ceramide in endothelial cells are still poorly understood. Recently, a novel mechanism involving membrane lipid rafts and their aggregation has been reported to participate in ceramide-mediated transmembrane and intracellular signaling. In this proposal, we hypothesize that ceramide production and lipid raft clustering aggregate different signaling molecules such as NADPH oxidase subunits to form a membrane signaling amplification platform and thereby contributes to the reduction of NO bioavailability and endothelial dysfunction induced by different injury factors. We will first demonstrate the formation of lipid raft platforms and associated aggregation of the receptors in response to Fas L or TNF-alpha in bovine coronary arterial endothelial cells using fluorescent and confocal microscopy and flotation of detergent resistant membranes. We will also determine the association of ceramide with lipid raft clustering and explore the mechanisms of ceramide production by selective inhibition of related enzymes or by silencing the genes coding these enzymes such as sphingomyelinases using siRNA. Then, we will determine whether lipid raft clustering co-aggregates and activates endothelial NADPH oxidase by confocal microscopy, enzyme kinetic analysis and subunit translocation detection. Finally, we will determine whether lipid raft clustering stimulates 02- production and thereby contributes to endothelial dysfunction associated with NO using fluorescence imaging analysis of NO or 02- in the intact endothelium of bovine coronary arteries. The results of these studies will clarify the role of lipid raft clustering and ceramide production in mediating the actions of Fas L and TNF-alpha and provide new insights into the mechanisms contributing to endothelial dysfunction under different pathological conditions such as ischemia/reperfusion, atherosclerosis and hypertension.