Ceramide Signaling in Coronary Endothelial Dysfunction

冠状动脉内皮功能障碍中的神经酰胺信号传导

• 批准号: 6709817
• 负责人: PinLan Li
• 金额: $ 32.42万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6709817
• 关键词: NAD(P)H dehydrogenase; RNA interference; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell membrane; ceramides; confocal scanning microscopy; coronary artery; enzyme inhibitors; fluorescence microscopy; gene induction /repression; intracellular; nitric oxide; polymerase chain reaction; small interfering RNA; sphingolipids; sphingomyelin phosphodiesterase; tissue /cell culture; tumor necrosis factor alpha; vascular endothelium; western blottings

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.

描述（由申请人提供）：最近的研究表明，神经酰胺（一种鞘脂）有助于不同因子（如TNF-α、Fas L和内皮抑制素）的有害作用，从而产生内皮功能障碍。然而，神经酰胺在内皮细胞中的产生和作用的机制仍然知之甚少。最近，一种新的机制，涉及膜脂筏和他们的聚集已被报道参与神经酰胺介导的跨膜和细胞内信号。在这个建议中，我们假设，神经酰胺的生产和脂筏聚集不同的信号分子，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的生物利用度和内皮功能障碍诱导的不同损伤因素。我们将首先证明形成的脂筏平台和相关的聚集的受体在牛冠状动脉内皮细胞Fas L或TNF-α，使用荧光和共聚焦显微镜和耐洗涤剂膜的浮选。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通过使用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簇是否共聚集并激活内皮NADPH氧化酶。最后，我们将使用牛冠状动脉的完整内皮中的NO或O2-的荧光成像分析来确定脂筏聚集是否刺激O2-产生，从而有助于与NO相关的内皮功能障碍。这些研究结果将阐明脂筏聚集和神经酰胺产生在介导Fas L和TNF-α作用中的作用，并为不同病理条件下（如缺血/再灌注、动脉粥样硬化和高血压）导致内皮功能障碍的机制提供新的见解。