Vascular Lipolysis: A Determinant of Atherosclerosis

描述(由申请人提供)：最近的研究清楚地证明了磷脂和鞘磷脂代谢物在动脉粥样硬化和 血管生物学。而负责磷脂和鞘磷脂的酶 已经确定了血管壁中的水解物，并证明它起了作用 对于动脉粥样硬化的形成，酶(S)负责降低其水平 水解物、生物活性溶血磷脂酰胆碱(LysoPC)和 神经酰胺，到目前为止还没有被表征。由此产生的结果 实验室发现存在胰型羧酸酯脂肪酶 (CEL)在人体血管壁中。这种蛋白是由主动脉内皮细胞合成的 氧化低密度脂蛋白诱导的细胞和单核细胞来源的巨噬细胞 (OxLDL)。重要的是，CEL表现出强烈的胆汁非依赖于盐的溶血酶C和 神经酰胺的水解性。人类CEL也是高度多态的，编码 蛋白质在羧基上富含脯氨酸重复单位的数量不同 终点站。基于这些新的观察结果，该应用程序建议测试 假设：(1)CEL在血管系统中的表达是一种 通过以下途径保护动脉粥样硬化早期的炎症反应机制 降解和降低溶血磷脂酶和神经酰胺的致动脉粥样硬化特性； CEL C末端富含脯氨酸的重复单位数为 对确定其降解溶血磷脂酶和神经酰胺的能力很重要；和(3) CEL基因多态是个体易感性的决定因素 修饰-低密度脂蛋白介导的动脉粥样硬化事件。这些假设VIII将通过以下方式进行检验 在三个具体目标下进行实验。特定目标1计划生产 血管特异性CEL转基因小鼠对血管CEL的影响 动脉粥样硬化斑块发生发展过程中的基因表达。 特定目标2将使用定点突变方法来测试 假设其C-末端富含脯氨酸的重复单位的数量为 CEL的溶血磷脂酶和神经酰胺酶活性的重要决定因素 因此，不同的CEL亚型在保护血管中的有效性 细胞对抗溶血磷脂和神经酰胺诱导的动脉粥样硬化事件。具体目标3 将确定修饰的低密度脂蛋白诱导CEL基因表达的机制 在人的巨噬细胞和内皮细胞中。实验将被设计用于测试 CEL基因激活是通过信号转导介导的假说 OxLDL与细胞上清道夫受体结合的机制 表面，或者需要特定脂类的内化 与氧化脂蛋白有关的成分。这些加在一起， 研究将确定有助于确定 动脉粥样硬化易感性。还可以设计基因筛查策略 识别易患动脉粥样硬化的受试者进行早期干预和 降低他们罹患这种疾病的风险。

DESCRIPTION (provided by applicant): Recent studies have clearly documented the importance of phospholipid and sphingolipid metabolites in atherosclerosis and vascular biology. While enzymes responsible for phospholipid and sphingomyelin hydrolysis in the vessel wall have been identified and were shown to contribute to atherogenesis, enzyme(s) responsible for lowering the level of their hydrolytic products, the bioactive lysophosphatidylcholine (LysoPC) and ceramide, have not been characterized to date. Results generated from this laboratory revealed the presence of the pancreatic-type carboxyl ester lipase (CEL) in human vascular wall. This protein is synthesized by aortic endothelial cells and monocyte-derived macrophages in a manner inducible by oxidized LDL (oxLDL). Importantly, CEL displayed avid bile salt-independent lysoPC and ceramide hydrolytic activities. Human CEL is also highly polymorphic, encoding proteins varying in the number of proline-rich repeating units at the carboxyl terminus. Based on these novel observations, this application proposes to test the following hypotheses: (1) CEL expression in the vasculature is an inflammatory response mechanism that protects early stages of atherogenesis by hydrolyzing and reducing the atherogenic properties of lysoPC and ceramide; (2) the number of proline-rich repeating units in the C-terminus of CEL is important in determining its ability to hydrolyze lysoPC and ceramide; and (3) CEL gene polymorphism is a determinant of individual susceptibility to modified-LDL mediated atherogenic events. These hypotheses viii be tested by experiments under 3 specific aims. Specific Aim 1 plans to produce vascular-specific CEL transgenic mice to evaluate the impact of vascular CEL gene expression on initiation and progression of the atherosclerotic plaque. Specific Aim 2 will use site-directed mutagenesis approach to test the hypothesis that the number of proline-rich repeating units at its C-terminus is an important determinant of lysoPC and ceramide hydrolytic activities of CEL, and thus the effectiveness of different CEL isoforms in protecting vascular cells against lysoPC- and ceramide-induced atherogenic events. Specific Aim 3 will identify the mechanism by which modified LDL induces CEL gene expression in human macrophages and endothelia cells. Experiments will be designed to test the hypothesis that CEL gene activation is mediated via signal transduction mechanisms as a consequence of oxLDL binding to scavenger receptors on the cell surface, or alternatively requires the internalization of specific lipid constituents associated with oxidized lipoproteins. Taken together, these studies will identify novel factors that contribute to determining atherosclerosis susceptibility. Genetic screening strategy may also be designed to identify subjects predispose to atherosclerosis for early intervention and reducing their risk of developing this disease.