A Novel Mechanism by which iPLA2 Links Diabetes to Cardiovascular Diseases

描述（由申请人提供）：iPLA 2（钙非依赖性磷脂酶A2）是磷脂酶A2超家族的一员，在血管平滑肌中表达，并表现出多种细胞功能。我们的初步数据显示，iPLA 2在2型糖尿病db/db小鼠和高脂饮食喂养的小鼠的血管系统中被激活/上调，并且在原代培养的血管平滑肌细胞（VSMC）中被高葡萄糖激活/上调。此外，通过药理学抑制剂BEL或基因缺失抑制iPLA 2可消除，而iPLA 2的过表达可加剧培养的VSMC中高糖诱导的NAD（P）H氧化酶介导的超氧化物产生。这些数据清楚地表明，iPLA 2是培养的VSMC中高糖诱导的NAD（P）H氧化酶介导的ROS产生所必需的，因此暗示iPLA 2在糖尿病诱导的血管壁中ROS增加中的潜在重要作用。虽然增加的ROS可能影响多种细胞功能，但我们的数据显示，RhoA/ROCK/PKC通路的下游参与者CPI-17受ROS调节，表明iPLA 2通过ROS调节RhoA/ROCK/CPI-17通路。因此，我们假设2型糖尿病激活血管平滑肌中的iPLA 2，导致增强的NAD（P）H氧化酶介导的ROS产生和随后的RhoA/ROCK/PKC/CPI-17途径激活，从而显著促进2型糖尿病相关的血管平滑肌过度收缩和高血压。三个具体目标是：1）检验iPLA 2是2型糖尿病诱导的血管平滑肌组织中NAD（P）H氧化酶产生ROS所必需的假设。2)为了检验iPLA 2是2型糖尿病诱导的血管平滑肌组织中RhoA/ROCK/CPI-17通过NAD（P）H氧化酶激活所必需的假设。3)确定iPLA 2在2型糖尿病相关的血管平滑肌过度收缩和高血压中的体内意义。将使用两种类型的糖尿病动物模型（高脂饮食喂养小鼠和db/db小鼠）与遗传修饰小鼠（iPLA 2转基因、iPLA 2敲除和p47 phox缺陷小鼠）的组合。将在分离的血管中测定iPLA 2、NAD（P）H氧化酶和RhoA/ROCK/PKC/CPI-17的活性和血管张力，并通过遥测测定动物的血压。虽然2型糖尿病高血压的发展似乎是多种适应不良途径的结果，但拟议研究的结果将阐明可能导致将iPLA 2鉴定为预防和治疗2型糖尿病相关心血管并发症的潜在新型治疗靶点的特定机制。

DESCRIPTION (provided by applicant): iPLA2 (calcium independent phospholipase A2) is a member of the phospholipase A2 superfamily that is expressed in vascular smooth muscle and exhibits diverse cellular functions. Our preliminary data show that iPLA2 is activated/up-regulated in the vasculature of type 2 diabetic db/db mice and high-fat diet-fed mice and by high glucose in primary cultured vascular smooth muscle cells (VSMC). Moreover, inhibition of iPLA2 by BEL, a pharmacological inhibitor, or genetic deletion abolishes, whereas overexpression of iPLA2 exacerbates high glucose-induced NAD(P)H oxidase-mediated superoxide production in cultured VSMCs. These data clearly show that iPLA2 is required for high glucose-induced NAD(P)H oxidase-mediated ROS production in cultured VSMCs and thus implicate an potentially important role of iPLA2 in the diabetes induced ROS increase in vascular wall. While the increased ROS may affect multiple cellular functions, our data show that CPI-17, a downstream player in RhoA/ROCK/PKC pathway, is regulated by ROS, suggesting that iPLA2 regulates RhoA/ROCK/CPI-17 pathway via ROS. Therefore, we hypothesize that type 2 diabetes activates iPLA2 in vascular smooth muscle, leading to enhanced NAD(P)H oxidase-mediated ROS production and consequent RhoA/ROCK/PKC/CPI-17 pathway activation, and thereby significantly contributes to type 2 diabetes- associated vascular smooth muscle hyper-contractility and hypertension. Three Specific Aims are: 1) To test the hypothesis that iPLA2 is required for type 2 diabetes-induced ROS production by NAD(P)H oxidase in vascular smooth muscle tissues. 2) To test the hypothesis that iPLA2 is required for the type 2 diabetes- induced activation of RhoA/ROCK/CPI-17 via NAD(P)H oxidase in vascular smooth muscle tissue. 3) To determine the in vivo significance of iPLA2 in type 2 diabetes-associated vascular smooth muscle hyper- contractility and hypertension. Two type diabetic animal models (the high-fat diet-fed mice and db/db mice) in combination with the genetic modified mice (the iPLA2 transgenic, iPLA2 knockout, and p47phox deficient mice) will be used. The activities of iPLA2, NAD(P)H oxidase, and RhoA/ROCK/PKC/CPI-17 and vascular tone will be assayed in isolated vasculatures and blood pressure will be determined in animals by telemetry. While the development of hypertension in type 2 diabetes seems to be the result of multiple maladaptive pathways, results from the proposed studies will elucidate specific mechanisms that could lead to the identification of iPLA2 as a potential novel therapeutic target for the prevention and treatment of cardiovascular complications associated with type 2 diabetes.