A Novel Mechanism by which iPLA2 Links Diabetes to Cardiovascular Diseases

描述(申请人提供)：iPLA2(钙非依赖性磷脂酶A2)是磷脂酶A2超家族中的一个成员，表达于血管平滑肌中，具有多种细胞功能。我们的初步数据显示，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，iPLA2被激活/上调，并在原代培养的血管平滑肌细胞(VSMC)中被高糖激活/上调。此外，药物抑制剂BEL对iPLA2的抑制或基因缺失被取消，而iPLA2的过表达则加剧了高糖诱导的NAD(P)H氧化酶介导的VSMC超氧化物歧化。这些数据清楚地表明，iPLA2是高糖诱导的NAD(P)H氧化酶介导的培养的VSMCs产生ROS所必需的，因此iPLA2在糖尿病引起的血管壁ROS增加中起着潜在的重要作用。虽然ROS的增加可能影响多种细胞功能，但我们的数据表明，RhoA/ROCK/PKC途径的下游成员CPI-17受到ROS的调节，这表明iPLA2通过ROS调节RhoA/ROCK/CPI-17途径。因此，我们假设2型糖尿病激活血管平滑肌iPLA2，导致NAD(P)H氧化酶介导的ROS产生增加，进而激活RhoA/ROCK/PKC/CPI-17通路，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)验证iPLA2是2型糖尿病诱导血管平滑肌组织中NAD(P)H氧化酶产生ROS所必需的假说。2)验证iPLA2是2型糖尿病通过NAD(P)H氧化酶激活RhoA/ROCK/CPI-17所必需的假说。3)探讨iPLA2在2型糖尿病相关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(高脂饮食喂养的小鼠和db/db小鼠)和转基因小鼠(iPLA2转基因、iPLA2基因敲除和p47Phox缺陷小鼠)。将在分离的血管中检测iPLA2、NAD(P)H氧化酶、RhoA/ROCK/PKC/CPI-17的活性和血管张力，并通过遥测测量动物的血压。虽然2型糖尿病的高血压的发展似乎是多种不适应途径的结果，但拟议的研究结果将阐明特定的机制，这些机制可能导致将iPLA2确定为预防和治疗与2型糖尿病相关的心血管并发症的潜在新的治疗靶点。

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.