A Novel Mechanism by which iPLA2 Links Diabetes to Cardiovascular Diseases

描述（由申请人提供）：iPLA2（钙独立磷脂酶A2）是磷脂酶A2超家族的成员，在血管平滑肌中表达，具有多种细胞功能。我们的初步数据表明，iPLA2在2型糖尿病小鼠和高脂肪饮食小鼠的血管中被激活/上调，并通过高糖原代培养血管平滑肌细胞（VSMC）被激活/上调。此外，药理抑制剂BEL对iPLA2的抑制或基因缺失会消除，而iPLA2的过表达会加剧高糖诱导的NAD(P)H氧化酶介导的VSMCs超氧化物的产生。这些数据清楚地表明，在培养的VSMCs中，高糖诱导的NAD(P)H氧化酶介导的ROS生成需要iPLA2，因此暗示iPLA2在糖尿病诱导的血管壁ROS增加中可能发挥重要作用。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调控，这表明iPLA2通过ROS调控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iPLA2，导致NAD(P)H氧化酶介导的ROS生成增强，从而导致RhoA/ROCK/PKC/CPI-17通路激活，从而显著促进2型糖尿病相关血管平滑肌超收缩和高血压。具体目的有三个：1)验证血管平滑肌组织中NAD(P)H氧化酶诱导2型糖尿病ROS生成需要iPLA2的假设。2)验证iPLA2在2型糖尿病诱导的血管平滑肌组织中通过NAD(P)H氧化酶激活RhoA/ROCK/CPI-17所需的假设。3)确定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。将使用两种型糖尿病动物模型（高脂肪饮食小鼠和db/db小鼠）与转基因小鼠（iPLA2转基因小鼠、iPLA2敲除小鼠和p47phox缺陷小鼠）联合使用。iPLA2、NAD(P)H氧化酶、RhoA/ROCK/PKC/CPI-17活性和血管张力将在离体血管中测定，动物血压将通过遥测测定。虽然2型糖尿病高血压的发展似乎是多种不适应途径的结果，但拟议研究的结果将阐明可能导致iPLA2作为预防和治疗2型糖尿病相关心血管并发症的潜在新治疗靶点的具体机制。

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.