PKC Isoenzymes and Diabetic Microvascular Hyperpermeability

描述(申请人提供)：微血管高通透性是许多炎症性疾病发展的重要损害过程，包括糖尿病并发症。我们研究计划的长期目标是了解生理和病理条件下微血管屏障功能调节的细胞和分子机制。作为该计划的一个组成部分，该项目旨在阐明糖尿病发展过程中导致微血管高通透性的信号通路和分子机制，糖尿病是一种影响大量人群的疾病，其发病率和死亡率较高，其并发症以微血管损伤为特征。我们的中心假说认为，糖尿病通过MAPK刺激的基因表达、PDK1增强的催化活性和DAG介导的激酶激活，在多个水平上上调微血管内皮细胞中的PKCβ。我们进一步认为，上调的PKCβ通过GDI-RhoA-ROCK级联反应诱导内皮细胞收缩，以及由β-连环蛋白磷酸化和VE-钙粘蛋白解离引发的细胞间连接解离，从而增加静脉内皮细胞的细胞旁通透性。提出了三个具体的目标：1)明确地确定PKCβ在糖尿病微血管渗漏中的作用；2)研究PKCβ上调糖尿病猪微血管通透性的信号机制；3)阐明PKCβ诱导的微血管高通透性的分子基础。这些目标将通过一种多方面的分子生理学方法实现，该方法将分子技术与微血管水平的功能分析相结合。人类相关的猪糖尿病模型将作为定量评估完整微血管内皮屏障功能的主要模型。这项研究的数据将为糖尿病微血管并发症的发病机制提供新的见解。明确PKC诱导的终末点损伤的确切分子机制可能为寻找治疗靶点提供新的途径。基于这项研究，我们未来的研究方向将是开发诊断和治疗与慢性炎症性疾病相关的微血管渗漏的分子探针和治疗方法。

DESCRIPTION (provided by applicant): Microvascular hyperpermeability represents an important injurious process underlying the development of many inflammatory diseases including diabetic complications. The long-term goal of our research program is to understand the cellular and molecular mechanisms in the regulation of microvascular barrier function under physiological and pathological conditions. As an integral component of the program, this project is designed to elucidate the signaling pathways and molecular mechanisms responsible for microvascular hyperpermeability during development of diabetes, a disease that affects a large population with high morbidity and mortality resulting from complications characterized by microvascular injury. Our central hypothesis states that diabetes upregulates PKCbeta in microvascular endothelium at multiple levels via MAPK-stimulated gene expression, PDK1-potentiated catalytic activity, and DAG-mediated kinase activation. We further propose that upregulated PKCbeta increases the paracellular permeability of venular endothelium by inducing endothelial cell contraction via the GDI-RhoA-ROCK cascade coupled with intercellular junction disorganization triggered by beta-catenin phosphorylation and VE-cadherin dissociation. Three specific aims are proposed: 1) to unequivocally establish the role of PKCbeta in microvascular leakage during diabetes; 2) to characterize the signaling mechanisms of PKCbeta upregulation in diabetic pigs; and 3) to elucidate the molecular basis of PKCbeta-elicited microvascular hyperpermeability. These aims will be accomplished through a multifaceted molecular physiology approach that incorporates molecular techniques with functional analyses at the microvascular level. A human-relevant pig model of diabetes will serve as the primary model for quantitative assessment of endothelial barrier function in intact microvessels. Data derived from this study will provide new insights into the pathogenesis of diabetic microvascular complications. Identification of the precise molecular mechanisms responsible for PKC-induced end-point injury may lead to a new avenue for searching therapeutic targets. Based on this study, a future direction of our research efforts will be directed to the development of molecular probes and therapies for diagnosis and treatment of microvascular leakage associated with chronic inflammatory diseases.