ENDOTHELIAL DYSFUNCTION: FORMATION OF MICROPARTICLES

描述(由申请人提供)： 拟议的项目将加强候选人以前在血管生物学和糖尿病方面的经验，并将候选人发展成为一名独立的研究人员。虽然PL在体内和体外实验技术方面的经验将有助于实施所提出的项目，但它的完成也将极大地丰富候选人在细胞生物学和生理学方面的知识。 内皮细胞功能障碍(ECD)是包括中风、高血压、心肌梗死和糖尿病在内的各种病理状态的共同前驱和分母。内皮细胞功能障碍的公认特征是一氧化氮(NO)的产生或生物可利用性不足，纤溶酶原激活物抑制物-1(PAL-1)水平升高，以及存在促凝血状态。我们先前已经证明，PAL-1水平的升高会导致培养的内皮细胞产生的NO减少，以及Annexin V染色的微小颗粒的产生增加-这是阴离子磷脂表达的证据。伴随而来的是微粒加速生成凝血酶。PAL-1基因敲除小鼠在注射PAL-1后，循环中微粒子数量显著减少，微粒子数量显著增加。结合最近对急性冠状动脉综合征和糖尿病患者循环内皮细胞微粒水平升高的研究结果，我们的数据提示：a)微粒可能是ECD的结果，并有助于伴随这些疾病的促凝状态；b)循环微粒水平升高是否有助于内皮细胞功能障碍的发生和维持。基于这些发现，我们假设：1)应激内皮细胞增加的微粒形成不仅是内皮功能障碍的一个新的标志，而且也是内皮功能障碍的一个新的标志；2)PAL-1(一个公认的内皮功能障碍的标志)水平的升高导致具有促凝血特性的微粒的形成增加。目前的建议旨在1)建立一种新的内皮功能障碍的病理生理学机制--应激内皮细胞形成微粒；2)获得糖尿病患者内皮细胞产生的循环微粒的数量信息；3)研究内皮功能障碍的已识别标记物之间的可能联系，即PAL-1水平升高和凝血活性增强。为了实现这些目标，将在体外(在高糖或PAL-1应激的内皮细胞中)和体内(在Zucker糖尿病大鼠中)使用荧光激活细胞分选来研究微粒的形成。将使用PAL-1基因敲除小鼠在体内和体外使用内皮细胞培养来研究PAL-1和凝血酶活性增加之间的联系。建议的研究可能提供关于微粒在内皮细胞功能障碍和凝血功能异常的病理生理学中的作用的新信息。

DESCRIPTION (provided by applicant): The proposed project should enhance candidate's previous experience in vascular biology and diabetes and develop the candidate into an independent researcher. While Pl's experience with in vivo and in vitro experimental techniques will help to conduct the presented project, its completion will significantly enrich candidate's knowledge in cell biology and physiology as well. Endothelial cell dysfunction (ECD) is a common precursor and denominator of various pathologic conditions, including stroke, hypertension, myocardial infarction and diabetes mellitus. The established hallmarks of endothelial cell dysfunction are a deficiency in the production or bioavailability of nitric oxide (NO), an increased level of plasminogen activator inhibitor-1 (PAl-1), and a presence of a pro-coagulant state. We have previously demonstrated that elevated level of PAl-1 results in a decreased production of NO by cultured endothelial cells, as well as an increased production of microparticies stained with Annexin V - evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin by microparticles. PAl-1 knockout mice presented with a significantly decreased number of circulating microparticles and a marked increase in the number of microparticles after PAl-1 injection. Combined with recent findings of elevated levels of circulating endothelial microparticles in patients with acute coronary syndrome and diabetes, our data suggest that a) microparticles may be a consequence of ECD and contribute to a procoagulant state accompanying these diseases and b) raise the question whether increased levels of circulating microparticles may contribute to the development and maintenance of endothelial cell dysfunction. Based on these findings we hypothesize that: 1) increased microparticle formation by stressed endothelial cells is not only a novel marker of, but also a contributor to endothelial dysfunction and 2) elevated level of PAl-1 (a recognized marker of endothelial dysfunction) leads to increased formation of microparticles with pro-coagulant properties. The current proposal seeks to 1) establish a novel pathophysiolgical mechanism of endothelial dysfunction - formation of microparticles by stressed endothelial cells; 2) obtain information on the population of circulating microparticles produced by endothelial cells in diabetes mellitus; 3) to investigate possible links between identified markers of endothelial dysfunction, namely elevated levels of PAl-1 and increased coagulant activity. To accomplish these goals, microparticle formation will be studied using fluorescence-activated cell sorting in vitro (in endothelial cells stressed with high glucose or PAl-1) and in vivo (in Zucker diabetic rats). The link between PAl-1 and increased coagulant activity will be studied in vivo using PAl-1 knockout mice and in vitro using endothelial cell cultures. The proposed studies may provide novel information on the contribution of microparticles to the pathophysiology of endothelial cell dysfunction and coagulation abnormalities.