NEUTROPHIL DEPENDENT REGULATION OF VENULAR PERMEABILIT

冠状动脉微血管交换在心肌缺血中起重要作用。 维持正常的心脏功能。 在急性炎症和 心肌缺血再灌注，中性粒细胞粘附于毛细血管后 微静脉内皮细胞，随后微静脉通透性增加 包括一个共同的病理生理学后果的发展， 心肌功能障碍虽然很多工作都致力于 对嗜中性粒细胞衍生的激动剂和第二信使的澄清， 对它们的分子靶点知之甚少。 在这方面， 活化的中性粒细胞是否通过 改变内皮的结构和功能完整性 仍然难以捉摸 因此，本研究的总体目标是 在分子水平上确定控制 内皮细胞对活化的中性粒细胞的高通透性反应。 与这一目标相对应的主要假设是中性粒细胞- 诱导的屏障功能障碍是通过一系列事件发生的， 内皮细胞从细胞骨架重组到连接 破坏，最终促进跨内皮流量， 液体和蛋白质。 具体而言，将检验三个假设：（1） 激活的中性粒细胞触发细胞骨架收缩过程 通过肌球蛋白轻链磷酸化，（2）激活中性粒细胞 通过磷酸化下调内皮粘附连接 和VE-钙粘蛋白/β-连环蛋白复合物的解离，和（3）激活 嗜中性粒细胞促进贴壁依赖性细胞收缩， 局部粘连的形成和再分布。 测试这些 假设，将采用几种先进的实验方法， 包括完整灌注的冠状小静脉标本、培养的 微静脉内皮细胞单层模型，western blot分析，荧光 免疫细胞化学、DNA和蛋白质转染以及显性阴性 抑制方法。 这项研究将为我们提供一个新的视角， 炎症和缺血性心脏病的分子病理学。 活化中性粒细胞的特异性靶蛋白的鉴定将 加强治疗策略的发展，以有效地抑制 有害的水肿过程，而不抑制有益的 炎症反应的影响。

Coronary microvascular exchange plays an essential role in the maintenance of normal cardiac function. In acute inflammation and myocardial ischemia-reperfusion, neutrophil adhesion to postcapillary venular endothelium followed by an increase in venular permeability comprises a common pathophysiological consequence in the development of myocardial dysfunction. Although much work has been dedicated to the clarification of neutrophil-derived agonists and second messengers, little is known about their molecular targets. Within this context, whether activated neutrophils affect microvascular barrier function by altering the structural and functional integrity of the endothelium remains elusive. Therefore, the overall goal of this study is to identify at molecular levels the signaling mechanism that controls the endothelial hyperpermeability response to activated neutrophils. Corresponding to this goal is the major hypothesis that neutrophil- induced barrier dysfunction occurs through a chain of events in the endothelium from cytoskeletal reorganization to junctional disorganization, which ultimately facilitate transendothelial flux of fluid and proteins. Specifically, three hypotheses will be tested: (1) activated neutrophils trigger the cytoskeletal contractile process through myosin light chain phosphorylation, (2) activated neutrophils downregulate the endothelial adherence junction through phosphorylation and dissociation of VE-cadherin/beta-catenin complex, and (3) activated neutrophils promote anchorage-dependent cell contraction through the formation and redistribution of focal adhesions. To test these hypotheses, several advanced experimental approaches will be employed, including the intact perfused coronary venule preparation, the cultured venular endothelial monolayer model, western blot analysis, fluorescence immunocytochemistry, DNA and protein transfection, and dominant negative inhibition approaches. The study will provide a new insight into the molecular pathobiology of inflammatory and ischemic heart diseases. Identification of specific target proteins of activated neutrophils will enhance the development of therapeutic strategies to effectively inhibit the deleterious process of edema without suppressing the beneficial effects of inflammatory responses.