VAMP8 regulates endothelial exocytosis and microvascular obstruction

描述(申请人提供)：在美国，冠状动脉疾病是主要的死亡原因。治疗心肌梗死的主要目标是通过开放闭塞的冠状动脉和再灌流心脏来恢复心脏的血流量。但即使在血管开放后，约25%的心肌梗死患者在组织水平上没有心肌灌注，他们的预后要差得多。治疗后缺乏心肌灌注被称为无复流现象。无复流现象是由微血管阻塞引起的：大的冠状动脉通畅，但小的毛细血管被成团的白细胞和血小板堵塞。微血管阻塞的发病机制复杂，目前尚不清楚。我们认为内皮细胞胞吐在微血管阻塞中起着关键作用。我们的理论是，缺血和再灌流会触发内皮释放颗粒。内皮颗粒分泌的内容物引起中性粒细胞黏附和血小板活化，导致微血管阻塞。胞吐作用是细胞中信使分子的调节分泌。胞吐作用是细胞生物学的基础，但我们对其他哺乳动物细胞的胞吐作用的了解是有限的。我的实验室的总体目标是了解人类血管系统中胞吐的途径。我们最近发现内皮细胞胞吐在缺血再灌注损伤中起重要作用。我们发明了针对胞吐机制的多肽，阻断内皮细胞的胞吐，限制微血管阻塞，减少缺血再灌注损伤。我们的新的初步数据现在表明，VAMP8调节内皮细胞胞吐。本项目的目标是确定VAMP8在胞吐和微血管阻塞中的作用。我们的假设是，VAMP8介导内皮细胞胞吐、血管炎症和微血管阻塞。在这项提案中，我们将定义VAMP8在内皮细胞胞吐中的作用。我们将确定VAMP8的亚细胞位置，并确定其在内皮细胞中的相互作用伙伴。接下来，我们将鉴定VAMP8的结构域，这些结构域介导与VAMP8伙伴的相互作用，并开发阻断这些结构域的多肽。这一目的将揭示VAMP8在内皮细胞胞吐机制中的分子作用。接下来，我们将确定VAMP8在体内血管炎症和微血管阻塞中的作用。我们将在体内研究VAMP8对内皮细胞胞吐的影响。我们将测量VAMP8对小鼠白细胞转运的影响。然后，我们将在心肌缺血再灌注模型中使用缺乏VAMP8的小鼠来测量VAMP8对微血管阻塞的影响。最后，我们将测试多肽阻断VAMP8和限制微血管阻塞的能力。这一目标将揭示VAMP8如何影响微血管阻塞。

DESCRIPTION (provided by applicant): Coronary artery disease is the leading cause of death in the USA. The major goal of treating myocardial infarction is to restore blood flow to the heart by opening the occluded coronary artery and reperfusing the heart. But even after the vessel is opened, about 25% of patients with a myocardial infarction do not have myocardial perfusion at the tissue level, and they have a much worse prognosis. Lack of myocardial perfusion after therapy is referred to as the no-reflow phenomenon. The no-reflow phenomenon is caused by microvascular obstruction: the large coronary artery is patent but the small capillaries are blocked with clumps of leukocytes and platelets. The pathogenesis of microvascular obstruction is complex and not well understood. We propose that endothelial exocytosis plays a pivotal role in microvascular obstruction. Our theory is that ischemia and reperfusion trigger endothelial release of granules. The secreted contents of endothelial granules then cause neutrophil adherence and platelet activation, leading to microvascular obstruction. Exocytosis is the regulated secretion of messenger molecules from cells. Exocytosis is fundamental to cell biology, but our understanding of exocytosis in other mammalian cells is limited. The overall goal of my lab is to understand pathways of exocytosis in the human vasculature. We recently discovered that endothelial exocytosis plays a major role in ischemia and reperfusion injury. We invented peptides that target the exocytic machinery, blocking endothelial exocytosis, limiting microvascular obstruction, and decreasing ischemia- reperfusion injury. Our new preliminary data now show that VAMP8 regulates endothelial exocytosis. The goal of this project is to characterize the role of VAMP8 in exocytosis and microvascular obstruction. Our hypothesis is that VAMP8 mediates endothelial exocytosis, vascular inflammation, and microvascular obstruction. In this proposal we will define the role of VAMP8 in endothelial exocytosis. We will determine the subcellular location of VAMP8 and identify its interaction partners in endothelial cells. Next we will characterize the domains of VAMP8 that mediate interactions with VAMP8 partners, and develop peptides which block these domains. This aim will reveal the molecular role of VAMP8 in the exocytic machinery of endothelial cells. Next we will define the role of VAMP8 in vascular inflammation and microvascular obstruction in vivo. We will characterize the effect of VAMP8 upon endothelial exocytosis in vivo. We will measure the effect of VAMP8 upon leukocyte trafficking in mice. We will then measure the effect of VAMP8 upon microvascular obstruction, using mice that lack VAMP8 in a model of myocardial ischemia-reperfusion. Finally we will test that ability of peptides to block VAMP8 and limit microvascular obstruction. This aim will reveal how VAMP8 affects microvascular obstruction.