The regulation of transendothelial migration by calcium signaling in endothelial cells

跨内皮细胞迁移是炎症反应中的关键步骤，白细胞离开血管进入炎症组织。大多数病理，包括缺血/再灌注(I/R)损伤，都是由于功能障碍的炎症。当缺氧的组织被复氧时，就会发生I/R损伤，产生活性氧物种，破坏细胞并引发局部炎症。I/R损伤是心肌梗死(MI；心脏病发作)发病率和死亡率的主要来源，使心肌缺血性损伤更加严重。限制I/R损伤是治疗心肌梗死和其他血管疾病的主要目标。TEM是由白细胞表面的血小板/内皮细胞黏附分子1(PECAM)与集中于内皮细胞(EC)边界的PECAM相互作用而启动的。有效的透射电子显微镜需要胞浆内游离钙离子浓度(↑[Ca~(2+)]i)的瞬时增加。TRPC6被发现是负责(↑[Ca~(2+)]i)的钙通道，并在PECAM-PECAM相互作用的下游促进透射电子显微镜的功能。然而，PECAM向TRPC6发出信号的机制尚不清楚。我将确定PECAM如何向TRPC6发出信号，TRPC6如何在体内调节↑[Ca~(2+)]i，以及TRPC6与心肌I/R损伤的相关性。内皮细胞具有由PECAM、血管内皮细胞(VE)-钙粘附素和血管内皮生长因子受体2(VEGFR2)组成的连接性机械感觉复合体。这种信号复合体对流体剪切作出反应。初步数据表明，在瞬变过程中，这个相同的复合体也可能具有将信号从PECAM传递到TRPC6的功能。目的我将确定PECAM如何发出信号激活TRPC6。我们将对该系统的每个组件使用特定的药物抑制剂以及基因敲除和重新表达策略。PECAM FRET张力传感器和VE-钙粘蛋白突变体将被用来检验这一假设，即EC PECAM使用相同的机械传感信号复合体来传递来自PECAM在白细胞伪足上接触的压力，就像它传递来自液体透明膜的压力一样。在AIM II中，活体显微镜将利用EC中表达的遗传编码钙传感器↑[CaMP3]i来量化小鼠体内GCaMP3[CaMP1]i的时间和强度。我们将培育TRPC6缺陷和足够的GCaMP3限制在EC的小鼠。这些小鼠将被照射并与TRPC6WT小鼠的骨髓重组，以比较携带和不携带TRPC6的小鼠的透射电子显微镜和↑[钙]i。目的III确定TRPC6在缺血/再灌注(I/R)损伤相关炎症过程中的作用。我们将通过活体显微镜在细胞水平和器官水平研究急性心肌梗死模型中的I/R。由于在炎症模型中，TRPC6基因敲除显著降低了透射电子显微镜，我们假设抑制TRPC6将提供对I/R损伤的保护。这些研究将对透射电子显微镜的机制提供深入的认识，并可能确定TRPC6作为一种新的治疗靶点，用于治疗由病理性炎症引起的多种疾病，包括心肌梗死的I/R损伤。

Transendothelial migration (TEM) is a critical step in the inflammatory response in which leukocytes leave the blood vessel to enter the inflamed tissues. Most pathology, including ischemia/reperfusion (I/R) injury, is due to dysfunctional inflammation. I/R injury occurs when tissues deprived of oxygen are reoxygenated, producing reactive oxygen species that damage cells and incite local inflammation. I/R injury is a major source of morbidity and mortality in myocardial infarction (MI; heart attack), making ischemic injury to the heart muscle even worse. Limiting I/R injury is major goal in treatment of MI and other vascular disorders. TEM is initiated by interactions between platelet/endothelial cell adhesion molecule 1 (PECAM) on the surface of leukocytes and PECAM concentrated at the endothelial cell (EC) borders. Efficient TEM requires a transient increase in cytosolic free calcium ion concentration (↑[Ca2+]i). TRPC6 was found to be the calcium channel responsible for (↑[Ca2+]i) and functions downstream of PECAM-PECAM interactions to promote TEM. However, the mechanism by which PECAM signals to TRPC6 is unknown. I will determine how PECAM signals to TRPC6, how TRPC6 regulates ↑[Ca2+]i in vivo, and the relevance of TRPC6 to I/R injury in MI. Endothelial cells have a junctional mechanosensory complex consisting of PECAM, vascular endothelial (VE)-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). This signaling complex responds to fluid shear. Preliminary data suggest that this same complex may also function to transmit signals from PECAM to TRPC6 during TEM. Aim I will determine how PECAM signals to activate TRPC6. We will use specific pharmacologic inhibitors as well as knockdown and re-expression strategies for each component of the system. PECAM FRET tension sensors and VE-cadherin mutants will be used to test the hypothesis that EC PECAM uses the same mechanosensory signaling complex to transmit stress from engagement of PECAM on the leukocyte pseudopod as it does to transmit stress from fluid sheer. In Aim II, intravital microscopy will be used to quantify the timing and intensity of ↑[Ca2+]i in mice with the genetically encoded calcium sensor GCaMP3 expressed in EC. We will breed TRPC6-deficient and sufficient mice with GCaMP3 restricted to EC. These mice will be irradiated and reconstituted with bone marrow from TRPC6 WT mice to compare TEM and ↑[Ca2+]i in mice with and without TRPC6. Aim III will determine the role of TRPC6 in the clinically relevant inflammatory setting of ischemia/reperfusion (I/R) injury. We will study I/R at the cellular level by intravital microscopy and at the organ level in an acute myocardial infarction model. As TRPC6 knockout significantly decreases TEM in models of inflammation, we hypothesize that TRPC6 inhibition will offer protection from I/R injury. These studies will provide insights into the mechanisms of TEM and may identify TRPC6 as a novel therapeutic target for a multitude of diseases caused by pathologic inflammation, including I/R injury in myocardial infarction.