The regulation of transendothelial migration by calcium signaling in endothelial cells

内皮细胞中钙信号传导对跨内皮迁移的调节

• 批准号: 9331824
• 负责人: Natalya Diaz
• 金额: $ 0.27万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2017-06-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9331824
• 关键词: Acute myocardial infarction; Adhesives; Anti-Inflammatory Agents; Anti-inflammatory; Atherosclerosis; Binding; Blood Vessels; Bone Marrow; CD31 Antigens; Calcium; Calcium Channel; Calcium Signaling; Calcium ion; Cause of Death; Cells; Clinical; Complex; Data; Defect; Disease; Emigrations; Endothelial Cells; Endothelium; Fluorescence Resonance Energy Transfer; Goals; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Ischemia; KDR gene; Knock-out; Leukocytes; Link; Liquid substance; Modeling; Molecular; Morbidity - disease rate; Mus; Myocardial; Myocardial Infarction; Myocardium; Organ; Oxygen; PLC gamma1; Pathologic; Pathology; Pharmacology; Reactive Oxygen Species; Recruitment Activity; Regulation; Reperfusion Injury; Reperfusion Therapy; Resolution; Role; Series; Signal Pathway; Signal Transduction; Source; Speed; Stress; Surface; System; Testing; Time; Tissues; United States; Vascular Diseases; Wound Healing; cadherin 5; cell injury; clinically relevant; cremaster muscle; fighting; in vivo; inhibitor/antagonist; insight; intravital microscopy; killings; knock-down; migration; monocyte; mortality; mutant; neutrophil; new therapeutic target; novel; pathogen; reconstitution; release of sequestered calcium ion into cytoplasm; sensor

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.

跨内皮迁移（TEM）是炎症反应中的关键步骤，其中白细胞离开血管进入发炎组织。大多数病理学，包括缺血/再灌注（I/R）损伤，都是由于功能失调的炎症引起的。当缺氧的组织重新充氧时，就会发生缺血再灌注损伤，产生活性氧，损害细胞并引发局部炎症。 I/R 损伤是心肌梗塞（MI；心脏病发作）发病率和死亡率的一个主要来源，使得心肌缺血性损伤更加严重。限制 I/R 损伤是治疗 MI 和其他血管疾病的主要目标。 TEM 是由白细胞表面的血小板/内皮细胞粘附分子 1 (PECAM) 与集中在内皮细胞 (EC) 边界的 PECAM 之间的相互作用引发的。有效的 TEM 需要短暂增加胞质游离钙离子浓度 (↑[Ca2+]i)。 TRPC6 被发现是负责 (↑[Ca2+]i) 的钙通道，并在 PECAM-PECAM 相互作用的下游发挥作用，以促进 TEM。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 ↑[Ca2+]i，以及 TRPC6 与 MI 中 I/R 损伤的相关性。 内皮细胞具有由 PECAM、血管内皮 (VE)-钙粘蛋白和血管内皮生长因子受体 2 (VEGFR2) 组成的连接机械感觉复合体。该信号复合物对流体剪切作出反应。初步数据表明，该复合体还可能在 TEM 期间将信号从 PECAM 传输到 TRPC6。 目标 我将确定 PECAM 如何发出信号来激活 TRPC6。我们将为系统的每个组件使用特定的药理学抑制剂以及敲低和重新表达策略。 PECAM FRET 张力传感器和 VE-钙粘蛋白突变体将用于测试以下假设：EC PECAM 使用相同的机械感觉信号复合物来传递 PECAM 与白细胞伪足接合产生的应力，就像它传递流体剪切产生的应力一样。在 Aim II 中，将使用活体显微镜来量化具有 EC 中表达的基因编码钙传感器 GCaMP3 的小鼠中 ↑[Ca2+]i 的时间和强度。我们将培育 TRPC6 缺陷且具有限制于 EC 的 GCaMP3 的小鼠。这些小鼠将接受 TRPC6 WT 小鼠的骨髓照射和重建，以比较有和没有 TRPC6 的小鼠的 TEM 和 ↑[Ca2+]i。目标 III 将确定 TRPC6 在缺血/再灌注 (I/R) 损伤的临床相关炎症环境中的作用。我们将通过活体显微镜在细胞水平上研究 I/R，并在急性心肌梗死模型中研究器官水平上的 I/R。由于 TRPC6 敲除显着降低炎症模型中的 TEM，因此我们假设 TRPC6 抑制将提供针对 I/R 损伤的保护。这些研究将深入了解 TEM 的机制，并可能将 TRPC6 确定为病理性炎症引起的多种疾病的新治疗靶点，包括心肌梗死中的 I/R 损伤。