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.

跨内皮细胞迁移（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，是由于功能障碍性炎症。I/R损伤发生在缺氧的组织再氧合时，产生损伤细胞并引发局部炎症的活性氧。I/R损伤是心肌梗死（MI;心脏病发作）的发病率和死亡率的主要来源，使心肌的缺血性损伤更加严重。限制I/R损伤是MI和其他血管疾病治疗的主要目标。 TEM是由白细胞表面的血小板/内皮细胞粘附分子1（PECAM）与内皮细胞（EC）边界处浓缩的PECAM之间的相互作用引发的。有效的TEM需要胞质游离钙离子浓度（↑[Ca 2 +]i）的瞬时增加。TRPC 6被发现是负责（↑[Ca 2 +]i）的钙通道，并在PECAM-PECAM相互作用的下游起作用以促进TEM。然而，PECAM向TRPC 6发信号的机制尚不清楚。我将确定PECAM如何信号TRPC 6，如何TRPC 6调节↑[Ca 2 +]i在体内，和相关的TRPC 6 I/R损伤MI。 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（VEGFR 2）组成的连接性机械感觉复合体。这种信号复合物对流体剪切力有反应。初步数据表明，在TEM过程中，这种复合物也可能起到从PECAM向TRPC 6传输信号的作用。 目的我将确定如何PECAM信号激活TRPC 6。我们将使用特定的药理学抑制剂，以及敲低和重新表达策略的每个组成部分的系统。PECAM FRET张力传感器和VE-钙粘蛋白突变体将用于测试EC PECAM使用相同的机械感觉信号传导复合物来传递来自PECAM在白细胞伪足上的接合的应力，正如它传递来自流体剪切的应力一样的假设。在目标II中，将使用活体显微镜对EC中表达的遗传编码钙传感器GCaMP 3的小鼠中↑[Ca 2 +]i的时间和强度进行定量。我们将培育TRPC 6缺陷和足够的小鼠与GCaMP 3限制EC。将对这些小鼠进行辐照，并用来自TRPC 6 WT小鼠的骨髓重建，以比较有和无TRPC 6的小鼠中的TEM和↑[Ca 2 +]i。目的III将确定TRPC 6在缺血/再灌注（I/R）损伤的临床相关炎症环境中的作用。我们将研究I/R在细胞水平的活体显微镜和器官水平的急性心肌梗死模型。由于TRPC 6敲除显著降低了炎症模型中的TEM，我们假设TRPC 6抑制将提供I/R损伤的保护。这些研究将为TEM的机制提供见解，并可能将TRPC 6确定为由病理性炎症引起的多种疾病（包括心肌梗死中的I/R损伤）的新治疗靶点。