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，是由于功能失调的炎症。I/R损伤发生在缺氧的组织再充氧时，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; heart attack）发病率和死亡率的主要来源，使心肌缺血损伤更加严重。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/内皮细胞粘附分子1 （PECAM）与内皮细胞（EC）边界集中的PECAM相互作用引发的。高效的透射电镜需要细胞质中游离钙离子浓度的瞬时增加（↑[Ca2+]i）。TRPC6被发现是钙通道，负责（↑[Ca2+]i）和PECAM-PECAM相互作用的下游功能，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与心肌梗死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物。这种信号复合物响应流体剪切。初步数据表明，在TEM过程中，该复合体也可能发挥从PECAM向TRPC6传递信号的作用。目的：确定PECAM信号如何激活TRPC6。我们将对系统的每个组成部分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EC PECAM使用相同的机械感觉信号复合体来传递PECAM与白细胞假足接触时的压力，就像它传递来自流体的压力一样。在Aim II中，活体显微镜将用于量化具有EC中表达的遗传编码钙传感器GCaMP3的小鼠中↑[Ca2+]i的时间和强度。我们将培养trpc6缺乏和充足的小鼠，GCaMP3仅限于EC。这些小鼠将被照射并与TRPC6 WT小鼠的骨髓重组，以比较携带和不携带TRPC6小鼠的TEM和↑[Ca2+]i。目的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和器官水平上研究急性心肌梗死模型的I/R。由于TRPC6敲除显著降低炎症模型中的TEM，我们假设TRPC6抑制将对I/R损伤提供保护。这些研究将为TEM的机制提供新的见解，并可能确定TRPC6作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。