Cytoskeletal Dynamics of Brain Pericytes and Impact on Capillary Flow

脑周细胞的细胞骨架动力学及其对毛细血管血流的影响

• 批准号: 9789063
• 负责人: Andy Y Shih
• 金额: $ 23.54万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2020-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789063
• 关键词: Actins; Actomyosin; Animals; Back; Basic Science; Biological Assay; Biology; Blood Vessels; Blood capillaries; Blood flow; Brain; Brain Pathology; Caliber; Cell Line; Cells; Cerebral cortex; Cerebrovascular Circulation; Cerebrovascular Disorders; Cerebrum; Chemicals; Coagulation Process; Contracts; Cytochalasins; Cytoskeleton; Data; Development; Endothelium; Erythrocytes; Excision; F-Actin; Fluorescence; Fluorescent Probes; G Actin; Human; Impairment; Individual; Investigation; Ischemia; Lead; Light; Location; Measures; Mediator of activation protein; Methodology; Methods; Microinjections; Mus; Myosin Light Chain Kinase; Optics; Pathologic; Pericytes; Pharmaceutical Preparations; Pharmacology; Pre-Clinical Model; Preparation; Public Health; Research; Resistance; Rho-associated kinase; Signal Pathway; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Source; Stroke; Techniques; Technology; Testing; Therapeutic; Translating; Vascular blood supply; arteriole; base; brain cell; capillary bed; cell type; cerebral capillary; cerebral microvasculature; cerebrovascular; constriction; drug testing; electric impedance; improved; in vivo; inhibitor/antagonist; jasplakinolide; monomer; mouse model; multiphoton imaging; novel; optogenetics; polymerization; pressure; prevent; response; stroke model; two-photon

Project summary. Cerebral pericytes are specialized mural cells that line the entire cerebrovascular capillary bed. The concept that pericytes are contractile and have the capacity control capillary flow dates back to their discovery in the 1890s. Yet, due to a lack of methods to target and manipulate pericytes in vivo, this facet of pericyte biology has remained highly understudied. It is imperative to understand pericyte contractility because many human and animal studies have demonstrated insufficiency in capillary flow during stroke, with aberrant constriction of capillaries being a likely contributor. Modulating the contractile ability of pericytes may represent a valuable therapeutic approach to improve cerebral blood supply during ischemia, and a means to further improve the efficacy of existing clot-removal treatments. Currently, little is known about the signaling pathways involved in pericyte contraction. Recent studies have shown that the vast majority of pericytes that line the brain capillary bed are negative for α- smooth muscle actin (α-SMA), which is central to actomyosin-based contraction of smooth muscle cells of arterioles. Yet, our preliminary data suggest that these α-SMA-negative pericytes retain the ability to contract in vivo and can impede capillary flow, pointing to an alternative contractile mechanism. Our central hypothesis is that brain capillary pericytes can contract through dynamic actin cytoskeleton reorganization, rather than actomyosin cross-bridge cycling. We test this hypothesis by combining pharmacology with a novel optogenetic assay to activate individual capillary pericytes in a “cause and effect” manner both in vivo and ex vivo. In Aim 1, we will test whether drugs that inhibit or promote actin polymerization can alter optogenetically-induced pericyte contraction in the brains of live mice. We will further test these drugs on pericyte contractility in an ex vivo, pressurized arteriole-to- capillary preparation to exclude indirect actions from non-vascular brain cells. In Aim 2, we directly visualize actin polymerization in capillary pericytes in the normal and ischemic brain in vivo. We will express Lifeact-GFP, a novel fluorescent probe for F-actin, specifically in vascular mural cells and examine whether F-actin content increases in pericytes prior to pathological capillary constriction. This project will shed light on capillary pericyte cytoskeletal dynamics and its relation to capillary flow, an aspect of pericyte biology that is highly understudied in the brain microvasculature in vivo. If successful, our findings will help to establish the rationale, methodologies, and mouse models for further investigations of how pericyte cytoskeletal dynamics are involved in capillary flow impairment during stroke and related brain pathologies.

项目摘要。 脑周细胞是整个脑血管毛细血管床的专门壁细胞。这 周细胞是收缩的概念，并具有能力控制毛细管流程的历史 1890年代的发现。但是，由于缺乏靶向和在体内操纵周细胞的方法，这 周围生物学的方面始终广为人知。必须了解周细胞 收缩力是因为许多人类和动物研究表明毛细血管流不足 在中风期间，毛细血管的异常收缩可能是可能的贡献者。调节 周细胞的收缩能力可能代表一种有价值的治疗方法来改善脑血液 缺血期间的供应，以及进一步提高现有凝块脱毛处理效率的手段。 目前，关于周围收缩涉及的信号通路知之甚少。最近的研究 已经表明，脑毛细管床的绝大多数周细胞对于α-均为阴性 平滑肌肌动蛋白（α-SMA），这对于平滑肌细胞的基于肌动蛋白的收缩至关重要 小动脉。然而，我们的初步数据表明，这些α-SMA阴性周细胞保留了能力 体内收缩并可能阻碍毛细管流，指出另一种收缩机制。我们的 中心假设是脑毛细管周细胞可以通过动态肌动蛋白收缩 细胞骨架重组，而不是肌动菌素跨桥循环。我们检验了这个假设 通过将药理学与新型的光遗传学测定相结合，以激活A中的单个毛细血管周细胞 体内和体内的“因果和效应”。在AIM 1中，我们将测试抑制或 促进肌动蛋白聚合可以改变现场大脑中的光源性周细胞收缩 老鼠。我们将进一步测试这些药物在离体中的周细胞收缩性上 毛细血管制备以排除非血管脑细胞的间接作用。在AIM 2中，我们直接 可视化体内正常和缺血性脑中毛细血管周细胞中的肌动蛋白聚合。我们将 Express LifeAct-GFP，一种用于F-肌动蛋白的新型荧光探针，特别是在血管壁细胞和 检查在病理毛细收缩之前，F-肌动蛋白含量是否增加。这 项目将阐明毛细血管周细胞骨架动力学及其与毛细管流的关系 周围生物学的方面在体内在脑微脉管系统中得到了高度理解。如果成功， 我们的发现将有助于建立基本原理，方法和鼠标模型，以进一步 对周围细胞骨架动力学如何参与毛细血管流动障碍的研究 中风和相关的大脑病理。