Cytoskeletal Dynamics of Brain Pericytes and Impact on Capillary Flow

项目总结。 脑周细胞是一种特殊的壁细胞，排列在整个脑血管毛细血管床上。这个 周细胞可收缩并具有控制毛细血管流动的能力的概念可以追溯到他们的 19世纪90年代的发现。然而，由于缺乏在体内靶向和操纵周细胞的方法，这 周细胞生物学方面的研究仍然很少。了解周细胞势在必行 收缩能力，因为许多人类和动物研究表明毛细血管血流不足 在卒中期间，毛细血管的异常收缩可能是原因之一。正在调整 周细胞的收缩能力可能是一种有价值的改善脑血液的治疗方法 这是一种在缺血期间补充血栓的方法，也是进一步提高现有血栓清除治疗效果的手段。 目前，对周细胞收缩所涉及的信号通路知之甚少。最新研究 已经表明，排列在大脑毛细血管床上的绝大多数周细胞对α呈阴性反应。 肌动蛋白(α-SMA)，它是肌动球蛋白引起的平滑肌细胞收缩的中枢 小动脉。然而，我们的初步数据表明，这些α-SMA阴性的周细胞保持着 在体内收缩，会阻碍毛细血管流动，表明有另一种收缩机制。我们的 中心假说是脑毛细血管周细胞可以通过动态肌动蛋白收缩 细胞骨架重组，而不是肌动蛋白跨桥循环。我们检验了这一假设 通过结合药理学和一种新的光遗传学方法来激活单个毛细血管周细胞 体内和体外的“因果”方式。在目标1中，我们将测试抑制或 促进肌动蛋白聚合可以改变活体大脑中光遗传诱导的周细胞收缩 老鼠。我们将进一步测试这些药物在体外加压小动脉对周细胞收缩的作用。 毛细血管制备以排除非血管脑细胞的间接作用。在《目标2》中，我们直接 在活体内观察正常和缺血脑毛细血管周细胞中肌动蛋白的聚合。我们会 表达Lifeact-GFP，一种新的F-肌动蛋白荧光探针，特异性地在血管壁细胞和 检查病理性毛细血管收缩前周细胞中的F-肌动蛋白含量是否增加。这 该项目将阐明毛细血管周细胞细胞骨架动力学及其与毛细血管流动的关系，以及 周细胞生物学的一个方面，在活体的脑微血管系统中研究得很少。如果成功， 我们的发现将有助于为进一步的研究建立理论基础、方法和老鼠模型 周细胞细胞骨架动力学如何参与毛细血管血流损害的研究 中风和相关的脑部病理。

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.