Cytoskeletal Dynamics of Brain Pericytes and Impact on Capillary Flow

项目摘要。 脑周细胞是分布于整个脑血管毛细血管床的特化壁细胞。的 周细胞是收缩性的，并具有控制毛细血管流动的能力，这一概念可以追溯到他们的 发现于1890年代。然而，由于缺乏体内靶向和操纵周细胞的方法， 周细胞生物学方面的研究仍然非常不足。必须了解周细胞 收缩性，因为许多人类和动物研究已经证明毛细血管流动不足 在中风期间，毛细血管的异常收缩是可能的贡献者。调制 周细胞的收缩能力可能代表一种有价值的治疗方法，以改善脑血 缺血期间的供应，以及进一步提高现有凝块去除治疗的功效的手段。 目前，很少有人知道的信号通路参与周细胞收缩。最近的研究 已经表明，绝大多数排列在脑毛细血管床上的周细胞对α- 平滑肌肌动蛋白（α-SMA），是平滑肌细胞基于肌动蛋白的收缩的中心 的小动脉然而，我们的初步数据表明，这些α-SMA阴性周细胞保留了 在体内收缩，并且可以阻碍毛细血管流动，指向另一种收缩机制。我们 中心假设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，而不是肌动球蛋白跨桥循环。我们检验这个假设 通过将药理学与新型光遗传学测定相结合来激活细胞中的单个毛细血管周细胞， “因果”的方式在体内和离体。在目标1中，我们将测试是否有药物抑制或 促进肌动蛋白聚合可以改变光遗传学诱导的肝细胞周细胞收缩 小鼠我们将进一步测试这些药物对周细胞收缩性的影响，在离体，加压小动脉- 毛细血管制备，以排除非血管脑细胞的间接作用。第二，我们直接 在正常和缺血的脑中，在活体中，使毛细血管周细胞中的肌动蛋白聚合可视化。我们将 表达Lifeact-GFP，一种新型的F-肌动蛋白荧光探针，特别是在血管壁细胞中， 检查在病理性毛细血管收缩之前周细胞中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骨架动力学及其与毛细血管流动的关系， 这是周细胞生物学的一个方面，在体内脑微血管系统中研究得非常不足。如果成功， 我们的发现将有助于建立进一步研究的理论基础、方法学和小鼠模型。 研究周细胞骨架动力学如何参与毛细血管血流损伤， 中风和相关的脑病理。

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.