Novel Signaling Pathways in Ischemic Stroke

描述（由申请人提供）：血小板活化是大多数缺血性中风的最终共同途径。在血管功能障碍和炎症的情况下，急性血栓形成引发一系列事件，最终导致神经元坏死死亡和神经血管单位支持结构损伤。然而，连接这些事件的信号通路还没有被很好地理解。Rho/Rho相关的卷曲卷曲形成激酶（ROCK1和ROCK2）是肌动蛋白细胞骨架的重要调节因子。由于肌动蛋白细胞骨架的变化是血小板聚集、血管收缩和炎症细胞募集的基础，因此Rho/ROCK通路可能在缺血性卒中中发挥核心作用。因此，本建议的总体目的是研究ROCK亚型在血小板中的作用，并确定它们如何可能导致血栓栓塞性中风。为了实现这一目标，我们将使用基因敲除（KO）、骨髓移植（BMT）和Cre/loxP技术靶向血小板中的ROCK缺失，并将研究血小板ROCK在血栓形成、凝块传播和局灶性脑缺血中的后续功能丧失。这些拟议研究的结果有望导致开发异型特异性ROCK抑制剂作为缺血性卒中患者的新疗法。具体目的1将确定岩石促进血小板功能和动脉血栓形成的机制。我们将检验岩石在调节血小板细胞骨架组装和调节血小板功能方面发挥不同作用的假设。为了确定和比较ROCK1和ROCK2对血栓形成的影响，我们将研究来自ROCK1-/-和ROCK2-/-骨髓移植（BMT）小鼠的血小板在被各种血小板激动剂激活后的聚集、粘附、异型和同型聚集形成，并通过电子显微镜直接观察。此外，我们将研究调节血小板肌动蛋白细胞骨架和功能的潜在下游信号通路。具体目的2将确定在血栓栓塞模型中，血小板ROCK缺失对血栓形成和传播的病理生理后果。我们将在体内验证岩石对血小板功能至关重要的假设，并且在血栓形成介导的局灶性脑缺血小鼠模型中，血小板缺失岩石可提供卒中保护。为此，我们将开发血小板特异性ROCK - KO小鼠（ROCK1Plt-/-和ROCK2Plt-/-小鼠），并利用(1)颈动脉损伤模型来测量动脉闭塞性血栓形成，(2)依赖于受体兴奋剂的血小板消耗模型来研究微血栓形成，以及(3)使用预形成血栓的血栓栓塞性中风模型来确定血栓形成和粘附血管系统的能力，介导血管闭塞，并导致脑缺血和梗死。

DESCRIPTION (provided by applicant): The activation of platelets is the final common pathway for most ischemic strokes. Acute thrombus formation in the setting of vascular dysfunction and inflammation initiates a cascade of events that culminates in necrotic death of neurons and injury to their supportive structures in the neurovascular unit. However, the signaling pathways that link these events are not well understood. The Rho/Rho-associated coiled-coil forming kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. Because changes in the actin cytoskeleton underlie platelet aggregation, vascular contractility, and inflammatory cell recruitment, it is likely that the Rho/ROCK pathway will play a central role in ischemic strokes. Accordingly, the overall aim of this proposal is to investigate the role of ROCK isoforms in platelets and to determine how they might contribute to thromboembolic strokes. To achieve this goal, we will target ROCK deletion in platelets using knockout (KO), bone marrow transplantation (BMT), and Cre/loxP technology and will investigate the subsequent loss-of-function of platelet ROCKs in thrombus formation, clot propagation, and focal cerebral ischemia. The results of these proposed studies will hopefully lead to the development of isoform-specific ROCK inhibitors as novel therapies for patients with ischemic strokes. Specific aim 1 will determine the mechanisms by which ROCKs contribute to platelet function and arterial thrombosis. We will test the hypothesis that ROCKs play differential roles in regulating the assembly of the platelet cytoskeleton and mediating platelet function. To determine and compare the effect of ROCK1 and ROCK2 on thrombosis, platelets derived from ROCK1-/- and ROCK2-/- bone marrow transplanted (BMT) mice will be studied for aggregation, adhesion, hetero- and homo-typic aggregate formation, and by direct visualization with electron microscopy after activation with various platelet agonists. Furthermore, we will investigate the potential downstream signaling pathways of ROCKs that regulates platelet actin cytoskeleton and function. Specific aim 2 will determine the pathophysiological consequences of platelet ROCK deletion on thrombus formation and propagation in a clot embolic model of stroke. We will test the hypothesis that ROCKs are critically important for platelet function in vivo, and that platelet deletion of ROCKs confers stroke protection in a mouse model of thrombosis-mediated focal cerebral ischemia. To do this, we will develop platelet-specific ROCK KO mice (ROCK1Plt-/- and ROCK2Plt-/- mice) and utilize (1) a carotid artery injury model for measurement of arterial occlusive thrombosis, (2) an agonist-dependent platelet consumptive model to study micro thrombi formation, and (3) a clot-embolic stroke model using preformed thrombi to determine the ability of a clot to form and adhere to the vasculature, mediate vascular occlusion, and cause cerebral ischemia and infarction. PUBLIC HEALTH RELEVANCE: Stroke is the 3rd leading cause of death and a major cause of disability in the Western society. The final common pathway of most strokes is platelet activation and aggregation. This research application proposes to investigate the role of an emerging signaling pathway, Rho kinase (ROCK), in platelets as a potential therapeutic target for preventing and treating ischemic strokes.