Novel Signaling Pathways in Ischemic Stroke

描述(申请人提供)：血小板的激活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背景下，急性血栓形成会引发一系列事件，最终导致神经元的坏死性死亡及其在神经血管单位的支持结构受到损害。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、血管收缩和炎性细胞募集的基础，Rho/ROCK通路很可能在缺血性中风中发挥核心作用。因此，这项建议的总体目标是调查岩石异构体在血小板中的作用，并确定它们可能如何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技术靶向删除血小板中的岩石，并将研究随后血小板岩石在血栓形成、血栓扩散和局灶性脑缺血中的功能丧失。这些拟议研究的结果将有望导致异构体特异性ROCK抑制剂的开发，作为治疗缺血性中风患者的新疗法。具体目标1将确定岩石促进血小板功能和动脉血栓形成的机制。我们将验证岩石在调节血小板细胞骨架的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ROCK1-/-和ROCK2-/-骨髓移植(BMT)小鼠的血小板的聚集性、粘附性、异型和同型聚集体的形成，以及在不同的血小板激动剂激活后用电子显微镜直接观察。此外，我们还将研究调节血小板肌动蛋白细胞骨架和功能的ROCK的潜在下游信号通路。具体目标2将确定在中风血栓栓塞性模型中，血小板岩石缺失对血栓形成和扩散的病理生理学后果。我们将在血栓介导局灶性脑缺血的小鼠模型中，测试岩石对体内血小板功能至关重要的假设，以及岩石中的血小板缺失提供中风保护的假设。为此，我们将建立血小板特异性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)激动剂依赖的血小板消耗模型来研究微血栓的形成，以及(3)使用预形成的血栓来确定血栓形成和粘连血管系统、介导血管闭塞以及导致脑缺血和梗塞的血栓栓塞性中风模型。 公共卫生相关性：在西方社会，中风是第三大死亡原因，也是导致残疾的主要原因。大多数中风的最终共同途径是血小板的激活和聚集。这项研究的应用建议调查一个新兴的信号通路，Rho激酶(ROCK)在血小板中的作用，作为预防和治疗缺血性中风的潜在治疗靶点。

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.