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卒中的最终共同途径。在血管功能障碍和炎症背景下的急性血栓形成引发了一系列事件，最终导致神经元坏死性死亡和神经血管单元中支持结构的损伤。然而，连接这些事件的信号通路并不清楚。Rho/Rho相关卷曲螺旋形成激酶（ROCK 1和ROCK 2）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、血管收缩性和炎性细胞募集的基础，因此Rho/ROCK通路可能在缺血性卒中中发挥核心作用。因此，本提案的总体目标是研究ROCK亚型在血小板中的作用，并确定它们如何导致血栓栓塞性卒中。为了实现这一目标，我们将使用敲除（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。这些研究的结果将有望导致亚型特异性ROCK抑制剂的开发，作为缺血性卒中患者的新疗法。 具体目标1将确定ROCK促进血小板功能和动脉血栓形成的机制。我们将检验ROCK在调节血小板细胞骨架组装和介导血小板功能中发挥不同作用的假设。为了确定和比较ROCK 1和ROCK 2对血栓形成的作用，将研究来自ROCK 1-/-和ROCK 2-/-骨髓移植（BMT）小鼠的血小板的聚集、粘附、异型和同型聚集体形成，并通过在用各种血小板激动剂活化后用电子显微镜直接观察。此外，我们将研究潜在的下游信号通路的ROCKs调节血小板肌动蛋白细胞骨架和功能。 具体目标2将确定血小板ROCK缺失对卒中血栓栓塞模型中血栓形成和传播的病理生理学后果。我们将测试的假设，ROCKs是至关重要的血小板功能在体内，和血小板ROCKs缺失赋予中风保护血栓介导的局灶性脑缺血小鼠模型。为此，我们将开发血小板特异性ROCK KO小鼠（ROCK 1 Plt-/-和ROCK 2 Plt-/-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）激动剂依赖性血小板消耗模型来研究微血栓形成，和（3）使用预先形成的血栓的血栓栓塞中风模型，以确定血栓形成并粘附到脉管系统，介导血管闭塞，并引起脑缺血和梗塞。 公共卫生相关性：中风是西方社会第三大死亡原因，也是残疾的主要原因。大多数中风的最后共同途径是血小板活化和聚集。这项研究申请提出了一个新兴的信号通路，Rho激酶（ROCK），在血小板作为预防和治疗缺血性中风的潜在治疗靶点的作用。

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.