Thrombin Mediated Cytotoxicity during Cerebral Ischemia

脑缺血期间凝血酶介导的细胞毒性

基本信息

批准号：
8448649
负责人：
Patrick D Lyden
金额：
$ 35.25万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8448649
关键词：
Adult Affect Americas Anatomy Animals Area Argatroban Behavioral Blood Proteins Blood Vessels Brain Injuries Brain Ischemia Cause of Death Cell Death Cells Cerebral Ischemia Cerebrum Cleaved cell Clinical Trials Coagulation Process Data Distal Dose Edema Extravasation Fluorescence Gene Expression Infarction Injury Ischemia Knockout Mice Left Mediating Methods Middle Cerebral Artery Occlusion Modeling Molecular Mus Neurologic Neuroprotective Agents Neurotoxins Outcome PAR-1 Receptor PAR-2 Receptor Parietal Parietal Lobe Pathway interactions Patients Peptide Hydrolases Peptides Play Process Proteinase-Activated Receptors RNA Interference Rattus Receptor Signaling Role Saline Scheme Serine Protease Signal Pathway Stroke Testing Therapeutic Thrombin Thrombolytic Therapy Thrombosis Tissues Toxic effect acute stroke behavior measurement behavioral impairment cell killing cytotoxicity design disability effective therapy inhibitor/antagonist insight knock-down lentiviral-mediated mutant neurotoxicity neurovascular unit preconditioning protease-activated receptor 3 protease-activated receptor 4 receptor research study translational study

项目摘要

DESCRIPTION (provided by applicant): Although thrombolytic therapy for acute stroke continues to gain wider acceptance and usage, there remains a compelling need for neuroprotective therapy and new treatment to reduce thrombolytic complications. Thrombin is a serine protease that plays a critical role in the coagulation cascade. Thrombin induces protection at low doses (thrombin preconditioning) but acts as a neurotoxin at high doses, killing cells via the protease activated receptors (PARs). We now propose to test the hypothesis that thrombin partially mediates edema and cell death during stroke via the PAR-1 receptor signaling pathway. Using a protease-cleavable cell-penetrating probe developed by Dr. Roger Tsien, we will determine directly whether thrombin activation mediates edema and tissue injury. Using a highly reproducible and quantifiable model of ischemic edema and tissue injury, we will test a variety of pharmacological manipulations of coagulation or PAR-1 and its presumed signaling pathways. Using lentiviral mediated RNA interference, we will reduce gene expression of PAR-1 in focal areas of the parietal cortex of adult mice prior to standard MCAo and then determine the effect of PAR-1 knock-down on tissue injury. Using knock-out mice deficient in PAR-1, PAR-2, PAR-3, or PAR-4 subjected to MCAo, we will determine whether each or all PAR receptors influence vascular disruption and tissue injury after MCAo. Finally, we will extend our previous studies to include behavioral measures of cerebral injury and seek to determine whether thrombin exacerbates-and whether argatroban ameliorates-behavioral deficits after MCAo. We will use intra-arterial mutant thrombin designed to activate PAR-1 receptor but not affect coagulation during ischemia. We will also test neuroprotective drugs such as mutant APC or others screened for effect on stroke outcome. Taken together, these studies will reveal whether thrombin cytotoxicity plays a role in stroke and whether agents active at PAR-1 signaling pathway represent an effective therapy. Relevance The search for effective neuroprotectants is hampered by many factors: we continue to lack a full understanding of the molecular mechanisms of ischemic cell death and vascular disruption, as well as the patho-anatomic mechanisms of ischemic edema, infarction, and behavioral impairment. Fundamental exploration of molecular and patho- anatomic mechanisms AND translational studies of therapeutics remain highly significant and critical to understanding and treating stroke, the most common cause of adult disability in the world.

描述（由申请人提供）：尽管急性中风的溶栓疗法继续获得更广泛的接受和使用，但仍然迫切需要神经保护疗法和新的治疗方法来减少溶栓并发症。凝血酶是一种丝氨酸蛋白酶，在凝血级联中发挥着关键作用。凝血酶在低剂量时可诱导保护（凝血酶预处理），但在高剂量时可充当神经毒素，通过蛋白酶激活受体 (PAR) 杀死细胞。我们现在提议检验凝血酶通过 PAR-1 受体信号通路部分介导中风期间水肿和细胞死亡的假设。使用Roger Tsien博士开发的蛋白酶可裂解细胞穿透探针，我们将直接确定凝血酶激活是否介导水肿和组织损伤。使用高度可重复和可量化的缺血性水肿和组织损伤模型，我们将测试凝血或 PAR-1 的各种药理学操作及其假定的信号通路。使用慢病毒介导的RNA干扰，我们将在标准MCAo之前降低成年小鼠顶叶皮层焦点区域中PAR-1的基因表达，然后确定PAR-1敲低对组织损伤的影响。使用接受 MCAo 的 PAR-1、PAR-2、PAR-3 或 PAR-4 缺陷的敲除小鼠，我们将确定每个或所有 PAR 受体是否影响 MCAo 后的血管破坏和组织损伤。最后，我们将扩展之前的研究，将脑损伤的行为测量纳入其中，并试图确定凝血酶是否会加剧 MCAo 后的行为缺陷，以及阿加曲班是否会改善行为缺陷。我们将使用动脉内突变型凝血酶，旨在激活 PAR-1 受体，但不影响缺血期间的凝血。我们还将测试神经保护药物，例如突变 APC 或其他经过筛选对中风结果有影响的药物。总而言之，这些研究将揭示凝血酶细胞毒性是否在中风中发挥作用，以及激活 PAR-1 信号通路的药物是否代表一种有效的治疗方法。相关性寻找有效的神经保护剂受到许多因素的阻碍：我们仍然缺乏对缺血性细胞死亡和血管破坏的分子机制，以及缺血性水肿、梗塞和行为障碍的病理解剖学机制的充分了解。分子和病理解剖机制的基础探索以及治疗的转化研究对于理解和治疗中风（世界上成人残疾的最常见原因）仍然非常重要和关键。