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）：尽管用于急性中风的溶栓治疗继续获得更广泛的接受和使用，但仍需对神经保护疗法和新疗法的迫切需求，以减少溶栓并发症。凝血酶是一种丝氨酸蛋白酶，在凝血级联反应中起关键作用。凝血酶以低剂量（凝血酶预处理）诱导保护，但在高剂量时充当神经毒素，通过蛋白酶活化受体（PARS）杀死细胞。现在，我们建议测试通过PAR-1受体信号传导途径在中风期间部分介导的凝血酶和细胞死亡的假设。使用Roger Tsien博士开发的可蛋白酶可转化的细胞探针，我们将直接确定凝血酶激活是否介导水肿和组织损伤。使用高度可再现和可量化的缺血性水肿和组织损伤模型，我们将测试各种凝结或PAR-1及其假定信号通路的药理操作。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的焦点区域中PAR-1的基因表达，然后确定PAR-1敲低对组织损伤的影响。使用缺乏MCAO的PAR-1，PAR-2，PAR-3或PAR-4缺乏的敲除小鼠，我们将确定每个或所有PAR受体是否会影响MCAO后的血管破坏和组织损伤。最后，我们将扩展以前的研究，以包括脑损伤的行为度量，并试图确定凝血酶是否加剧，以及Argatroban是否在MCAO之后会改善行为不足。我们将使用旨在激活PAR-1受体的动脉内突变体凝血酶，但不会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）或其他对中风结果影响的神经保护药物。综上所述，这些研究将揭示凝血酶细胞毒性是否在中风中起作用，以及在PAR-1信号通路上活跃的药物是否代表了一种有效的治疗。许多因素阻碍了寻找有效神经保护剂的相关性：我们继续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，梗死和行为障碍的致病动态机制的分子机制充分了解。分子和病原体机制和治疗剂的转化研究的基本探索仍然是高度重要的，对于理解和治疗中风，这是世界上成人残疾的最常见原因。