Regulation of Brain Thrombosis in Stroke Models

中风模型中脑血栓形成的调节

• 批准号: 7907785
• 负责人: Berislav V Zlokovic
• 金额: $ 47.71万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907785
• 关键词: Acute; American; Angiography; Anti-Inflammatory Agents; Anti-inflammatory; Anticoagulants; Apoptosis; Apoptotic; Area; Blood - brain barrier anatomy; Blood capillaries; Brain; Brain Injuries; Brain Thrombosis; Bromodeoxyuridine; Capillary Endothelial Cell; Cell Proliferation; Cells; Cessation of life; Clinical Trials; Data; Dendritic Spines; Deposition; Dose; Endothelial Cells; Event; Extravasation; Fibrin; Figs - dietary; Glycogen Synthase Kinases; Goals; Hemorrhage; Human; Image; Immunoglobulin G; In Vitro; Injury; Ischemia; Ischemic Stroke; Knockout Mice; Lectin; Length; Lilly brand of drotrecogin alfa activated; Mediating; Mitochondria; Modeling; Morbidity - disease rate; Mouse Protein; Mus; Natural regeneration; Nature; Neuronal Plasticity; Neurons; Optics; Outcome; PAR-1 Receptor; PECAM1 gene; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Phase; Photons; Pilot Projects; Process; Protein C; Recombinants; Recovery; Recovery of Function; Regimen; Regulation; Reporting; Risk; Role; Safety; Sensory; Signal Transduction; Sirolimus; Sphingosine-1-Phosphate Receptor; Staining method; Stains; Stroke; Structure; Testing; Therapeutic; Thrombotic Stroke; Time; Translations; Tube; Tubulin; Vascular Endothelial Growth Factors; Vascular blood supply; Vascular remodeling; Wild Type Mouse; Work; activated Protein C; activated protein C receptor; analog; angiogenesis; base; behavior test; bench to bedside; brain repair; capillary; effective therapy; functional outcomes; hemodynamics; human FRAP1 protein; human NOS3 protein; in vivo; minimally invasive; mortality; mutant; neovascularization; nerve stem cell; nestin protein; neuroblast; neurogenesis; neuroprotection; public health relevance; receptor; relating to nervous system; response

DESCRIPTION (provided by applicant): The vast majority of strokes are ischemic/thrombotic in nature. There is no effective therapy for stroke. Our earlier work showed early post-ischemic neuroprotection by activated protein C (APC), a protease with anti- thrombotic, anti-apoptotic and anti-inflammatory activities, as well as that protease activated receptor 1 (PAR1) and endothelial protein C receptor (EPCR) on brain endothelial cells (BEC), and PAR1 and PAR3 on neurons mediate APC's neuroprotection and inhibition of mitochondria-dependent and death receptor-dependent apoptosis. This work led to a Phase 1/2a clinical trial with APC (Xigris) for acute ischemic stroke with a 6 h therapeutic window (APCAST). Our recent pilot studies indicated that recombinant wtAPC was neuroprotective when given 24 h after ischemia onset suggesting a much larger therapeutic window for APC. Late post- ischemic 6-72 and 72-144 h multi-dosing therapies with wtAPC stimulated neovascularization and neurogenesis. Multi-dosing therapy with 3K3A-APC, an APC mutant with reduced anticoagulant activity, was neuroprotective when initiated 12 h after ischemia onset and stimulated neurogenesis. The cytoprotective- selective 5A-APC mutant with < 10% anticoagulant activity, but not the anticoagulant-selective E149A-APC with 400% increased anticoagulant activity and no cytoprotective activity, was neuroprotective after stroke in mice and increased post-ischemic in vivo dendritic spine formation. Our pilot data show human 3K3A-APC mediates angiogenesis from human BEC and neurogenesis from human neuronal progenitor cells (NPC) in processes that require EPCR, PAR1 and sphingosine 1-phosphate receptor 1 (S1P1), and PAR1, PAR3 and S1P1, respectively. Our central hypothesis is that late therapy with recombinant wtAPC and its cytoprotective- selective 5A-APC mutant, but not the anticoagulant-selective E149A-APC mutant, is neuroprotective and promotes brain regeneration after ischemic/thrombotic stroke by modifying neuronal plasticity, vascular remodeling and stroke-induced neurogenesis through PAR1, EPCR and S1P1 on BEC, and PAR1, PAR3 and S1P1 on NPC. Recombinant murine wtAPC, 5A-APC and E149A-APC will be studied in murine stroke models and human APCs on human BEC and NPC. We propose to test first safety and efficacy of late therapies with wtAPC, 5A-APC and E149A-APC using models of cortical ischemic stroke or photothrombotic ministroke in control mice (aim 1); and in mice with PAR1-4 deletions, EPCR depletion and neural-specific loss of S1P1 (aim 2). The effects of APCs on in vivo neuronal plasticity, vascular remodeling (aim 1), neovascularization (aim 3) and neurogenesis (aim 4) and the receptors requirements for these APC-mediated processes in vivo will be determined. Human BEC (aim 3) and NPC (aim 4) will be used to determine in vitro receptors requirements and downstream Akt targets critical for APC's in vitro angiogenesis and neurogenesis. The proposed studies will bridge the field of ischemic stroke to the field of protein C with an emphasis on late neuroprotection and brain repair therapies that is promising for translation from bench to bedside. PUBLIC HEALTH RELEVANCE: Stroke is a major cause of morbidity and mortality for Americans caused by an acute block in blood supply to the brain resulting often in an irreversible brain damage. There is no effective therapy for stroke today. The proposed studies should help develop the rationale for clinical trials of activated protein C (APC) and its analogs with a substantially extended therapeutic window for stroke to help stroke patients over an extended period of time after stroke occurred with two primary goals: (1) to protect brain during periods of abnormally low blood supply; and (2) to support regeneration and brain repair process during a recovery phase after stroke.

描述（由申请人提供）：绝大多数中风本质上都是缺血性/血栓形成。没有有效的中风疗法。 Our earlier work showed early post-ischemic neuroprotection by activated protein C (APC), a protease with anti- thrombotic, anti-apoptotic and anti-inflammatory activities, as well as that protease activated receptor 1 (PAR1) and endothelial protein C receptor (EPCR) on brain endothelial cells (BEC), and PAR1 and PAR3 on neurons mediate APC's neuroprotection and inhibition of线粒体依赖性和死亡受体依赖性凋亡。这项工作导致使用APC（XIGRIS）进行了1/2A临床试验，用于急性缺血性中风，带有6小时的治疗窗口（APCAST​​）。我们最近的试点研究表明，缺血发作后24小时给予重组WTAPC是神经保护性的，这表明APC的治疗窗口更大。 WTAPC刺激了新血管形成和神经发生的缺血后6-72和72-144 H多剂量疗法。当缺血发作和刺激神经发生后12小时开始时，用3K3A-APC（一种具有降低抗凝抗凝活性的APC突变体）进行多剂量疗法是神经保护性的。具有<10％抗凝活性的细胞保护性 - 选择性5A-APC突变体，但没有抗凝性抗凝凝胶活性增加，没有抗凝性活性增加，而抗凝性抗凝性E149A-APC则是卒中症状的抗凝活性和无细胞保护活性的，是小鼠中的神经保护作用，在小鼠中增加了神经保护作用，并增加了美见后的摄入型齿状形式。我们的试点数据显示，人类3K3A-APC在需要EPCR，PAR1和1-磷酸受体1（S1P1）以及PAR1，PAR1，PAR1，PAR3和S1P1的过程中介导了人类BEC和人类神经祖细胞（NPC）的神经发生的血管生成。我们的中心假设是，重组WTAPC的晚期治疗及其细胞保护性选择性5A-APC突变体，但不是抗凝作用E149A-APC突变体的抗凝剂e149a-apc突变体，是神经保护性的，是神经保护性的，可通过修饰神经元塑料塑料，脱发和Strokular and Crodcular and Strocling and rodcrockular and Strockular and rodcrocring and rodcrocring and rodcrockular and Strockular and rodcrockular and rodcrocring and rodcrodins-促进脑再生，并促进脑再生S1P1在BEC上，NPC上的PAR1，PAR3和S1P1。重组鼠WTAPC，5A-APC和E149A-APC将在人类BEC和NPC上的鼠类中风模型和人类APC中进行研究。我们建议使用WTAPC，5A-APC和E149A-APC使用皮质缺血性中风或光胞菌病变型在对照小鼠中测试后期疗法的首次安全性和功效（AIM 1）；在具有PAR1-4缺失的小鼠中，S1P1的EPCR耗竭和神经特异性损失（AIM 2）。 APC对体内神经元可塑性，血管重塑（AIM 1），新血管形成（AIM 3）和神经发生（AIM 4）的影响以及体内这些APC介导过程的受体要求。人类BEC（AIM 3）和NPC（AIM 4）将用于确定体外受体需求，而下游AKT目标对APC的体外血管生成和神经发生至关重要。拟议的研究将桥接缺血性中风的领域到蛋白质C领域，重点是神经保护和脑修复疗法，这有望从长凳上翻译为床边。公共卫生相关性：中风是由于急性血液供应对大脑的急性障碍而导致的美国人发病率和死亡率的主要原因，这通常导致不可逆的脑部损伤。今天没有有效的中风疗法。拟议的研究应有助于发展激活蛋白C（APC）临床试验及其类似物的基本原理，并具有大大扩展的中风治疗窗口的临床试验，以帮助中风后的长时间中卒中患者，这是两个主要目标：（1）在异常低血液供应期间保护大脑； （2）在中风后的恢复阶段支持再生和脑修复过程。