Treatment of stroke with a clinically approved proteasome inhibitor

描述(申请人提供)：大脑中动脉闭塞导致进行性血管功能障碍，这有助于脑损伤的演变。使用组织纤溶酶原激活剂(TPA)溶栓可促进不良血管事件的发生，从而将中风的治疗窗口限制在3小时内。高龄加重卒中后血管功能障碍，限制了tPA的应用。蛋白酶体抑制剂可促进内皮型一氧化氮合酶(ENOS)的表达，改善内皮功能。我们的初步研究表明，蛋白酶体抑制剂VELCADE是一种临床上用于治疗癌症的药物，它能有效地减少幼年大鼠的脑梗塞，并同时减少继发性血栓形成和微血管通透性。此外，VELCADE与tPA的联合治疗将治疗窗口延长到中风后至少6小时，而不会增加出血转化。然而，中风是老年人死亡和残疾的主要原因。为了模拟临床情况，我们建议研究VECLADE对老龄大鼠的影响。在目标1中，我们假设VELCADE治疗可剂量依赖性地减少卒中后老年大鼠的脑梗塞体积和神经功能缺陷。最佳剂量的VELCADE延长了中风的治疗窗口。目的2观察VELCADE和tPA联合治疗对老年大鼠脑梗塞、神经功能、溶栓、微血管血栓形成、血管通畅性和完整性的影响。通过减少不良血管事件，VELCADE增强了tPA的溶栓作用，并允许减少tPA的有效治疗剂量。在目标3中，利用eNOS基因敲除小鼠和NOS抑制剂，我们将研究VELCADE单独或与tPA联合治疗中风的有益效果的机制。我们认为eNOS通过下调促凝血基因和基质金属蛋白酶(MMPs)来介导VELCADE的神经保护作用，从而引发血栓形成和BBB损伤。VELCADE可抵消tPA延迟给药对血管功能的不利影响，从而改善微循环和血管完整性。我们的研究可能对VELCADE以及VELCADE和tPA联合应用对卒中的有益作用的机制提供基本的见解，并可能导致一种新的卒中治疗策略。公共卫生相关性：中风引起进行性血管功能障碍，这有助于脑损伤的演变。作为FDA批准的唯一治疗急性中风的药物，组织纤溶酶原激活剂(TPA)可增强将中风的治疗窗口限制在3小时内的不良血管事件。高龄加重卒中后血管功能障碍，限制了tPA的应用。蛋白酶体抑制剂促进血管内皮细胞一氧化氮合酶(ENOS)的表达，eNOS是血管稳态的重要调节因子。使用一种有效的蛋白酶体抑制剂VELCADE进行治疗，这是一种临床上用于治疗癌症的药物，有效地减少了不良血管事件的发生，并同时减少了脑梗塞。因此，在目前的应用中，我们建议研究VELCADE单独以及与tPA联合应用对老年大鼠栓塞性卒中后的神经保护作用及其机制。

DESCRIPTION (provided by applicant): Occlusion of the middle cerebral artery elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. Thrombolysis with tissue plasminogen activator (tPA) promotes adverse vascular events that limit the therapeutic window of stroke to three hours. Advanced age exacerbates vascular dysfunction after stroke which limits the utilization of tPA. Proteasome inhibitors enhance endothelial nitric oxide synthase (eNOS) expression and improve endothelial function. Our preliminary studies demonstrate that treatment with a proteasome inhibitor, VELCADE, an agent in clinical use for the treatment of cancer, effectively reduces cerebral infarction, and concomitantly reduces secondary thrombosis and microvascular permeability in young rats. In addition, treatment with VELCADE in combination with tPA extends the therapeutic window to at least 6 hours after stroke without increasing hemorrhagic transformation. However, stroke is a major cause of death and disability in the elderly. To mimic clinical situation, we propose to investigate the effect of VECLADE on aged rats. In Aim 1, we hypothesize that treatment with VELCADE dose dependently reduces infarct volume and neurological functional deficit in aged rats after stroke. Optimal doses of VELCADE extend the therapeutic window for stroke. In Aim 2, we will investigate the effects of combination treatment with VELCADE and tPA on cerebral infarction, neurological function, thrombolysis, microvascular thrombus formation, vascular patency and integrity in aged rats after embolic stroke. By reducing the adverse vascular events, VELCADE amplifies the thrombolytic effect of tPA, and permits a reduction in the effective therapeutic dose of tPA. In Aim 3, using eNOS knockout mice and NOS inhibitors, we will examine the mechanisms that underlie the beneficial effects of VELCADE alone or in combination with tPA in the treatment of stroke. We propose that eNOS mediates the neuroprotective effect of VELCADE by down-regulation of pro- coagulation genes and matrix metalloproteinases (MMPs), which provoke thrombosis, and BBB damage. VELCADE counteracts the detrimental effects of delayed administration of tPA on vascular function and consequently improves microcirculation and vascular integrity. Our study may provide fundamental insights into the mechanisms underlying beneficial effects of VELCADE and combination of VELCADE and tPA in embolic stroke, and may lead to a novel treatment strategy for stroke. PUBLIC HEALTH RELEVANCE: Stroke elicits a progressive vascular dysfunction, which contributes to the evolution of brain injury. As the only FDA approved drug for the treatment of acute stroke, tissue plasminogen activator (tPA) potentiates adverse vascular events that limit the therapeutic window of stroke to three hours. Advanced age exacerbates vascular dysfunction after stroke which limits the utilization of tPA. Proteasome inhibitors enhance endothelial nitric oxide synthase (eNOS, an important regulator of vascular homeostasis) expression. Treatment with a potent proteasome inhibitor, VELCADE, an agent in clinical use for the treatment of cancer, effectively reduces the development of adverse vascular events, and concomitantly reduces cerebral infarction. Therefore, in the current application, we propose to investigate the neuroprotective effects of VELCADE alone and incombination with tPA and the mechanisms underlying the beneficial effects in aged rats after embolic stroke.