Treatment of stroke with a clinically approved proteasome inhibitor

描述（由申请人提供）：大脑中动脉闭塞导致进行性血管功能障碍，这有助于脑损伤的进展。组织型纤溶酶原激活剂（tPA）溶栓可促进不良血管事件的发生，从而将卒中的治疗时间窗限制在3小时内。高龄加重卒中后血管功能障碍，限制了tPA的使用。蛋白酶体抑制剂增强内皮一氧化氮合酶（eNOS）表达并改善内皮功能。我们的初步研究表明，蛋白酶体抑制剂万珂（一种临床上用于治疗癌症的药物）治疗可有效减少幼龄大鼠的脑梗死，同时降低继发性血栓形成和微血管通透性。此外，万珂联合tPA治疗可将治疗窗延长至卒中后至少6小时，且不会增加出血性转化。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对老年大鼠的影响。在目标1中，我们假设万珂剂量依赖性地减少老年大鼠卒中后的梗死体积和神经功能缺损。万珂的最佳剂量可延长卒中的治疗窗。在目标2中，我们将研究万珂和tPA联合治疗对栓塞性卒中后老年大鼠脑梗死、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，万珂增强了tPA的溶栓作用，并允许减少tPA的有效治疗剂量。在目标3中，我们将使用eNOS敲除小鼠和NOS抑制剂，研究万珂单药或与tPA联合治疗卒中的获益作用机制。我们认为，eNOS通过下调促凝血基因和基质金属蛋白酶（MMPs）介导万珂的神经保护作用，而促凝血基因和MMPs可引起血栓形成和BBB损伤。万珂可抵消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。我们的研究可能为万珂以及万珂与tPA联合治疗栓塞性卒中的潜在获益机制提供基本见解，并可能为卒中提供新的治疗策略。公共卫生相关性：卒中是一种进行性血管功能障碍，可导致脑损伤的进展。作为FDA批准的唯一一种用于治疗急性卒中的药物，组织纤溶酶原激活剂（tPA）可增强不良血管事件，将卒中的治疗窗限制在3小时内。高龄加重卒中后血管功能障碍，限制了tPA的使用。蛋白酶体抑制剂增强内皮型一氧化氮合酶（eNOS，血管稳态的重要调节剂）表达。万珂是一种有效的蛋白酶体抑制剂，临床上用于治疗癌症，可有效减少血管不良事件的发生，同时减少脑梗死。因此，在本申请中，我们建议在栓塞性卒中后的老年大鼠中研究万珂单药和与tPA联合用药的神经保护作用以及有益作用的机制。

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.