Treatment of stroke with a clinically approved proteasome inhibitor

描述（由申请人提供）：大脑中动脉闭塞引起进行性血管功能障碍，这有助于脑损伤的演变。组织型纤溶酶原激活剂（tPA）溶栓促进不良血管事件，将卒中的治疗窗口限制在3小时。高龄加重脑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）表达，改善内皮功能。我们的初步研究表明，用一种蛋白酶体抑制剂VELCADE（一种临床用于治疗癌症的药物）治疗可以有效地减少脑梗死，并同时减少年轻大鼠的继发性血栓形成和微血管通透性。此外，VELCADE联合tPA治疗将治疗窗口延长至卒中后至少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，我们拟研究VECLADE对老年大鼠的影响。在Aim 1中，我们假设VELCADE剂量依赖性地减少脑卒中后老年大鼠的梗死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期。在Aim 2中，我们将研究VELCADE和tPA联合治疗对老年大鼠栓塞性脑卒中后脑梗死、神经功能、溶栓、微血管血栓形成、血管通畅和完整性的影响。通过减少不良血管事件，VELCADE放大了tPA的溶栓作用，并允许降低tPA的有效治疗剂量。在Aim 3中，我们将使用eNOS敲除小鼠和NOS抑制剂，研究VELCADE单独或与tPA联合治疗中风的有益作用机制。我们认为eNOS通过下调促凝基因和基质金属蛋白酶（MMPs）介导VELCADE的神经保护作用，从而引起血栓形成和血脑屏障损伤。VELCADE抵消延迟给药tPA对血管功能的有害影响，从而改善微循环和血管完整性。我们的研究可能为VELCADE和VELCADE与tPA联合治疗栓塞性卒中的有益作用机制提供基础见解，并可能导致卒中的新治疗策略。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唯一批准的治疗急性中风的药物，组织纤溶酶原激活剂（tPA）会加剧血管不良事件，将中风的治疗窗口期限制在3小时内。高龄加重脑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，一种重要的血管稳态调节因子）的表达。使用一种有效的蛋白酶体抑制剂VELCADE（一种临床用于治疗癌症的药物）进行治疗，有效地减少了不良血管事件的发生，并同时减少了脑梗死。因此，在当前的应用中，我们建议研究VELCADE单独和联合tPA对老年大鼠栓塞性卒中后的神经保护作用及其有益作用的机制。

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.