Translating Endogenous Vascular Protective Cascades into Therapy for Aneurysmal Subarachnoid Hemorrhage

 描述(申请人提供)：动脉瘤性蛛网膜下腔出血(SAH)是一种高度病态的疾病，大部分是由迟发性脑缺血(DCI)引起的，DCI是由大动脉血管痉挛和微循环障碍(包括自我调节功能障碍、微血管血栓形成和血脑屏障(BBB)破坏)共同引起的。条件反射是指大脑暴露在亚致命刺激下，使其对随后的致命刺激更具抵抗力的现象。大多数人都研究了条件反射对神经元的保护作用，但我们和其他人已经确定，脑血管也是一个目标。鉴于DCI是由广泛的血管缺陷(从大动脉到微循环)引起的，而且条件化似乎能够预防这些缺陷，应用于SAH的基于条件反射的治疗策略具有很大的前景。在我们过去的研究中，我们发现对DCI存在强大的内源性保护机制，可以由条件性刺激(SAH前的短暂缺氧；称为低氧预适应或HPreC)诱导，并依赖于内皮型一氧化氮合酶(ENOS)。在一项先导性研究中，我们表明，当在临床相关的时间点(SAH后3h；称为低氧后适应或HPostC)启动相同的刺激(低氧)时，可提供类似的强大的DCI保护。然而，这种保护的分子诱导剂(S)尚不清楚。我们认为Sirtuin1(SIRT1)是一个关键的诱导因子，因为缺氧增加了SIRT1的表达，SIRT1抑制剂EX527失去了HPreC诱导的DCI保护，SIRT1激活剂白藜芦醇模拟了HPreC和HPostC提供的DCI保护。我们项目的长期目标是验证假设，即HPostC诱导强大的和多方面的DCI保护，SIRT1是这种保护的关键诱导因素，SIRT1激活是治疗SAH的一种新策略，具有特殊的翻译潜力。其具体目的是1)确定HPostC诱导的DCI保护的广度和可持续性；2)测试SIRT1是否是HPostC诱导的DCI保护的关键诱导因素；以及3)确定SIRT1在SAH中激活的翻译潜力。所使用的方法包括：a)评估SIRT1、eNOS、MMP-9、TIMP-1和TF的表达和活性；b)评估SAH诱导的小鼠血管痉挛、微循环障碍(自动调节功能障碍、微血管血栓形成和血脑屏障破坏)和神经功能障碍；c)评估SAH诱导的血管痉挛、微循环缺陷、神经功能障碍、神经细胞死亡和长期神经行为缺陷；d)SIRT1的药理和遗传抑制；以及g)SIRT1的药理和遗传增强。如果成功，这些研究将提高对HPostC诱导的SAH神经血管保护的广度、机制和可持续性的理解，将SIRT1确定为DCI的新的可药物治疗靶点，将确定SIRT1指导的治疗DCI的可译性，并将确定可用于在未来的人类研究中直接测试药效的脑脊液和血清生物标记物。

 DESCRIPTION (provided by applicant): Aneurysmal subarachnoid hemorrhage (SAH) is a highly morbid condition - much of which is due to delayed cerebral ischemia (DCI), which is caused by a combination of large-artery vasospasm and microcirculatory deficits including autoregulatory dysfunction, microvascular thrombosis, and blood-brain- barrier (BBB) breakdown. Conditioning refers to the phenomenon whereby exposure of the brain to a sub- lethal stimulus renders it more resistant to a subsequent lethal stimulus. Most have examined the protective effects of conditioning on neurons, but we and others have established that the cerebral vessels are also a target. Given that DCI is caused by wide-ranging vascular deficits (from large arteries to the microcirculation) and that conditioning appears capable of preventing these deficits, a conditioning- based therapeutic strategy applied to SAH holds great promise. In our past study, we showed that powerful endogenous protective mechanisms against DCI exist, can be induced by a conditioning stimulus (brief hypoxia prior to SAH; termed hypoxic preconditioning or HPreC), and are dependent on endothelial nitric oxide synthase (eNOS). In a pilot study, we show that the same stimulus (hypoxia) when initiated at a clinically relevant time point (3h after SAH; termed hypoxic postconditioning or HPostC) provides similarly strong DCI protection. The molecular inducer(s) of this protection, however, are unknown. We believe sirtuin1 (SIRT1) is a key inducer, as hypoxia augments SIRT1 expression, HPreC-induced DCI protection is lost with the SIRT1 inhibitor EX527, and the SIRT1 activator resveratrol mimics the DCI protection afforded by HPreC and HPostC. The long-term goal of our project is to test the hypotheses that HPostC induces robust and multifaceted DCI protection, SIRT1 is a key inducer of this protection, and SIRT1 activation is a novel strategy for SAH that has exceptional translational potential. The Specific Aims are 1) Determine the breadth and sustainability of HPostC-induced DCI protection; 2) Test whether SIRT1 is a key inducer of HPostC-induced DCI protection; and 3) Determine the translational potential of SIRT1 activation in SAH. Methods used include: a) assessment of SIRT1, eNOS, MMP-9, TIMP-1, and TF expression and activity; b) assessment of SAH-induced vasospasm, microcirculatory deficits (autoregulatory impairment, microvessel thrombosis, and BBB breakdown), and neurological deficits in mice; c) assessment of SAH-induced vasospasm, microcirculatory deficits, neurological deficits, neuronal cell death, and long-term neurobehavioral deficits in rats; d) pharmacologic and genetic inhibition of SIRT1; and g) pharmacologic and genetic augmentation of SIRT1. If successful, these studies will result in an improved understanding of the breadth, mechanism, and sustainability of HPostC-induced neurovascular protection in SAH, will identify SIRT1 as a novel and druggable therapeutic target for DCI, will determine the translatability of SIRT1-directed therapeutics for DCI, and will identify CSF and serum biomarkers that can be used to directly test for pharmacodynamic efficacy in future human studies.