Heme oxygenase-1 VS eNOS gene transfer to cerebral arteries to prevent vasospasm

脑血管痉挛引起的脑缺血是蛛网膜下腔出血患者发病率和死亡率的主要原因。我们的长期目标是开发一种安全有效的预防和治疗脑血管痉挛的基因治疗方法。我们以前的研究重点是对腺病毒介导的内皮型一氧化氮合酶(ENOS)基因进入脑循环的转移进行开发和优化。在这些研究中，我们证明了eNOS基因转移增加了局部一氧化氮的产生，并显着增强了转导的脑动脉的血管扩张活性。然而，确切的保护机制尚不清楚，似乎涉及痉挛动脉中一氧化氮产生的恢复。对于这一应用来说，最重要的是，先前的一些研究表明，一氧化氮增加了血红素氧合-1(HO-1)的表达。此外，现有证据表明，血红蛋白和血红素释放到蛛网膜下腔引起的血管氧化损伤在脑血管痉挛的发病机制中起着关键作用。血红素从蛛网膜下腔的清除取决于HO-1的酶活性，HO-1是血红素分解代谢中的限速酶。HO-1的酶活性增加可能通过刺激抗氧化剂胆红素和胆绿素的形成以及血管扩张剂气体一氧化碳的形成来保护大脑动脉免受血管痉挛的影响。因此，这一应用的中心假设是，重组HO-1在脑动脉中的表达可以防止蛛网膜下腔出血后脑血管痉挛的发展。为了验证这一假设，我们将通过腺病毒介导的基因转移在脑动脉过表达HO-1。我们的初步研究结果表明，表达重组HO-1可以保护脑动脉免受血红蛋白诱导的损伤。我们建议通过免疫组织化学、电子显微镜、HO-1酶活性测定、环鸟苷酸放射免疫分析以及转导动脉血管运动反应性的体外和体内分析来研究重组HO-1在脑动脉中的表达和功能。采用犬双出血脑血管痉挛模型，研究重组HO-1表达在防治脑血管痉挛中的治疗价值。HO-1的作用将与重组eNOS的作用进行比较。我们预计，本申请中提出的实验将确定重组HO-1是否可以用于脑血管痉挛的基因治疗。我们的研究还将扩大关于重组eNOS对血管作用的潜在机制以及HO-1在保护脑动脉免受血红蛋白诱导的氧化应激中的作用的知识基础。

Brain ischemia due to cerebral vasospasm is a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Our long-term goal is to develop a safe and effective gene therapy approach for the prevention and treatment of cerebral vasospasm. The focus of our previous studies was on the development and optimization of adeno-virus mediated transfer of endothelial nitric oxide synthase (eNOS) gene into the cerebral circulation. In these studies we demonstrated that eNOS gene transfer increases local production of nitric oxide and significantly enhances vasodilator activity of transduced cerebral arteries. However, the exact mechanism of protection is unclear and appears to involve restoration of nitric oxide production in spastic arteries. Most importantly for this application, a number of previous studies demonstrated that nitric oxide increases expression of heme oxygenation-1 (HO-1). Furthermore, existing evidence suggests that vascular oxidative injury caused by hemoglobin and heme released into the subarachnoid space plays a key role in the pathogenesis of cerebral vasospasm. Clearance of heme from the subarachnoid space is dependent on the enzymatic activity of HO-1, the rate-limiting enzyme in heme catabolism. Increased enzymatic activity of HO-1 may protect cerebral arteries from vasospasm by stimulating formation of the antioxidants, bilirubin and biliverdin, as well as the vasodilator gas, carbon monoxide. Thus, the central hypothesis of this application is that expression of recombinant HO-1 in cerebral arteries may prevent development of cerebral vasospasm after subarachnoid hemorrhage. To test this hypothesis we will overexpress HO-1 in cerebral arteries by adenovirus-mediated gene transfer. Our preliminary findings indicate that expression of recombinant HO-1 protects cerebral arteries from hemoglobin-induced injury. We propose to study the expression and function of recombinant HO-1 in cerebral arteries by immunohistochemistry, electron microscopy, measurements of HO-1 enzymatic activity, radioimmunoassay of cyclic GMP and both in vitro and in vivo analysis of vasomotor reactivity of transduced arteries. The "double hemorrhage" canine model of cerebral vasospasm will be used to study the therapeutic value of recombinant HO-1 expression in the prevention and treatment of cerebral vasospasm. The effects of HO-1 will be compared with the effects of recombinant eNOS. We anticipate that experiments proposed in this application will determine whether recombinant HO-1 can be used in the development of gene therapy for cerebral vasospasm. Our studies will also expand the knowledge base concerning the mechanisms underlying the vascular effects of recombinant eNOS and the role of HO-1 in protections of cerebral arteries against hemoglobin-induced oxidative stress.