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导致蛛网膜下腔出血患者发病和死亡的主要原因。我们的长期目标是开发一种安全有效的基因治疗方法来预防和治疗脑血管痉挛。我们之前的研究重点是开发和优化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。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，并显著增强了转导的脑动脉的血管舒张活性。然而，确切的保护机制尚不清楚，似乎涉及恢复痉挛动脉中一氧化氮的产生。最重要的是，许多先前的研究表明，一氧化氮增加血红素氧合-1 （HO-1）的表达。此外，已有证据表明，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是血红素分解代谢的限速酶。HO-1酶活性的增加可能通过刺激抗氧化剂、胆红素和胆绿素以及血管舒张气体一氧化碳的形成来保护脑动脉免受血管痉挛。因此，本研究的中心假设是，重组HO-1在脑动脉中的表达可能会阻止蛛网膜下腔出血后脑血管痉挛的发生。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中过表达HO-1。我们的初步研究结果表明，重组HO-1的表达可以保护脑动脉免受血红蛋白诱导的损伤。我们拟通过免疫组化、电镜、HO-1酶活性测定、环GMP放射免疫测定和体内外血管舒缩性分析等方法研究重组HO-1在脑动脉中的表达和功能。利用犬脑血管痉挛“双出血”模型，研究重组HO-1表达在预防和治疗脑血管痉挛中的治疗价值。将HO-1的作用与重组eNOS的作用进行比较。我们期望在本应用中提出的实验将确定重组HO-1是否可以用于开发脑血管痉挛的基因治疗。我们的研究还将扩大重组eNOS血管效应机制的知识基础，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。

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.