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.
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