Chronic cerebral vasospasm is a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Pathogenesis of vasospasm is not completely understood, but existing evidence suggests that impairment of nitric oxide vasodilator function is an important mechanism underlying narrowing of arteries exposed to autologous blood. We hypothesize that increased biosynthesis of nitric oxide in adventitia of cerebral arteries may cause vasodilatation and prevent development of vasospasm. To test this hypothesis, we over-expressed recombinant endothelial nitric oxide synthase (eNOS) gene in adventitial fibroblasts of cerebral arteries by adenoviral-mediated gene transfer. This model provides unique opportunity to characterize the effects of nitric oxide released from adventitia on regulation of vasomotor reactivity, as well as signal transduction pathways coupled to activation of recombinant eNOS protein. For instance, preliminary findings indicate that in our model, vasoconstrictor effect of a putative mediator of vasospasm, endothelin-1, is reversed to vasodilatation. This reversal is apparently due to endothelin-1-induced activation of recombinant eNOS protein expressed in adventitia. We propose to study expression and function of recombinant eNOS gene in adventitia of cerebral arteries by immunohistochemistry,. measurements of eNOS enzymatic activity, radioimmunoassay of cyclic GMP and in vitro and in vivo analysis of vasomotor reactivity of tansduced arteries.""""""""Double hemorrhage"""""""" canine model of cerebral vasospasm will be used to study effects of the recombinant eNOS gene on vascular tone of spastic arteries. We anticipate that experiments propose in this application will determine whether eNOS over-expression in the adventitia can serve as a substitute source of nitric oxide, and whether this approach can be used to restore nitric oxide biosynthesis in cerebral arterial wall. Our studies will also expand the knowledge base concerning the vascular biology of adventitia, and its role as a target for transfer and expression of therapeutically beneficial recombinant proteins.
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