Expression and Function of Recombinant Endothelial Nitric Oxide Synthase Gene in Canine Basilar Artery After Experimental Subarachnoid Hemorrhage

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Abstract
Background and Purpose —Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH . Methods —Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli β-galactosidase (AdCMVβ-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) β-galactosidase protein measurement, and (3) isometric tension recording. Results —Transduction with AdCMVβ-Gal and AdCMVeNOS resulted in the expression of recombinant β-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of β-galactosidase protein was ≈2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVβ-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. Conclusions —These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide–mediated relaxations through local (adventitial) production of nitric oxide.