Adventitial Expression of Recombinant eNOS Gene Restores NO Production in Arteries Without Endothelium

From the Departments of Anesthesiology and Pharmacology (M.T., A.F.Y.C., Z.S.K.) and the Divisions of Endocrinology and Metabolism (T.O'B.) and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn.

Correspondence to Zvonimir S. Katusic, MD, PhD, Associate Professor, Departments of Anesthesiology and Pharmacology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail Katusic.Zvonimir{at}mayo.edu

Abstract—The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37°C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the ß-galactosidase reporter gene (AdCMVß-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVß-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3x10^-11 to 10^-9 mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVß-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3x10^-11 to 10^-9 mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, N^G-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

Key Words: fibroblasts • adenoviral vector • gene transfer • gene therapy • bradykinin

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