Adventitial Expression of Recombinant Endothelial Nitric Oxide Synthase Gene Reverses Vasoconstrictor Effect of Endothelin-1

Abstract

The present study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on reactivity of canine basilar arteries to endothelin-1 (ET-1). Experiments were performed ex vivo. The arteries were exposed (30 minutes at 37 degrees C) to adenoviral vectors encoding eNOS gene (AdCMVeNOS) or beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the vascular adventitia. Rings of control (nontransduced), AdCMVbeta-Gal- and AdCMVeNOS-transduced arteries with and without endothelium were suspended for isometric tension recording. Levels of guanosine 3',5'-cyclic monophosphate (cGMP) were measured by radioimmunoassay. During contractions to uridine 5'-triphosphate, ET-1 (10(-10) to 3x10(-9) mol/L) caused further increase in tension in control and AdCMVbeta-Gal-transduced arteries. In contrast, ET-1 caused concentration-dependent relaxations of AdCMVeNOS-transduced arteries. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were endothelium-independent. They were abolished by N(G)-nitro-L-arginine methyl ester or by chemical treatment of adventitia with paraformaldehyde before gene transfer. ET-1 (10(-9) mol/L) significantly increased intracellular cGMP levels in AdCMVeNOS-transduced arteries without endothelium. In arteries transduced with AdCMVeNOS, higher concentrations (10(-9) to 3x10(-8) mol/L) of ET-2 also caused relaxations, whereas ET-3 and sarafotoxin, a selective ET(B) receptor agonist, did not produce any relaxations. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were strongly reduced by BQ-123 (10(-7) mol/L), an ET(A) receptor antagonist, but were not affected by BQ-788 (3x10(-7) mol/L), an ET(B) receptor antagonist. These results suggest that genetically modified adventitia can produce nitric oxide and cause relaxations in response to ET-1 via activation of ET(A) receptors. Our findings support a novel concept that successful transfer and expression of recombinant eNOS gene can lead to a qualitative change in responsiveness to vasoconstrictor substances.

Expression and Function of Recombinant S1179D Endothelial Nitric Oxide Synthase in Canine Cerebral Arteries

“…[3][4][5][6] This observation was confirmed in the present study. In AdCMVS1179DeNOS-transduced arteries we detected high expression of recombinant protein by Western analysis.…”

“…1,2 Previous studies demonstrated that the recombinant eNOS gene can be transferred with the use of adenoviral vectors into the adventitial layer of cerebral [3][4][5][6] and peripheral [7][8][9][10] arteries. The expression of eNOS gene in the adventitia can augment the vasodilator effect of bradykinin [3][4][5] and inhibit the vasoconstrictor effect of endothelin-1 (ET-1) 6 in canine cerebral arteries.…”

Protective Vasomotor Effects of In Vivo Recombinant Endothelial Nitric Oxide Synthase Gene Expression in a Canine Model of Cerebral Vasospasm

“…A similar finding was reported in an ex vivo basilar artery gene transfer study by Onoue et al 6 who postulated that because activation of fibroblast ET A receptors causes increased intracytosolic calcium levels, ET-1 may stimulate recombinant eNOS (a calcium-activated enzyme) through an ET A receptor-mediated pathway, thereby facilitating NO production by adventitial fibroblasts and paradoxically protecting against contractions. It is therefore apparent that functional vasoprotection conferred by recombinant eNOS expression in AdeNOS-transduced arteries is mediated through both augmentation of relaxation and attenuation of contraction.…”

“…T he expression and function of recombinant genes in cerebral arteries after adenovirus-mediated gene transfer have been extensively characterized, [1][2][3][4][5][6][7] although the ability of recombinant endothelial nitric oxide synthase (eNOS) gene expression to ameliorate a disease involving intracranial arteries has not been demonstrated. The present study, representing a continuation of efforts to translate cerebrovascular gene transfer technology closer to the clinical arena, 8 was performed to characterize this ability, particularly in view of the important role played by NO in normal vasomotor function and the pathogenesis of cardiovascular diseases, including post-subarachnoid hemorrhage (SAH) cerebral vasospasm.…”

Translational Paradigms in Cerebrovascular Gene Transfer

“…First, NO may be only one of a number of important mediators underlying the pathogenesis of cerebrovascular disease; other candidates include calcitonin gene-related peptide (CGRP), endothelin-1 (ET-1), and altered enzymatic activities of cyclooxygenase, superoxide dismutase (SOD), and heme oxygenase-1 (HO-1) (Chen et al, 1997a;De Belder and Radomski, 1994;Dietrich and Dacey, 2000;Furchgott and Zawadzki, 1980;Khurana and Besser, 1997;Luders et al, 2000;Ono et al, 2002;Onoue et al, 1998Onoue et al, , 1999Toyoda et al, 2000c;Vanhoutte, 1998; Human Pial (intact arteries) Adenovirus iNOS ex vivo Function * All animal disease models were related to subarachnoid hemorrhage (SAH) and post-SAH cerebral vasospasm, with the exception of the study by Lund et al (1998), in which the disease model was atherosclerosis.…”