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Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a
M Y Alexander1, M J Brosnan, C A Hamilton
1Department of Medicine and Therapeutics, University of Glasgow, UK. yalexander@clinmed.gla.ac.uk
Objective:
We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function.
Methods:
Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control beta-galactosidase-containing virus, (2 x 10(9) pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo.
Results:
Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for beta-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10(-9) to 3 x 10(-6) M) in the absence and presence of NG-nitroarginine methyl ester (100 microM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n = 6 for each; P = 0.0069; 95% CI, 0.864 to 3.277).
Conclusions:
Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.