© 1997 American Heart Association, Inc.
Articles |
Expression and Function of Recombinant Endothelial NO Synthase in Coronary Artery Smooth Muscle Cells
From the Divisions of Cardiovascular Disease (I.J.K., R.S.S., V.J.P.), the Departments of Anesthesiology and Pharmacology (M.T., Z.S.K.) and of Endocrinology and Metabolism (L.A.F., T.O.), Mayo Clinic, Rochester, Minn; and the Laboratory of Cell Biology, National Institutes of Mental Health, National Institutes of Health (S.M.), Bethesda, Md.
Correspondence to Timothy O'Brien, MD, Senior Associate Consultant, Department of Endocrinology and Metabolism, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail obrien.timothy{at}mayo.edu
Abstract Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and ß-galactosidase (AdßGal). In contrast to AdßGal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.
Key Words: nitric oxide • gene transfer • nitric oxide synthase • adenovirus • vascular smooth muscle cells
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