EXPRESSION AND FUNCTION OF RECOMBINANT ENDOTHELIAL NO SYNTHASE IN CORONARY-ARTERY SMOOTH-MUSCLE CELLS

Smooth muscle cells (SMCs) play a key role in the pathogenesis of vasc ular 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 expressi on of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosid ase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cell s, CSMCs transduced with AdeNOS stained positive with the NADPH-diapho rase stain, acquired calcium-dependent NOS activity (measured by the c onversion of [H-3]L-arginine to [H-3]-citrulline), had increasing cycl ic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-tran sduced cells was augmented by increasing intracellular levels of the e NOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine upt ake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as a ssessed by cell counts 3 and 6 days after serum stimulation of quiesce nt CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional e nzyme whose activity can be augmented by increasing intracellular leve ls of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs res ults in inhibition of serum-stimulated DNA synthesis and cell prolifer ation. These findings imply that eNOS gene transfer to SMCs may be a u nique mode of increasing local NO production in the arterial wall.