Adenoviral transfer of the eNOS gene to rat aortic smooth muscle cells in vitro: Effect on proliferation and differentiation
Description
Vascular injury, following surgical techniques such as balloon angioplasty, results in endothelial damage. The endothelium is the principal physiological source of nitric oxide in the vasculature. Nitric oxide (NO) has been shown to have an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation. NO has also been implicated in phenotypic modulation in VSMCs. Following disruption of NO production in vivo, VSMCs are modulated from their normal contractile phenotype, to a proliferative phenotype. This results in coronary artery restenosis. Endothelial nitric oxide synthase (eNOS) is responsible for the production of NO in the vasculature. We conducted an adenoviral transfer of the eNOS gene into rat aortic smooth muscle cells. We used a replication deficient, recombinant adenovirus encoding either the eNOS gene (AdCMV eNOS), or nuclear-targeted beta galactosidase (AdCMV betagal). We determined the effect of eNOS gene transfer on proliferation and phenotypic modulation in rat VSMCs. Immunocytochemistry indicated the presence of eNOS in eNOS transfected cells, but not in nontrasfected or beta gal transfected cells. Western blot analysis for eNOS showed similar results. eNOS transfection caused an inhibition of proliferation in rat VSMCs. A release from the inhibition of proliferation in eNOS transfected cells was seen in the presence of the PKA inhibitor (PKAi), Rp-8-Bromo-cAMPS. Levels of p21 and p53 increased in eNOS transfected cells, and this increase was abolished in the presence of PKAi. This indicates that p21 and p53 play a role in the NO-induced inhibition of proliferation in VSMCs, and that their involvement is mediated by PKA. Apoptosis was not involved in the upregulation of p53, as levels of Bax were not elevated in eNOS transfected cells as compared to non transfected cells. Flow cytometry analysis further confirmed these results, as there was no increase in hypodiploidy in eNOS transfected cells as compared to non transfected cells. Levels of contractile proteins, alpha actin and calponin increased in eNOS transfected cells. These increases were no longer seen in the presence of the PKG inhibitor, Rp-8-Bromo-cGMPS. This indicated that PKG may play a role in the maintenance of VSMCs in the contractile state