Drug-eluting stents for coronary artery disease results in inhibition of smooth muscle cell (SMC) and endothelial cells which may increase the risk of stent thrombosis. In this study, we attempted to enhance re-endothelialization of deployed stents while simultaneously inhibiting intimal hyperplasia by overexpression of endothelial nitric oxide synthase (eNOS) delivery in the vasculature using an adenovirus gene-eluting stent. Re-endothelialization was significantly greater in vessels obtained from normocholesterolemic animals at day 14 (85.34% +/- 7.38 versus 62.66% +/- 10.49; P < 0.05) and day 28 (91.1% +/- 10 versus 63.1% +/- 22; P < 0.05) and hypercholesterolemic animals (96.97% +/- 3.2 versus 28.33% +/- 38.76; P < 0.05) at day 28 with AdeNOS-eluting stents. At day 28, there was a significant increase in the lumen size [AdeNOS 2.73 mm(2) +/- 1.18, AdbetaGal 0.98 mm(2) +/- 0.98, phosphorylcholine (PC) 1.87 mm(2) +/- 1.18; P < 0.05], and a significant reduction in neointimal formation (AdeNOS 2.32 mm(2) +/- 1.13, AdbetaGal 3.73 mm(2) +/- 0.95, PC 3.2 mm(2) +/- 0.94; P < 0.05), and percent restenosis (AdeNOS 45.23 +/- 20.81, AdbetaGal 79.6 +/- 20.31, PC 70.16 +/- 22.2; P < 0.05) in AdeNOS-stented vessels in comparison with controls from hypercholesterolemic animals, assessed by morphometry and quantitative coronary angiography (AdeNOS 15.95% +/- 7.63, AdbetaGal 56.9% +/- 38.6, PC 58 +/- 34.6; P < 0.05). Stent-based delivery of AdeNOS results in enhanced endothelial regeneration and reduction in neointimal formation as compared with controls. This seems to be a promising treatment strategy for preventing in-stent restenosis (ISR) while simultaneously reducing the risk of stent thrombosis.

Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 Â 10 9 PFU of adenoviral(Ad)eNOS, AdiNOS or Adb-galactosidase (Adb-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR.The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.0770.044) and iNOS (0.08770.086) transduced arteries compared with Adb-Gal (0.33270.14) transduced arteries (n ¼ 7). In addition, AdeNOS treatment (4.2173.12% deendothelialized area) enhanced endothelial regeneration compared to Adb-Gal treatment (10.0574.98), while treatment with AdiNOS (25.17711.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n ¼ 7-8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.

Objective: Many vascular diseases are associated with reduced nitric oxide (NO) bioavailability. Nitric oxide synthase (NOS) gene therapy to the vasculature is a possible treatment for vascular disease as a means of increasing NO bioavailability, and this may be achieved using any of the NOS isoforms. The aim of our study was to compare the effects of adenoviral-mediated overexpression of the most commonly used NOS isoforms eNOS and iNOS on vascular cell proliferation. Methods: Human coronary artery smooth muscle cells (HCSMCs) and human umbilical vein endothelial cells (HUVECs) were transduced with adenoviral vectors encoding eNOS or iNOS at a multiplicity of infection of 100. Control cells were exposed to AdNull (empty vector) or diluent alone. Transgene expression was sought by Western blotting. The Greiss assay was used to measure nitrite levels. Cell proliferation was assessed by cell counting on days 0, 3 and 6. Apoptosis was sought using FACS analysis. Angiogenesis was measured using a commercially available in vitro kit. Results: Expression of both isoforms was detected in transduced cells by Western blot at all three time points. NOS transduction resulted in increased nitrite levels with higher levels seen in iNOS- compared to eNOS-transduced cells. Cell proliferation was diminished in AdeNOS- and AdiNOS-transduced cells compared with non-transduced cells on days 3 and 6 in both HCSMCs and HUVECs. Apoptosis was not detected in either cell line with either of the isoforms at any timepoint studied. Both eNOS and iNOS gene transfer caused a reduction in angiogenesis. Conclusions: NOS gene transfer to both endothelial and vascular smooth muscle cells is antiproliferative and antiangiogenic. The biological effect is identical with both isoforms and there is no evidence to support a differential effect on endothelial and vascular smooth muscle cell biology.