No abstract

Endothelial cell seeding of synthetic small diameter vascular grafts (SSDVG) has been shown to diminish thrombosis and intimal hyperplasia, resulting in improved graft patency. However, endothelial cell retention on seeded grafts when exposed to physiological shearing conditions remains poor. We report that the genetic engineering of endothelial cells to overexpress endothelial nitric oxide synthase (eNOS), may create improved anti-thrombotic and anti-hyperplastic endothelial cell phenotypes for SSDVG seeding. eNOS-overexpressing endothelial cells may potentially overcome the biochemical loss due to shear induced reduction in endothelial cell coverage on SSDVG. Bovine aortic endothelial cells (BAEC) were transfected with the human eNOS gene, and co-incubated with either human whole blood or bovine aortic smooth muscle cells (BASMC) in vitro. eNOS-transfected BAEC significantly overexpressed eNOS compared to control beta-Gal-transfected and untransfected BAEC up to 120 h post transfection. In co-incubation and co-culture assays, human platelet aggregation decreased by 46% and BASMC proliferation decreased by 67.2% when compared to incubation with untransfected BAEC.

Hydrogen peroxide (H2O2) is produced by inflammatory and vascular cells and induces oxidative stress, which may contribute to vascular disease and endothelial cell dysfunction. In smooth muscle cells, H2O2 induces production of O2 by activating NADPH oxidase. However, the mechanisms whereby H2O2 induces oxidative stress in endothelial cells are not well understood, although O2 may play a role. Recent studies have documented increased O2 in endothelial cells exposed to H2O2 via uncoupled nitric oxide synthase (NOS) and NADPH oxidase under static conditions. To assess responses to H2O2 in porcine aortic endothelial cells (PAEC) under shearing conditions, a constant flow rate of 24. 4 ml/min was applied to produce physiologically relevant shear stress (8. 2 dynes/cm). Here we demonstrate that treatment with 100 muM H2O2 increases intracellular O2 levels in PAEC. In addition, we demonstrate that l-NAME, an inhibitor of NOS, and apocynin, an inhibitor of NADPH oxidase, reduced O2 levels in PAEC treated with H2O2 under physiologic shear suggesting that both NOS and NADPH oxidase contribute to H2O2-induced O2 in PAEC. Co-inhibition of NOS and NADPH oxidase also reduced intracellular O2 levels under shear. We conclude that H2O2-induced oxidative stress in endothelial cells exhibits increased intracellular O2 levels through NOS and NADPH oxidase under shear. The inhibition of NOS and NADPH with H2O2 exposure is nonlinear, suggesting some interdependent or compensating system within endothelial cells. These findings suggest a complex interaction between H2O2 and oxidant-generating enzymes that may contribute to endothelial dysfunction in cardiovascular diseases.