Enhanced superoxide anion production in experimental venous bypass grafts is mediated by NAD(P)H oxidase

Background: Enhanced superoxide (SO) production and its interaction with nitric oxide (NO) is an important feature of vascular disease states. Accelerated atherosclerosis in vein graft (VG) disease is a major clinical problem, resulting from intimal hyperplasia and deficient NO-mediated endothelial function. SO's role in VG pathobiology remains unclear. Methods: We compared jugular-carotid interposition bypass grafts with native jugular veins in normocholesterolemic New Zealand White rabbits (n=16). Some animals underwent sham surgical isolation of the ungrafted jugular vein (JV) as added controls. VG and JV were harvested at 28 days and analysed by lucigenin-enhanced chemiluminescence to measure SO production, and Western blotting and immunohistochemistry to detect the p22phox subunit of the NAD(P)H oxidase enzyme. Results: Basal SO production was increased in VG (n=9) compared with both native and sham-operated JV (n=12; p<0.05). The flavin oxidase inhibitor diphenyleneiodonium reduced SO generation in VG by 63% (p<0.05) although other oxidase enzyme inhibitors had no significant effect. Furthermore, NADH stimulated SO production from VG significantly more than from JV in the same animal (6.3±0.9 vs 2.3±0.6 RLU/s/μg protein; p<0.005). Subcellular fractionation revealed >95% of the NADH-dependent activity in VG was in the particulate (membrane-bound) fraction. Western blotting demonstrated increased amounts of the p22phox subunit of the NAD(P)H oxidase in VG compared with paired JV, and this was localised by immunohistochemical staining to smooth muscle cells in the hyperplastic intima of VG. Conclusions: These data indicate (1) SO production is increased in VG compared with ungrafted and sham-operated JV in normocholesterolemic animals; (2) the major source of enhanced SO production in VG is the NAD(P)H oxidase enzyme, which is present in increased amounts in VG; and (3) the major cell type expressing p22phox is intimal smooth muscle cells. These findings suggest a possible role for SO production in VG pathobiology and in mediating deficient NO bioactivity.