Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells

Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the duration of expo sure. Recent studies indicate that O-2(-) is involved in cell cycle regulat ion and that OxLDL stimulates endothelial cells to produce O-2(-). This stu dy examined the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a potential source for O-2(-) in the proliferation-inducing act ivity of OxLDL in cultured human umbilical vein endothelial cells (HUVEC). Human LDL was oxidized by Cu++, and proliferation of HUVEC was detected by 3H-thymidine incorporation. OxLDL (5 mu g/ml) caused an increase in prolife ration of HUVEC of 250 to 300%. OxLDL-induced proliferation was blocked by addition of the antioxidants superoxide dismutase and catalase, suggesting that enhanced O-2(-) formation was involved. Diphenylene iodonium (DPI, 1 m u M), an inhibitor of NADPH oxidase, also prevented OxLDL-induced prolifera tion of HUVEC, indicating that NADPH oxidase was the source for enhanced O- 2(-) formation. The OxLDL effect was mimicked by lysophosphatidylcholine (L PC, 10 mu M), a compound formed during oxidation of LDL. LPC-induced prolif eration was also prevented by coincubation with DPI. Treatment of HUVEC wit h O-2(-) generated by the xanthine/xanthine oxidase reaction resulted in pr oliferation as did treatment with OxLDL. As expected, this stimulation coul d not be blocked by DPI. With the use of the cytochrome c-assay, it was dem onstrated that OxLDL and LPC enhanced O-2(-) formation in HUVEC (by factor 3.2 and by factor 3.5, respectively). Supporting the assumption that NADPH oxidase was the enzyme responsible for O-2(-) formation, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly re duced O-2(-) formation after stimulation with OxLDL and LPC. OxLDL and its compound LPC induce proliferation of HUVEC through activation of NADPH oxid ase. The active NADPH oxidase generates O-2(-), which mediates the prolifer ative effects.