Stimulation of NADPH Oxidase by Oxidized Low-Density Lipoprotein Induces Proliferation of Human Vascular Endothelial Cells

Abstract

Abstract. Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the duration of exposure. Recent studies indicate that O2- is involved in cell cycle regulation and that OxLDL stimulates endothelial cells to produce O2-. This study examined the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a potential source for O2- in the proliferation-inducing activity 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 μg/ml) caused an increase in proliferation of HUVEC of 250 to 300%. OxLDL-induced proliferation was blocked by addition of the antioxidants superoxide dismutase and catalase, suggesting that enhanced O2- formation was involved. Diphenylene iodonium (DPI, 1 μM), an inhibitor of NADPH oxidase, also prevented OxLDL-induced proliferation of HUVEC, indicating that NADPH oxidase was the source for enhanced O2- formation. The OxLDL effect was mimicked by lysophosphatidylcholine (LPC, 10 μM), a compound formed during oxidation of LDL. LPC-induced proliferation was also prevented by coincubation with DPI. Treatment of HUVEC with O2- generated by the xanthine/xanthine oxidase reaction resulted in proliferation as did treatment with OxLDL. As expected, this stimulation could not be blocked by DPI. With the use of the cytochrome c-assay, it was demonstrated that OxLDL and LPC enhanced O2- formation in HUVEC (by factor 3.2 and by factor 3.5, respectively). Supporting the assumption that NADPH oxidase was the enzyme responsible for O2- formation, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly reduced O2- formation after stimulation with OxLDL and LPC. OxLDL and its compound LPC induce proliferation of HUVEC through activation of NADPH oxidase. The active NADPH oxidase generates O2-, which mediates the proliferative effects.