Dual effect of oxidized LDL on cell cycle in human endothelial cells through oxidative stress

Background. Oxidized low-density lipoprotein (OxLDL) exerts proliferation and apoptosis in vascular cells, depending on its concentration and the: exposure time. Various steps in the cell cycle and in the apoptotic signaling cascade are modulated by O-2(-), and OxLDL stimulates vascular O-2(-) formation. Here we studied the role of NADPH oxidase, a potential source for O-2(-) formation after OxLDL stimulation, in cell proliferation, and we investigated whether OxLDL influences anti-apoptotic genes in cultured human umbilical vein endothelial cells (HUVEC). Methods and Results. OxLDL dose-dependently (10 to 300 mug/mL) stimulated O-2(-) formation in HUVEC (detected by cytochrome c assay and by chemiluminescence of lucigenin). Low OxLDL concentrations (5 to 10 mug/mL) induced proliferation (detected by H-3-thymidine incorporation), while higher concentrations (50 to 300 mug/mL) induced apoptotic cell death (detected by Annexin assay and DNA fragmentation). Proliferation was blocked by the antioxidants SOD and catalase and by diphenyleneiodonium (10 mu mol/L), an inhibitor of the O-2(-) generating NADPH oxidase. In addition, cells transfected with antisense oligonucleotides for NADPH oxidase showed a significantly reduced O-2(-) formation after stimulation with OxLDL. The OxLDL effect on apoptosis was also blocked by antioxidants. Since endothelial cells are protected against apoptosis through anti-apoptotic genes, we investigated whether OxLDL overcomes protection against apoptosis through suppression of the anti-apoptotic gene A20, a zinc-finger protein. OxLDL suppressed the expression of A20 in a dose-dependent manner. Conclusion. These data indicate that OxLDL has a dual effect on cell cycle in HUVEC, inducing proliferation at low and apoptosis at higher concentrations. Both effects are mediated by O-2(-) formation, with NADPH oxidase being a major source for O-2(-). Thus, OxLDL contributes importantly to vascular cellular turnover through the induction of oxidative stress.