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 exposure. Recent studies indicate that [O(2)](-) is involved in cell cycle regulation and that OxLDL stimulates endothelial cells to produce [O(2)](-). This study examined the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a potential source for [O(2)](-) 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 microg/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 [O(2)](-) formation was involved. Diphenylene iodonium (DPI, 1 microM), an inhibitor of NADPH oxidase, also prevented OxLDL-induced proliferation of HUVEC, indicating that NADPH oxidase was the source for enhanced [O(2)](-) formation. The OxLDL effect was mimicked by lysophosphatidylcholine (LPC, 10 microM), a compound formed during oxidation of LDL. LPC-induced proliferation was also prevented by coincubation with DPI. Treatment of HUVEC with [O(2)](-) 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 [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 reduced [O(2)](-) 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 [O(2)](-), which mediates the proliferative effects.
Heinloth
Alexandra
A
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
Heermeier
Kathrin
K
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
Raff
Ulrike
U
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
Wanner
Christoph
C
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
Galle
Jan
J
Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, Germany.
D002374:Catalase / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D026901:Membrane Transport Proteins; D009252:NADPH Dehydrogenase / Q000235:genetics*; D019255:NADPH Oxidases / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D008077:Lipoproteins, LDL; D026901:Membrane Transport Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D009861:Onium Compounds; D010750:Phosphoproteins; C099252:oxidized low density lipoprotein; D013481:Superoxides; C007517:diphenyleneiodonium; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase