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

J Am Soc Nephrol. 2000 Oct;11(10):1819-1825. doi: 10.1681/ASN.V11101819.

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 [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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalase / pharmacology
  • Cell Division / drug effects
  • Cells, Cultured
  • Endothelium, Vascular / cytology*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Lipopolysaccharides / pharmacology
  • Lipoproteins, LDL / pharmacology*
  • Membrane Transport Proteins*
  • NADPH Dehydrogenase / genetics*
  • NADPH Oxidases / metabolism*
  • Oligonucleotides / genetics
  • Oligonucleotides, Antisense / genetics
  • Onium Compounds / pharmacology
  • Phosphoproteins / genetics*
  • Superoxide Dismutase / pharmacology
  • Superoxides / metabolism
  • Transfection

Substances

  • Enzyme Inhibitors
  • Lipopolysaccharides
  • Lipoproteins, LDL
  • Membrane Transport Proteins
  • Oligonucleotides
  • Oligonucleotides, Antisense
  • Onium Compounds
  • Phosphoproteins
  • oxidized low density lipoprotein
  • Superoxides
  • diphenyleneiodonium
  • Catalase
  • Superoxide Dismutase
  • NADPH Oxidases
  • CYBA protein, human
  • NADPH Dehydrogenase