Exp Mol Med.  2015 Jun;47(6):e168. 10.1038/emm.2015.30.

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells

• Park IH
• Hwang HM
• Jeon BH
• Kwon HJ
• Hoe KL
• Kim YM
• Ryoo S

• KMID: 2155422
• DOI: http://doi.org/10.1038/emm.2015.30

Abstract

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-theta (PKCtheta) and protein kinase C-beta (PKCbeta) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCtheta and PKCbeta stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox-/- mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.

MeSH Terms

Animals
Aorta/*cytology
Cell Line
Cell Proliferation
Cells, Cultured
Humans
Lipoproteins, LDL/*metabolism
Mice, Inbred C57BL
Mitogen-Activated Protein Kinases/metabolism
Muscle, Smooth, Vascular/cytology
Myocytes, Smooth Muscle/*cytology
NADPH Oxidase/*metabolism
Phosphorylation
Protein Kinase C/metabolism
Signal Transduction
Superoxides/metabolism
Lipoproteins, LDL
Mitogen-Activated Protein Kinases
NADPH Oxidase
Protein Kinase C
Superoxides