title: NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells.
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-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ 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.
authors.affiliation: Department of Cardiothoracic Surgery, Yonsei University Wonju College of Medicine, Wonju, Korea.
authors.forename: Il Hwan
authors.initials: IH
authors.lastname: Park
authors.affiliation: Department of Biology, College of Natural Sciences, Kangwon National University, Chuncheon, Korea.
authors.forename: Hye Mi
authors.initials: HM
authors.lastname: Hwang
authors.affiliation: Infectious Signaling Network Research Center, Department of Physiology, School of Medicine, Chungnam National University, Daejeon, Korea.
authors.forename: Byeong Hwa
authors.initials: BH
authors.lastname: Jeon
authors.affiliation: Department of Microbiology, School of Medicine, Hallym University, Chuncheon, Korea.
authors.forename: Hyung-Joo
authors.initials: HJ
authors.lastname: Kwon
authors.affiliation: Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea.
authors.forename: Kwang Lae
authors.initials: KL
authors.lastname: Hoe
authors.affiliation: Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Young Myeong
authors.initials: YM
authors.lastname: Kim
authors.affiliation: Department of Biology, College of Natural Sciences, Kangwon National University, Chuncheon, Korea.
authors.forename: Sungwoo
authors.initials: S
authors.lastname: Ryoo
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology*; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism
chemical_list: D008077:Lipoproteins, LDL; D013481:Superoxides; D019255:NADPH Oxidases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases