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.