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.

Genetic Deletion of NADPH Oxidase 1 Rescues Microvascular Function in Mice With Metabolic Disease

“…13 Further, gene silencing techniques reveal cellular events involved in neointima formation to be driven by Nox1 activation in cell lines of vascular smooth muscle cells, namely, phenotypic modulation and proliferation in response to cyclic stretch 14 and low density lipoprotein. 15 This putative role of Nox1 in diabetes-induced atherosclerosis holds true in the intact animal, as demonstrated by the seminal paper by Gray et al . In this study, genetic deletion of Nox1, but not Nox4, abolished atherosclerotic lesion development in aortae of ApoE −/− mice made diabetic by daily injections of streptozotocin.…”

“…12 Nox1, expressed in endothelial cells and vascular smooth muscle cells 11 , may be a key driver of oxidative stress within an aberrant metabolic state given that its activity in vitro is induced in the presence of high glucose or saturated fatty acids. 12–15 Indeed, our laboratory previously demonstrated that restoring glycemic control via deletion of PTP1B or myostatin, improved microvascular function and attenuated Nox1 expression. 16 These findings suggest that the metabolic milieu of obesity is transduced to the vasculature through Nox1.…”

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

“…We previously demonstrated that nLDL induced VSMC proliferation through activation of NADPH oxidase and that phosphorylation of both PKCβ and ERK1/2 was responsible for p47phox translocation to the membrane. 3 Therefore, we tested whether limonin prevented nLDL-induced activation of PKCβII and ERK1/2. Interestingly, limonin abolished PKCβII phosphorylation at 10 min in nLDL-stimulated VSMCs ( Fig.…”

“…The chemiluminescence intensity in all experiments was measured for 5 min, and the values are presented as relative lucigenin units/min. 3 …”

“…The activation of these kinases has been shown to be associated with NADPH oxidase activation through p47phox translocation to p22phox and subsequent superoxide generation, which contributes to VSMC proliferation. 3 …”