Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy

Aims Reactive oxygen species (ROS)-mediated intracellular signalling is well described in the vasculature, yet the precise roles of ROS in paracrine signalling are not known. Studies implicate interstitial ROS hydrogen peroxide (H₂O₂) in vascular disease, and plasma H₂O ₂ levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H₂O₂ was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H₂O₂ activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H ₂O₂ induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1.Methods and resultsTreatment of rat aortic SMCs (rASMC) with exogenous H₂O₂ resulted in a concentration-dependent increase in Nox-derived superoxide (O₂ ^•-), determined by L-012 chemiluminescence, cytochrome c and electron paramagnetic resonance. Nox1 was verified as the source of O ₂^·- by siRNA. Aqp1 siRNA attenuated H ₂O₂ cellular entry and H₂O₂-induced O₂^•- production. H₂O₂ treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H₂O ₂-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it.ConclusionOur results demonstrate for the first time that extracellular H₂O₂, at pathophysiological concentrations, stimulates rASMC Nox1-derived O₂^•-, subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H₂O₂ enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.