title: Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy.
abstract: OBJECTIVE
  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 (H(2)O(2)) in vascular disease, and plasma H(2)O(2) levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H(2)O(2) was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H(2)O(2) activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H(2)O(2) induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1.
  
  
  RESULTS
  Treatment of rat aortic SMCs (rASMC) with exogenous H(2)O(2) resulted in a concentration-dependent increase in Nox-derived superoxide (O(2)(•-)), 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(2)O(2) cellular entry and H(2)O(2)-induced O(2)(•-) production. H(2)O(2) treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H(2)O(2)-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it.
  
  
  CONCLUSIONS
  Our results demonstrate for the first time that extracellular H(2)O(2), at pathophysiological concentrations, stimulates rASMC Nox1-derived O(2)(•-), subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H(2)O(2) 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.
authors.affiliation: Vascular Medicine Institute and Department of Pharmacology and Chemical Biology,  University of Pittsburgh, Pittsburgh, PA,  USA.
authors.forename: Imad
authors.initials: I
authors.lastname: Al Ghouleh
authors.forename: Giovanna
authors.initials: G
authors.lastname: Frazziano
authors.forename: Andres I
authors.initials: AI
authors.lastname: Rodriguez
authors.forename: Gábor
authors.initials: G
authors.lastname: Csányi
authors.forename: Salony
authors.initials: S
authors.lastname: Maniar
authors.forename: Claudette M
authors.initials: CM
authors.lastname: St Croix
authors.forename: Eric E
authors.initials: EE
authors.lastname: Kelley
authors.forename: Loreto A
authors.initials: LA
authors.lastname: Egaña
authors.forename: Gyun Jee
authors.initials: GJ
authors.lastname: Song
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bisello
authors.forename: Yong J
authors.initials: YJ
authors.lastname: Lee
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Pagano
mesh_terms: D000818:Animals; D051398:Aquaporin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005434:Flow Cytometry; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006984:Hypertrophy; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D016877:Oxidants / Q000494:pharmacology*; D010766:Phosphorylation; D034622:RNA Interference; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: C496022:Aqp1 protein, rat; D016877:Oxidants; D013481:Superoxides; D051398:Aquaporin 1; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614712:NOX1 protein, rat; D048848:MAP Kinase Kinase Kinase 5