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

Abstract

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 (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.

Methods and 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.

Conclusion: 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.

Figure 1

Exogenous H₂O₂ increases O₂^•– production in rASMC. (A) Representative trace of L-012 chemiluminescence over time after rASMC treatment with indicated concentrations of H₂O₂ ± superoxide dismutase (SOD, 300 U/mL). (B) Quantification of all the areas under the curve (AUC) in (A) in relative light units (RLU). (C) Quantification of first-peak amplitudes of CM^• radical EPR spectra of rASMC treated with vehicle or 50 μM H₂O₂ ± SOD (300 U/mL). Inset: representative EPR spectrum of rASMC treated with 50 μM H₂O₂. Means ± SEM, n = 3, *P < 0.05 vs. vehicle; ^#P < 0.05 vs. H₂O₂.

Figure 2

Nox1 is the enzymatic source of H₂O₂-induced O₂^•– in rASMC. (A) AUC of L-012 chemiluminescence of rASMC transfected with scrambled (Scr.) or Nox1 siRNA and treated with vehicle or 50 μM H₂O₂. Means ± SEM, n = 12, *P < 0.001 vs. Scr. siRNA vehicle, #P < 0.001 vs. Scr. siRNA H₂O₂. (B) O₂^•– production in pmol/min/mg protein quantified by SOD-inhibitable cytochrome c reduction in membrane fractions of rASMC transfected and treated as in (A). Means ± SEM, n = 4, *P < 0.05 vs. Scr. siRNA vehicle, ^#P < 0.05 vs. Scr. siRNA H₂O₂.

Figure 3

Aqp1 is important for H₂O₂ entry into rASMC and induction of O₂^•–. (A) Live-cell imaging of changes in HyPer fluorescence of rASMC expressing HyPer plus Scr. or Aqp1 siRNA before (pre) or after (post) treatment with vehicle or 50 μM H₂O₂. Right panel shows the quantification of HyPer signal vs. time. Data shown as means ± SEM of 10 stage positions per condition, representative of three independent experiments, *P < 0.001 vs. Scr. siRNA. (B) AUC of L-012 chemiluminescence expressed as fold change from Scr. siRNA vehicle of rASMC transfected with Scr. or Aqp1 siRNA and treated with vehicle or 50 μM H₂O₂. Means ± SEM, n = 40, *P < 0.001 vs. Scr. siRNA vehicle, ^#P < 0.01 vs. H₂O₂ Scr. siRNA.

Figure 4

H₂O₂ induces Ask1 phosphorylation in rASMC in a Nox1-dependent mechanism. (A) Representative Western blot for phospho-Ask1 (threonine-845) and total-Ask1 of lysates of rASMC transfected with Scr. or Nox1 siRNA and treated with 50 μM H₂O₂ (UT, untransfected cells). (B) The quantification of fluorescence intensity of phospho-Ask1 bands obtained in (A) normalized to total-Ask1. Means ± SEM, n = 6, *P < 0.05 vs. vehicle; #P < 0.05 vs. H₂O₂ Scr. siRNA.

Figure 5

H₂O₂ induces increase in rASMC hypertrophy, but not proliferation, in an Aqp1- and Nox1-dependent mechanism. (A) Quantification of the protein:DNA ratio of lysates from rASMC transfected with Scr. or Nox1 siRNA and treated with vehicle or 50 μM H₂O₂ for 24 h expressed as fold from Scr. vehicle. (B) FACS analysis of % enlarged cells of total rASMC treated as in (A) expressed as fold from Scr. vehicle. (C) Representative FACS analysis image of B. (D) FACS analysis quantified as in (B) of rASMC transfected with Scr. or Aqp1 siRNA and treated with vehicle or 50 μM H₂O₂ for 24 h. (E) [³H]thymidine incorporation measurement in counts per minute (CPM) of rASMC treated as in (A). Means ± SEM, n = 3–4. *P < 0.05 vs. Scr. siRNA vehicle. ^#P < 0.05 vs. H₂O₂ Scr. siRNA.

Figure 6

H₂O₂-induced Nox1-dependent increase in rASMC hypertrophy is augmented by Ask1 overexpression and attenuated by dominant-negative Ask1 expression. (A and B) Quantification of the protein:DNA ratio of lysates from rASMC transduced with adenoviruses expressing GFP control (Ad-GFP), dominant-negative Ask1 (Ad-DN-Ask1), or transgenic Ask1 (Ad-Tg-Ask1) and treated with vehicle or 50 μM H₂O₂ for 24 h expressed as fold from Ad-GFP vehicle. Means ± SEM, n = 5, *P < 0.05 vs. Ad-GFP vehicle. **P < 0.001 vs. Ad-Tg-Ask1 vehicle. ^†P < 0.05 vs. Ad-GFP H₂O₂. (C) Quantification of the protein:DNA ratio of lysates from rASMC transduced with transgenic Ask1 (Ad-Tg-Ask1), transfected with Scr. or Nox1 siRNA and treated with vehicle or 50 μM H₂O₂ for 24 h expressed as fold from Scr. siRNA vehicle Ad-Tg-Ask1. Means ± SEM, n = 5, *P < 0.01 vs. Scr. siRNA vehicle Ad-Tg-Ask1, ^#P < 0.001 vs. H₂O₂ Nox1 siRNA Ad-Tg-Ask1.