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. 2012 Nov;47(5):718-26.
doi: 10.1165/rcmb.2011-0418OC. Epub 2012 Aug 16.

The Nox4 inhibitor GKT137831 attenuates hypoxia-induced pulmonary vascular cell proliferation

David E Green  1 , Tamara C Murphy, Bum-Yong Kang, Jennifer M Kleinhenz, Cédric Szyndralewiez, Patrick Page, Roy L Sutliff, C Michael Hart
Affiliations

The Nox4 inhibitor GKT137831 attenuates hypoxia-induced pulmonary vascular cell proliferation

David E Green et al. Am J Respir Cell Mol Biol. 2012 Nov.

Abstract

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.

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Figures

Figure 1.
Figure 1.
Hypoxia-induced human pulmonary artery smooth muscle cell (HPASMC) and human pulmonary artery endothelial cell (HPAEC) proliferation was attenuated by GKT137831. HPAECs and HPASMCs were exposed to control (21% O2) or hypoxic (1% O2) environments for 72 hours. Cells were treated with vehicle or with GKT137831 (0.1–20 μM) for the final 24 hours (A) or for the entire 72-hour exposure (B). Proliferation was then determined by MTT assay. Each bar represents mean ± SEM cell proliferation expressed as fold-change relative to control samples (n = 3 HPAECs; n = 3 HPASMCs). $P < 0.05 versus 0-Control, +P < 0.01 versus 0-Control, *P < 0.001 versus 0-Control, #P < 0.05 versus 0-Hypoxia, ##P < 0.01 versus 0-Hypoxia, and ∧P < 0.001 versus 0-Hypoxia.
Figure 2.
Figure 2.
Nox4 RNA interference inhibits hypoxia-induced HPAEC and HPASMC proliferation. Selected HPAECs and HPASMCs were transfected with control (−) or Nox4 (+) siRNA and propogated for 96 hours. (A) The siNox4 concentration was titrated to achieve a 50% reduction in target protein expression. Each bar represents mean ± SEM Nox4 protein expressed relative to control samples (n = 3). *P < 0.001 versus control. (B) After transfection, HPASMCs and HPAECs were exposed to normoxic or hypoxic conditions for 72 hours, and proliferation was determined by MTT assay. Each bar represents mean ± SEM cell proliferation expressed as fold-change relative to normoxic control samples (n = 7 HPAECs; n = 3 HPASMCs). *P < 0.001 versus (−) normoxia; #P < 0.001 versus (−) hypoxia.
Figure 3.
Figure 3.
Hypoxia-induced increases in HPASMC and HPAEC H2O2 production were attenuated by GKT137831, and catalase attenuated cell proliferation. HPAECs (A) and HPASMCs (B) were exposed to control (21% O2) or hypoxic (1% O2) environments for 72 hours. Cells were treated with vehicle or GKT137831 (0.1, 5, or 20 μM) for the final 24 hours of exposure. H2O2 production was measured by Amplex Red assay. Each bar represents mean ± SEM concentration of H2O2 expressed as fold-change relative to control samples (n = 3). *P < 0.001 versus control; #P < 0.05 versus hypoxia. (C) HPASMCs and HPAECs were treated with polyethylene glycol-catalase (1,000 U/ml) during the final 24 hours of exposure to hypoxic or normoxic environments, and proliferation was measured by MTT assay. Each bar represents mean ± SEM proliferation expressed as fold-change relative to normoxic control samples (n = 3 HPAECs; n = 5 HPASMCs). *P < 0.01 versus normoxia; #P < 0.05 versus hypoxia.
Figure 4.
Figure 4.
Treatment with GKT137831 attenuated hypoxic reductions of peroxisome proliferator-activated receptor (PPAR)γ expression in HPASMCs and HPAECs. HPAECs and HPASMCs were exposed to control (21% O2) or hypoxic (1% O2) environments for 72 hours. Selected HPAECs (A) and HPASMCs (B) were then treated with vehicle or with GKT137831 (5–20 μM) during the final 24 hours of exposure. Cells were then collected, and proteins were isolated for Western blot analysis of PPARγ and CDK4. Each bar represents mean ± SEM density of PPARγ bands relative to CDK4 expressed as fold-change relative to control values (n = 4–6). *P < 0.05 versus control. (C and D) Pulmonary artery endothelial cells were isolated from the lungs of control subjects or patients with idiopathic pulmonary arterial hypertension (IPAH). Quantitative real-time PCR was performed on deidentified lysates for Nox4 (C) and PPARγ (D). Each bar represents mean ± SEM of Nox4 and PPARγ mRNA expressed relative to 9S (n = 3). *P < 0.05 versus control.
Figure 5.
Figure 5.
GKT137831 attenuated hypoxia-induced increases in right ventricular hypertrophy but not elevations in right ventricular systolic pressure (RVSP). Mice were exposed to hypoxia (10% O2) or normoxia (control, 21% O2) for 3 weeks. Vehicle, rosiglitazone (Rosi, 10 mg/kg/d), or GKT137831 (GKT, 30 or 60 mg/kg/d) was given daily by oral gavage for the final 10 days of exposure. (A) RVSP was measured by advancing a pressure transducer into the right ventricle. Each bar represents the mean RVSP ± SEM in mm Hg (n = 4–8). *P < 0.05 versus normoxic control mice; #P < 0.01 versus normoxic control mice. (B) Hearts were dissected into the right ventricle and left ventricle plus septum. Each bar represents the mean ± SEM ratio of the weight of the right ventricle to left ventricle plus septum (n = 8). *P < 0.05 versus normoxic control mice; #P < 0.05 versus hypoxic control mice.
Figure 6.
Figure 6.
GKT137831 or rosiglitazone attenuated hypoxia-induced vascular remodeling and proliferating cell nuclear antigen expression in vivo. Mice were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. Vehicle control (Veh), rosiglitazone (Rosi, 10 mg/kg/d), or GKT137831 (GKT, 30 or 60 mg/kg/d) were given daily by oral gavage for the final 10 days of exposure. Tissue sections generated from mouse lungs were stained with antibodies to α-SMA, and the vessel wall thickness and vessel density were measured for vessels with diameter < 100 μm. (A) Bars represent the mean ± SEM vessel wall thickness relative to normoxic control samples (n = 3–4). ***P < 0.001 versus normoxia; #P < 0.001 versus hypoxia. (B) Representative photomicrographs of α-SMA–stained vessels exposed to normoxia or hypoxia with or without GKT137831 are demonstrated. Labeling is shown (C, control; C+GKT, control + GKT137831; H, hypoxia; H + GKT, hypoxia + GKT137831), and the scale bar in each image = 50 μm. (C) Bars represent the mean ± SEM number of α-SMA staining vessels per mm2 relative to normoxic control samples (n = 3–4). **P < 0.01 versus normoxia-veh; *P < 0.05 versus normoxia-veh. (GKT-30 = GKT-30 mg/kg; GKT-60 = GKT-60 mg/kg; Rosi = rosiglitazone; Veh = vehicle). (D) Whole lung lysates were isolated for Western blot analysis of PCNA and CDK4 to determine cell proliferation. Each bar represents mean ± SEM density of PCNA bands relative to CDK4 expressed as fold-change relative to control values (n = 8). *P < 0.05 versus control-vehicle; #P < 0.05 versus hypoxia-vehicle.
Figure 7.
Figure 7.
GKT137831 or rosiglitazone attenuated hypoxic alterations in PPARγ in vivo and TGF-β1 in vivo and in vitro. Animals were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. Control (Vehicle), rosiglitazone (Rosi, 10 mg/kg/d), or GKT137831 (GKT, 30 or 60 mg/kg/d) were given daily by oral gavage for the final 10 days of exposure. Protein was prepared from whole lung homogenate for Western blot analysis. Each bar represents the mean ± SEM density of PPARγ (A) or TGF-β1 (B) bands relative to CDK4 expressed as fold-change relative to control samples (n = 8). *P < 0.05 versus normoxic control lung; #P < 0.05 versus hypoxic vehicle-treated lung. (C) HPAECs were exposed to hypoxia for 72 hours with or without GKT137831 (20 μM) or polyethylene glycol (PEG) catalase (1,000 U/ml) administered during the last 24 hours. Each bar represents the mean ± SEM TGF-β1 mRNA expression relative to control samples (n = 3). *P < 0.01 versus (–) normoxia; **P < 0.001 versus (–) normoxia; #P < 0.01 versus (–) hypoxia.
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