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

ABSTRACT: Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor ? (PPAR?) 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, PPAR?, and transforming growth factor (TGF)?1 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 PPAR? and TGF-?1 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-?1 expression and blunted reductions in PPAR? in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-?1 and reductions in PPAR? 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.

SUBMITTER: Green DE

PROVIDER: S-EPMC3547100 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Green David E DE Murphy Tamara C TC Kang Bum-Yong BY Kleinhenz Jennifer M JM Szyndralewiez Cédric C Page Patrick P Sutliff Roy L RL Hart C Michael CM

American journal of respiratory cell and molecular biology 20120816 5

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 ...[more]

PMID: 22904198

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Project description:BackgroundPulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH.MethodsOxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH.ResultsCyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling.ConclusionsA novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.

Project description:The Notch signaling pathway participates in pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis. Astragaloside IV (AS‑IV) is an effective antiproliferative treatment for vascular diseases. The present study aimed to investigate the protective effects and mechanisms underlying AS‑IV on hypoxia‑induced PASMC proliferation and pulmonary vascular remodeling in pulmonary arterial hypertension (PAH) model rats. Rats were divided into the following four groups: i) normoxia; ii) hypoxia (10% O2); iii) treatment, hypoxia + intragastrical administration of AS‑IV (2 mg/kg) daily for 28 days; and iv) DAPT, hypoxia + AS‑IV treatment + subcutaneous administration of DAPT (10 mg/kg) three times daily. The effects of AS‑IV treatment on the development of hypoxia‑induced PAH, right ventricle (RV) hypertrophy and pulmonary vascular remodeling were examined. Furthermore, PASMCs were treated with 20 µmol/l AS‑IV under hypoxic conditions for 48 h. To determine the effect of Notch signaling in vascular remodeling and the potential mechanisms underlying AS‑IV treatment, 5 mmol/l γ‑secretase inhibitor [N‑[N‑(3,5‑difluorophenacetyl)‑L‑alanyl]‑S‑phenylglycine t‑butyl ester (DAPT)] was used. Cell viability and apoptosis were determined by performing the MTT assay and flow cytometry, respectively. Immunohistochemistry was conducted to detect the expression of proliferating cell nuclear antigen (PCNA). Moreover, the mRNA and protein expression levels of Notch‑3, Jagged‑1, hes family bHLH transcription factor 5 (Hes‑5) and PCNA were measured via reverse transcription‑quantitative PCR and western blotting, respectively. Compared with the normoxic group, hypoxia‑induced PAH model rats displayed characteristics of PAH and RV hypertrophy, whereas AS‑IV treatment alleviated PAH and prevented RV hypertrophy. AS‑IV also inhibited hypoxia‑induced pulmonary vascular remodeling, as indicated by reduced wall thickness and increased lumen diameter of pulmonary arterioles, and decreased muscularization of distal pulmonary vasculature in hypoxia‑induced PAH model rats. Compared with normoxia, hypoxia promoted PASMC proliferation in vitro, whereas AS‑IV treatment inhibited hypoxia‑induced PASMC proliferation by downregulating PCNA expression in vitro and in vivo. In hypoxia‑treated PAH model rats and cultured PASMCs, AS‑IV treatment reduced the expression levels of Jagged‑1, Notch‑3 and Hes‑5. Furthermore, Notch signaling inhibition via DAPT significantly inhibited the pulmonary vascular remodeling effect of AS‑IV in vitro and in vivo. Collectively, the results indicated that AS‑IV effectively reversed hypoxia‑induced pulmonary vascular remodeling and PASMC proliferation via the Notch signaling pathway. Therefore, the present study provided novel insights into the mechanism underlying the use of AS‑IV for treatment of vascular diseases, such as PAH.

Project description:Background: Doxorubicin (DOX)-induced cardiotoxicity is a highly concerning issue, and the mechanism by which DOX induces cardiotoxicity is likely to be multifactorial. NADPH oxidase (NOX) is associated with DOX-induced cardiotoxicity. Setanaxib (GKT137831), a preferential direct inhibitor of NOX1 and NOX4, can delay or prevent the progression of many cardiovascular disorders by inhibiting reactive oxygen species (ROS) generation. In this study, we investigated the role of GKT137831 in ameliorating DOX-induced cardiotoxicity and the potential mechanisms of its action. Methods and Results: The mice model of cardiotoxicity induced by DOX was established, and GKT137831 treatment was performed at the same time. Neonatal rat cardiomyocytes (NRCMs) were treated with DOX or GKT137831 for in vitro experiments. We found that DOX administration impaired cardiac function in vivo, reflected by decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS%). DOX also impaired the viability of NRCMs in vitro. In addition, DOX increased the levels of NOX1 and NOX4 expression and ROS production and the cardiomyocyte apoptosis rate, both in vivo and in vitro. GKT137831 improved cardiac function, as indicated by the increased LVEF and FS%. In vitro, GKT137831 improved NRCM viability. It also decreased ROS production and the cardiomyocyte apoptosis rate. Apoptotic indices, such as cleaved PARP (c-PARP), cleaved caspase 3 (CC3) and BAX expression levels, were decreased, and the antiapoptotic index of Bcl-2 expression was increased. DOX markedly activated phosphorylated JNK, ERK and p38 proteins in NRCMs. Specific inhibitors of JNK (SP600125), ERK (PD98059) or p38 (SB203580) inhibited DOX-induced apoptosis of NRCMs. GKT137831 pretreatment inhibited excessive DOX-induced MAPK pathway activation. Conclusion: This study revealed that GKT137831 can alleviate DOX-induced cardiomyocyte apoptosis by inhibiting NOX1/4-driven ROS production. The upregulation of MAPK pathway induced by NOX1/4-derived ROS production may be the potential mechanism of GKT137831 action. GKT137831 may be a potential drug candidate to ameliorate DOX-induced cardiotoxicity.

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

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

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