RESUMO
Chronic exposure to hypoxia induces a pronounced remodelling of the pulmonary vasculature leading to pulmonary hypertension (PH). The remodelling process also entails increased proliferation and decreased apoptosis of pulmonary arterial smooth muscle cells (PASMC), processes regulated by the cytoskeletal protein paxillin. In this study, we aimed to examine the molecular mechanisms leading to deregulation of paxillin in PH. We detected a time-dependent increase in paxillin tyrosine 31 (Y31) and 118 (Y118) phosphorylation following hypoxic exposure (1 % O2) or platelet-derived growth factor (PDGF)-BB stimulation of primary human PASMC. In addition, both, hypoxia- and PDGF-BB increased the nuclear localisation of phospho-paxillin Y31 as indicated by immunofluorescence staining in human PASMC. Elevated paxillin tyrosine phosphorylation in human PASMC was attenuated by hypoxia-inducible factor (HIF)-1α depletion or by treatment with the PDGF-BB receptor antagonist, imatinib. Moreover, we observed elevated paxillin Y31 and Y118 phosphorylation in the pulmonary vasculature of chronic hypoxic mice (21 days, 10 % O2) which was reversible by imatinib-treatment. PDGF-BB-dependent PASMC proliferation was regulated via the paxillin-Erk1/2-cyclin D1 pathway. In conclusion, we suggest paxillin up-regulation and phosphorylation as an important mechanism of vascular remodelling underlying pulmonary hypertension.
Assuntos
Anti-Hipertensivos/farmacologia , Benzamidas/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Paxilina/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-sis/farmacologia , Pirimidinas/farmacologia , Remodelação Vascular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Apoptose/efeitos dos fármacos , Becaplermina , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mesilato de Imatinib , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Paxilina/genética , Fosforilação , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Interferência de RNA , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , TirosinaRESUMO
An accumulating body of evidence incriminates Rho kinase (ROCK) in the pathogenesis of pulmonary hypertension (PH). The therapeutic efficacy of azaindole-1, a novel highly selective and orally active ROCK inhibitor, has not yet been investigated in PH. This study aimed to investigate the effects of azaindole-1 on 1) acute hypoxic pulmonary vasoconstriction (HPV), 2) proliferation of pulmonary arterial smooth muscle cells (PASMCs) and 3) animal models of PH. Azaindole-1 significantly inhibited HPV in isolated, ventilated and buffer-perfused murine lungs and proliferation of primary rat PASMCs in vitro. Azaindole-1 was administered orally from 21 to 35 days after monocrotaline (MCT) injection in rats and hypoxic exposure in mice. Azaindole-1 (10 and 30 mg per kg body weight per day in rats and mice, respectively) significantly improved haemodynamics and right ventricular hypertrophy. Moreover, the medial wall thickness and muscularisation of peripheral pulmonary arteries were ameliorated. Azaindole-1 treatment resulted in a decreased immunoreactivity for phospho-myosin phosphatase target subunit 1 and proliferating cell nuclear antigen in pulmonary vessels of MCT-injected rats, suggesting an impaired ROCK activity and reduced proliferating cells. Azaindole-1 provided therapeutic benefit in experimental PH, and this may be attributable to its potent vasorelaxant and antiproliferative effects. Azaindole-1 may offer a useful approach for treatment of PH.