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1.
Am J Respir Cell Mol Biol ; 68(5): 537-550, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36724371

RESUMO

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.


Assuntos
Hipertensão Arterial Pulmonar , Ratos , Camundongos , Animais , Hipertensão Arterial Pulmonar/tratamento farmacológico , Remodelação Vascular , Proliferação de Células , Hipertensão Pulmonar Primária Familiar , Modelos Animais de Doenças , Miócitos de Músculo Liso , Artéria Pulmonar
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33803922

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteoma/genética , Hipertensão Arterial Pulmonar/genética , Transcriptoma/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Ciclo do Ácido Cítrico/genética , Epigênese Genética/genética , Feminino , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/crescimento & desenvolvimento , Artéria Pulmonar/patologia , Ratos
3.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805714

RESUMO

Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.


Assuntos
Fármacos Cardiovasculares/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/prevenção & controle , Miócitos de Músculo Liso/efeitos dos fármacos , Trifluoperazina/farmacologia , Animais , Antipsicóticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipóxia/induzido quimicamente , Hipóxia/genética , Hipóxia/fisiopatologia , Indóis/administração & dosagem , Monocrotalina/administração & dosagem , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Pirróis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Survivina/genética , Survivina/metabolismo
4.
Am J Respir Crit Care Med ; 203(5): 614-627, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33021405

RESUMO

Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.


Assuntos
Enzimas Reparadoras do DNA/genética , DNA/metabolismo , Estresse Oxidativo/genética , Monoéster Fosfórico Hidrolases/genética , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , Remodelação Vascular/genética , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Adulto , Idoso , Animais , Apoptose/genética , Western Blotting , Estudos de Casos e Controles , Proliferação de Células/genética , Cromatografia Líquida , Ensaio Cometa , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box M1/metabolismo , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/genética , Pirofosfatases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Espectrometria de Massas em Tandem , Regulação para Cima
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