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1.
JACC Basic Transl Sci ; 7(4): 384-403, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35540097

RESUMO

The authors show that increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) and pyruvate kinase muscle isozyme 2 (PKM2) expression is a common feature of a decompensated right ventricle in patients with pulmonary arterial hypertension and animal models. The authors find in vitro that overactivated PARP1 promotes cardiomyocyte dysfunction by favoring PKM2 expression and nuclear function, glycolytic gene expression, activation of nuclear factor κB-dependent proinflammatory factors. Pharmacologic and genetic inhibition of PARP1 or enforced tetramerization of PKM2 attenuates maladaptive remodeling improving right ventricular (RV) function in multiple rodent models. Taken together, these data implicate the PARP1/PKM2 axis as a critical driver of maladaptive RV remodeling and a new promising target to directly sustain RV function in patients with pulmonary arterial hypertension.

2.
Thorax ; 77(3): 247-258, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34226205

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterised by exuberant tissue remodelling and associated with high unmet medical needs. Outcomes are even worse when IPF results in secondary pulmonary hypertension (PH). Importantly, exaggerated resistance to cell death, excessive proliferation and enhanced synthetic capacity are key endophenotypes of both fibroblasts and pulmonary artery smooth muscle cells, suggesting shared molecular pathways. Under persistent injury, sustained activation of the DNA damage response (DDR) is integral to the preservation of cells survival and their capacity to proliferate. Checkpoint kinases 1 and 2 (CHK1/2) are key components of the DDR. The objective of this study was to assess the role of CHK1/2 in the development and progression of IPF and IPF+PH. METHODS AND RESULTS: Increased expression of DNA damage markers and CHK1/2 were observed in lungs, remodelled pulmonary arteries and isolated fibroblasts from IPF patients and animal models. Blockade of CHK1/2 expression or activity-induced DNA damage overload and reverted the apoptosis-resistant and fibroproliferative phenotype of disease cells. Moreover, inhibition of CHK1/2 was sufficient to interfere with transforming growth factor beta 1-mediated fibroblast activation. Importantly, pharmacological inhibition of CHK1/2 using LY2606368 attenuated fibrosis and pulmonary vascular remodelling leading to improvement in respiratory mechanics and haemodynamic parameters in two animal models mimicking IPF and IPF+PH. CONCLUSION: This study identifies CHK1/2 as key regulators of lung fibrosis and provides a proof of principle for CHK1/2 inhibition as a potential novel therapeutic option for IPF and IPF+PH.


Assuntos
Hipertensão Pulmonar , Fibrose Pulmonar Idiopática , Animais , Fibroblastos/metabolismo , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Miócitos de Músculo Liso/metabolismo
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.
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
5.
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
6.
Arterioscler Thromb Vasc Biol ; 40(3): 783-801, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31969012

RESUMO

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by the narrowing of pulmonary arteries (PAs). It is now established that this phenotype is associated with enhanced PA smooth muscle cells (PASMCs) proliferation and suppressed apoptosis. This phenotype is sustained in part by the activation of several DNA repair pathways allowing PASMCs to survive despite the unfavorable environmental conditions. PIM1 (Moloney murine leukemia provirus integration site) is an oncoprotein upregulated in PAH and involved in many prosurvival pathways, including DNA repair. The objective of this study was to demonstrate the implication of PIM1 in the DNA damage response and the beneficial effect of its inhibition by pharmacological inhibitors in human PAH-PASMCs and in rat PAH models. Approach and Results: We found in vitro that PIM1 inhibition by either SGI-1776, TP-3654, siRNA (silencer RNA) decreased the phosphorylation of its newly identified direct target KU70 (lupus Ku autoantigen protein p70) resulting in the inhibition of double-strand break repair (Comet Assay) by the nonhomologous end-joining as well as reduction of PAH-PASMCs proliferation (Ki67-positive cells) and resistance to apoptosis (Annexin V positive cells) of PAH-PASMCs. In vivo, SGI-1776 and TP-3654 given 3× a week, improved significantly pulmonary hemodynamics (right heart catheterization) and vascular remodeling (Elastica van Gieson) in monocrotaline and Fawn-Hooded rat models of PAH. CONCLUSIONS: We demonstrated that PIM1 phosphorylates KU70 and initiates DNA repair signaling in PAH-PASMCs and that PIM1 inhibitors represent a therapeutic option for patients with PAH.


Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , Hipertensão Pulmonar/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Apoptose , Proliferação de Células , Células Cultivadas , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Histonas/metabolismo , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Autoantígeno Ku/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genética , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Remodelação Vascular
7.
Circ Res ; 125(4): 449-466, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31154939

RESUMO

RATIONALE: Pulmonary hypertension (PH) due to left heart disease (LHD), or group 2 PH, is the most prevalent form of PH worldwide. PH due to LHD is often associated with metabolic syndrome (MetS). In 12% to 13% of cases, patients with PH due to LHD display vascular remodeling of pulmonary arteries (PAs) associated with poor prognosis. Unfortunately, the underlying mechanisms remain unknown; PH-targeted therapies for this group are nonexistent, and the development of a new preclinical model is crucial. Among the numerous pathways dysregulated in MetS, inflammation plays also a critical role in both PH and vascular remodeling. OBJECTIVE: We hypothesized that MetS and inflammation may trigger the development of vascular remodeling in group 2 PH. METHODS AND RESULTS: Using supracoronary aortic banding, we induced diastolic dysfunction in rats. Then we induced MetS by a combination of high-fat diet and olanzapine treatment. We used metformin treatment and anti-IL-6 (interleukin-6) antibodies to inhibit the IL-6 pathway. Compared with sham conditions, only supracoronary aortic banding+MetS rats developed precapillary PH, as measured by both echocardiography and right/left heart catheterization. PH in supracoronary aortic banding+MetS was associated with macrophage accumulation and increased IL-6 production in lung. PH was also associated with STAT3 (signal transducer and activator of transcription 3) activation and increased proliferation of PA smooth muscle cells, which contributes to remodeling of distal PA. We reported macrophage accumulation, increased IL-6 levels, and STAT3 activation in the lung of group 2 PH patients. In vitro, IL-6 activates STAT3 and induces human PA smooth muscle cell proliferation. Metformin treatment decreased inflammation, IL-6 levels, STAT3 activation, and human PA smooth muscle cell proliferation. In vivo, in the supracoronary aortic banding+MetS animals, reducing IL-6, either by anti-IL-6 antibody or metformin treatment, reversed pulmonary vascular remodeling and improve PH due to LHD. CONCLUSIONS: We developed a new preclinical model of group 2 PH by combining MetS with LHD. We showed that MetS exacerbates group 2 PH. We provided evidence for the importance of the IL-6-STAT3 pathway in our experimental model of group 2 PH and human patients.


Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/patologia , Síndrome Metabólica/complicações , Disfunção Ventricular/complicações , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Masculino , Síndrome Metabólica/etiologia , Olanzapina/toxicidade , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos Wistar , Remodelação Vascular
8.
Arterioscler Thromb Vasc Biol ; 39(8): 1667-1681, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31092016

RESUMO

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of distal pulmonary arteries leading to elevation of pulmonary artery pressure, right ventricular hypertrophy, and death. Although presenting high levels of DNA damage that normally jeopardize their viability, pulmonary artery smooth muscle cells (PASMCs) from patients with PAH exhibit a cancer-like proproliferative and apoptosis-resistant phenotype accounting for vascular lumen obliteration. In cancer cells, overexpression of the serine/threonine-protein kinase CHK1 (checkpoint kinase 1) is exploited to counteract the excess of DNA damage insults they are exposed to. This study aimed to determine whether PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome DNA damage, allowing cell survival and proliferation. Approach and Results: We demonstrated that CHK1 expression is markedly increased in isolated PASMCs and distal PAs from patients with PAH compared with controls, as well as in multiple complementary animal models recapitulating the disease, including monocrotaline rats and the simian immunodeficiency virus-infected macaques. Using a pharmacological and molecular loss of function approach, we showed that CHK1 promotes PAH-PASMCs proliferation and resistance to apoptosis. In addition, we found that inhibition of CHK1 induces downregulation of the DNA repair protein RAD 51 and severe DNA damage. In vivo, we provided evidence that pharmacological inhibition of CHK1 significantly reduces vascular remodeling and improves hemodynamic parameters in 2 experimental rat models of PAH. CONCLUSIONS: Our results show that CHK1 exerts a proproliferative function in PAH-PASMCs by mitigating DNA damage and suggest that CHK1 inhibition may, therefore, represent an attractive therapeutic option for patients with PAH.


Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Hipertensão Arterial Pulmonar/tratamento farmacológico , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/fisiologia , Dano ao DNA , Modelos Animais de Doenças , Humanos , Masculino , MicroRNAs/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Sprague-Dawley
9.
Am J Respir Crit Care Med ; 200(7): 910-920, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042405

RESUMO

Rationale: Pulmonary arterial hypertension (PAH) is a degenerative arteriopathy that leads to right ventricular (RV) failure. BRD4 (bromodomain-containing protein 4), a member of the BET (bromodomain and extra-terminal motif) family, has been identified as a critical epigenetic driver for cardiovascular diseases.Objectives: To explore the therapeutic potential in PAH of RVX208, a clinically available BET inhibitor.Methods: Microvascular endothelial cells, smooth muscle cells isolated from distal pulmonary arteries of patients with PAH, rats with Sugen5416 + hypoxia- or monocrotaline + shunt-induced PAH, and rats with RV pressure overload induced by pulmonary artery banding were treated with RVX208 in three independent laboratories.Measurements and Main Results: BRD4 is upregulated in the remodeled pulmonary vasculature of patients with PAH, where it regulates FoxM1 and PLK1, proteins implicated in the DNA damage response. RVX208 normalized the hyperproliferative, apoptosis-resistant, and inflammatory phenotype of microvascular endothelial cells and smooth muscle cells isolated from patients with PAH. Oral treatment with RVX208 reversed vascular remodeling and improved pulmonary hemodynamics in two independent trials in Sugen5416 + hypoxia-PAH and in monocrotaline + shunt-PAH. RVX208 could be combined safely with contemporary PAH standard of care. RVX208 treatment also supported the pressure-loaded RV in pulmonary artery banding rats.Conclusions: RVX208, a clinically available BET inhibitor, modulates proproliferative, prosurvival, and proinflammatory pathways, potentially through interactions with FoxM1 and PLK1. This reversed the PAH phenotype in isolated PAH microvascular endothelial cells and smooth muscle cells in vitro, and in diverse PAH rat models. RVX208 also supported the pressure-loaded RV in vivo. Together, these data support the establishment of a clinical trial with RVX208 in patients with PAH.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , Quinazolinonas/farmacologia , Fatores de Transcrição/metabolismo , Remodelação Vascular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Reparo do DNA , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Proteína Forkhead Box M1/genética , Regulação da Expressão Gênica , Humanos , Inflamação , Microvasos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/citologia , Ratos , Fatores de Transcrição/antagonistas & inibidores , Quinase 1 Polo-Like
10.
Am J Respir Crit Care Med ; 198(1): 90-103, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29394093

RESUMO

RATIONALE: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival. OBJECTIVES: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis. METHODS: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats. MEASUREMENTS AND MAIN RESULTS: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat). CONCLUSIONS: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.


Assuntos
Anti-Hipertensivos/uso terapêutico , Proteínas de Choque Térmico HSP90/análise , Proteínas de Choque Térmico HSP90/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Mitocôndrias/metabolismo , Remodelação Vascular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Ratos
11.
J Mol Med (Berl) ; 96(2): 223-235, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29290032

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive vascular remodeling disease characterized by a persistent elevation of pulmonary artery pressure, leading to right heart failure and premature death. Exaggerated proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is a key component of vascular remodeling. Despite major advances in the field, current therapies for PAH remain poorly effective in reversing the disease or significantly improving long-term survival. Because the transcription factor FOXM1 is necessary for PASMC proliferation during lung morphogenesis and its overexpression stimulates proliferation and evasion of apoptosis in cancer cells, we thus hypothesized that upregulation of FOXM1 in PAH-PASMCs promotes cell expansion and vascular remodeling. Our results showed that FOXM1 was markedly increased in distal pulmonary arteries and isolated PASMCs from PAH patients compared to controls as well as in two preclinical models. In vitro, we showed that miR-204 expression regulates FOXM1 levels and that inhibition of FOXM1 reduced cell proliferation and resistance to apoptosis through diminished DNA repair mechanisms and decreased expression of the pro-remodeling factor survivin. Accordingly, inhibition of FOXM1 with thiostrepton significantly improved established PAH in two rat models. Thus, we show for the first time that FOXM1 is implicated in PAH development and represents a new promising target. KEY MESSAGES: FOXM1 is overexpressed in human PAH-PASMCs and PAH animal models. FOXM1 promotes PAH-PASMC proliferation and resistance to apoptosis. Pharmacological inhibition of FOXM1 improves established PAH in the MCT and Su/Hx rat models. FOXM1 may be a novel therapeutic target in PAH.


Assuntos
Proteína Forkhead Box M1/fisiologia , Hipertensão Pulmonar/metabolismo , Miócitos de Músculo Liso/fisiologia , Artéria Pulmonar/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Proteína Forkhead Box M1/antagonistas & inibidores , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertrofia Ventricular Direita/metabolismo , Masculino , MicroRNAs/metabolismo , Artéria Pulmonar/citologia , Ratos Sprague-Dawley , Tioestreptona/uso terapêutico , Remodelação Vascular
12.
Sci Rep ; 7(1): 4546, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28674407

RESUMO

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with limited therapeutic options. Although exposed to stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) exhibit a "cancer-like" pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase regulating multiple pro-survival mechanisms and overexpressed in response to stress in cancer cells. Due to the similarities between cancer and PAH, we hypothesized that HDAC6 expression is increased in PAH-PASMCs to face stress allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH. We found that HDAC6 is significantly up-regulated in lungs, distal PAs, and isolated PASMCs from PAH patients and animal models. Inhibition of HDAC6 reduced PAH-PASMC proliferation and resistance to apoptosis in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 maintains Ku70 in a hypoacetylated state, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo, pharmacological inhibition of HDAC6 improved established PAH in two experimental models and can be safely given in combination with currently approved PAH therapies. Moreover, Hdac6 deficient mice were partially protected against chronic hypoxia-induced pulmonary hypertension. Our study shows for the first time that HDAC6 is implicated in PAH development and represents a new promising target to improve PAH.


Assuntos
Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Acetilação , Animais , Apoptose/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Imuno-Histoquímica , Autoantígeno Ku/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Miócitos de Músculo Liso/metabolismo , Ratos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
13.
Pulm Circ ; 7(2): 285-299, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28597757

RESUMO

Pulmonary arterial hypertension (PAH) remains a mysterious killer that, like cancer, is characterized by tremendous complexity. PAH development occurs under sustained and persistent environmental stress, such as inflammation, shear stress, pseudo-hypoxia, and more. After inducing an initial death of the endothelial cells, these environmental stresses contribute with time to the development of hyper-proliferative and apoptotic resistant clone of cells including pulmonary artery smooth muscle cells, fibroblasts, and even pulmonary artery endothelial cells allowing vascular remodeling and PAH development. Molecularly, these cells exhibit many features common to cancer cells offering the opportunity to exploit therapeutic strategies used in cancer to treat PAH. In this review, we outline the signaling pathways and mechanisms described in cancer that drive PAH cells' survival and proliferation and discuss the therapeutic potential of antineoplastic drugs in PAH.

14.
Arterioscler Thromb Vasc Biol ; 37(8): 1513-1523, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28473439

RESUMO

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a vascular disease not restricted to the lungs. Many signaling pathways described in PAH are also of importance in other vascular remodeling diseases, such as coronary artery disease (CAD). Intriguingly, CAD is 4× more prevalent in PAH compared with the global population, suggesting a link between these 2 diseases. Both PAH and CAD are associated with sustained inflammation and smooth muscle cell proliferation/apoptosis imbalance and we demonstrated in PAH that this phenotype is, in part, because of the miR-223/DNA damage/Poly[ADP-ribose] polymerase 1/miR-204 axis activation and subsequent bromodomain protein 4 (BRD4) overexpression. Interestingly, BRD4 is also a trigger for calcification and remodeling processes, both of which are important in CAD. Thus, we hypothesize that BRD4 activation in PAH influences the development of CAD. APPROACH AND RESULTS: PAH was associated with significant remodeling of the coronary arteries in both human and experimental models of the disease. As observed in PAH distal pulmonary arteries, coronary arteries of patients with PAH also exhibited increased DNA damage, inflammation, and BRD4 overexpression. In vitro, using human coronary artery smooth muscle cells from PAH, CAD and non-PAH-non-CAD patients, we showed that both PAH and CAD smooth muscle cells exhibited increased proliferation and suppressed apoptosis in a BRD4-dependent manner. In vivo, improvement of PAH by BRD4 inhibitor was associated with a reduction in coronary remodeling and interleukin-6 expression. CONCLUSIONS: Overall, this study demonstrates that increased BRD4 expression in coronary arteries of patient with PAH contributes to vascular remodeling and comorbidity development.


Assuntos
Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Epigênese Genética , Hipertensão Pulmonar/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Remodelação Vascular , Animais , Apoptose , Estudos de Casos e Controles , Proteínas de Ciclo Celular , Proliferação de Células , Células Cultivadas , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Dano ao DNA , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Interleucina-6/genética , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/genética , Fenótipo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Remodelação Vascular/genética
15.
Int J Mol Sci ; 17(6)2016 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-27338373

RESUMO

Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis.


Assuntos
Dano ao DNA , Hipertensão Pulmonar/metabolismo , Animais , Reparo do DNA , Genes Mitocondriais , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Estresse Oxidativo
16.
Am J Respir Crit Care Med ; 194(10): 1273-1285, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27149112

RESUMO

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This is sustained in time by the down-regulation of microRNA (miR)-204. In systemic vascular diseases, reduced miR-204 expression promotes vascular biomineralization by augmenting the expression of the transcription factor Runt-related transcription factor 2 (RUNX2). Implication of RUNX2 in PAH-related vascular remodeling and presence of calcified lesions in PAH remain unexplored. OBJECTIVES: We hypothesized that RUNX2 is up-regulated in lungs of patients with PAH, contributing to vascular remodeling and calcium-related biomineralization. METHODS: We harvested human lung tissues in which we assessed calcification lesions and RUNX2 expression. We also isolated PASMCs from these tissues for in vitro analyses. Using a bidirectional approach, we investigated the role for RUNX2 in cell proliferation, apoptosis, and calcification capacity. Ectopic delivery of small interfering RNA against RUNX2 was used in an animal model of PAH to evaluate the therapeutic potential of RUNX2 inhibition in this disease. MEASUREMENTS AND MAIN RESULTS: Patients with PAH display features of calcified lesions within the distal pulmonary arteries (PAs). We show that RUNX2 is up-regulated in lungs, distal PAs, and primary cultured human PASMCs isolated from PAH and compared with patients without PAH. RUNX2 expression histologically correlates with vascular remodeling and calcification. Using in vitro gain- and loss-of-function approaches, we mechanistically demonstrate that miR-204 diminution promotes RUNX2 up-regulation and that sustained RUNX2 expression activates hypoxia-inducible factor-1α, leading to aberrant proliferation, resistance to apoptosis, and subsequent transdifferentiation of PAH-PASMCs into osteoblast-like cells. In the PAH Sugen/hypoxia rat model, molecular RUNX2 inhibition reduces PA remodeling and prevents calcification, thus improving pulmonary hemodynamic parameters and right ventricular function. CONCLUSIONS: RUNX2 plays a pivotal role in the pathogenesis of PAH, contributing to the development of proliferative and calcified PA lesions. Inhibition of RUNX2 may therefore represent an attractive therapeutic strategy for PAH.


Assuntos
Proliferação de Células/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Calcificação Vascular/genética , Calcificação Vascular/fisiopatologia , Adulto , Proliferação de Células/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
17.
Eur Urol ; 69(4): 734-744, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26433571

RESUMO

BACKGROUND: Clear-cell renal cell carcinoma (ccRCC) exhibits suppressed mitochondrial function and preferential use of glycolysis even in normoxia, promoting proliferation and suppressing apoptosis. ccRCC resistance to therapy is driven by constitutive hypoxia-inducible factor (HIF) expression due to genetic loss of von Hippel-Lindau factor. In addition to promoting angiogenesis, HIF suppresses mitochondrial function by inducing pyruvate dehydrogenase kinase (PDK), a gatekeeping enzyme for mitochondrial glucose oxidation. OBJECTIVE: To reverse mitochondrial suppression of ccRCC using the PDK inhibitor dichloroacetate (DCA). DESIGN, SETTING, AND PARTICIPANTS: Radical nephrectomy specimens from patients with ccRCC were assessed for PDK expression. The 786-O ccRCC line and two animal models (chicken in ovo and murine xenografts) were used for mechanistic studies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Mitochondrial function, proliferation, apoptosis, HIF transcriptional activity, angiogenesis, and tumor size were measured in vitro and in vivo. Independent-sample t-tests and analysis of variance were used for statistical analyses. RESULTS: PDK was elevated in 786-O cells and in ccRCC compared to normal kidney tissue from the same patient. DCA reactivated mitochondrial function (increased respiration, Krebs cycle metabolites such as α-ketoglutarate [cofactor of factor inhibiting HIF], and mitochondrial reactive oxygen species), increased p53 activity and apoptosis, and decreased proliferation in 786-O cells. DCA reduced HIF transcriptional activity in an FIH-dependent manner, inhibiting angiogenesis in vitro. DCA reduced tumor size and angiogenesis in vivo in both animal models. CONCLUSIONS: DCA can reverse the mitochondrial suppression of ccRCC and decrease HIF transcriptional activity, bypassing its constitutive expression. Its previous clinical use in humans makes it an attractive candidate for translation to ccRCC patients. PATIENT SUMMARY: We show that an energy-boosting drug decreases tumor growth and tumor blood vessels in animals carrying human kidney cancer cells. This generic drug has been used in patients for other conditions and thus could be tested in kidney cancer that remains incurable.


Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Ácido Dicloroacético/farmacologia , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Renais/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos Nus , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Neovascularização Patológica , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
PLoS One ; 10(3): e0120257, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25768019

RESUMO

Successful stem cell therapy requires the optimal proliferation, engraftment, and differentiation of stem cells into the desired cell lineage of tissues. However, stem cell therapy clinical trials to date have had limited success, suggesting that a better understanding of stem cell biology is needed. This includes a better understanding of stem cell energy metabolism because of the importance of energy metabolism in stem cell proliferation and differentiation. We report here the first direct evidence that human bone marrow mesenchymal stem cell (BMMSC) energy metabolism is highly glycolytic with low rates of mitochondrial oxidative metabolism. The contribution of glycolysis to ATP production is greater than 97% in undifferentiated BMMSCs, while glucose and fatty acid oxidation combined only contribute 3% of ATP production. We also assessed the effect of physiological levels of fatty acids on human BMMSC survival and energy metabolism. We found that the saturated fatty acid palmitate induces BMMSC apoptosis and decreases proliferation, an effect prevented by the unsaturated fatty acid oleate. Interestingly, chronic exposure of human BMMSCs to physiological levels of palmitate (for 24 hr) reduces palmitate oxidation rates. This decrease in palmitate oxidation is prevented by chronic exposure of the BMMSCs to oleate. These results suggest that reducing saturated fatty acid oxidation can decrease human BMMSC proliferation and cause cell death. These results also suggest that saturated fatty acids may be involved in the long-term impairment of BMMSC survival in vivo.


Assuntos
Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Células-Tronco Mesenquimais/fisiologia , Análise de Variância , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Imunofluorescência , Glicólise/fisiologia , Humanos , Mitocôndrias/fisiologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Sais de Tetrazólio , Tiazóis
20.
Circ Res ; 116(1): 56-69, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25287062

RESUMO

RATIONALE: Right ventricular (RV) failure is a major cause of morbidity and mortality in pulmonary hypertension, but its mechanism remains unknown. Myocyte enhancer factor 2 (Mef2) has been implicated in RV development, regulating metabolic, contractile, and angiogenic genes. Moreover, Mef2 regulates microRNAs that have emerged as important determinants of cardiac development and disease, but for which the role in RV is still unclear. OBJECTIVE: We hypothesized a critical role of a Mef2-microRNAs axis in RV failure. METHODS AND RESULTS: In a rat pulmonary hypertension model (monocrotaline), we studied RV free wall tissues from rats with normal, compensated, and decompensated RV hypertrophy, carefully defined based on clinically relevant parameters, including RV systolic and end-diastolic pressures, cardiac output, RV size, and morbidity. Mef2c expression was sharply increased in compensating phase of RVH tissues but was lost in decompensation phase of RVH. An unbiased screening of microRNAs in our model resulted to a short microRNA signature of decompensated RV failure, which included the myocardium-specific miR-208, which was progressively downregulated as RV failure progressed, in contrast to what is described in left ventricular failure. With mechanistic in vitro experiments using neonatal and adult RV cardiomyocytes, we showed that miR-208 inhibition, as well as tumor necrosis factor-α, activates the complex mediator of transcription 13/nuclear receptor corepressor 1 axis, which in turn promotes Mef2 inhibition, closing a self-limiting feedback loop, driving the transition from compensating phase of RVH toward decompensation phase of RVH. In our model, serum tumor necrosis factor-α levels progressively increased with time while serum miR-208 levels decreased, mirroring its levels in RV myocardium. CONCLUSIONS: We describe an RV-specific mechanism for heart failure, which could potentially lead to new biomarkers and therapeutic targets.


Assuntos
Insuficiência Cardíaca/metabolismo , Hipertensão Pulmonar/metabolismo , Fatores de Transcrição MEF2/biossíntese , MicroRNAs/biossíntese , Função Ventricular Direita/fisiologia , Animais , Células Cultivadas , Insuficiência Cardíaca/patologia , Hipertensão Pulmonar/patologia , Masculino , Miócitos Cardíacos/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
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