RESUMO
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
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Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.
Assuntos
Hipertensão Arterial Pulmonar , Transdução de Sinais , Remodelação Vascular , Animais , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/fisiologia , Ratos , Humanos , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Masculino , Modelos Animais de Doenças , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Camundongos , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A , Proliferação de Células/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/metabolismo , Lisina/análogos & derivadosRESUMO
BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.
Assuntos
Insuficiência Cardíaca , Hipertensão Arterial Pulmonar , Ratos , Camundongos , Animais , Remodelação Ventricular , beta Catenina , Cateninas , Monocrotalina/toxicidade , Transdução de Sinais , Modelos Animais de Doenças , Função Ventricular DireitaRESUMO
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 PulmonarRESUMO
Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.
Assuntos
Hipertensão Pulmonar , Animais , Cromatina/metabolismo , Feto/metabolismo , Humanos , Pulmão/patologia , Camundongos , Artéria Pulmonar/patologia , Interferência de RNA , Fatores de Transcrição/metabolismo , Remodelação Vascular/genéticaRESUMO
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.
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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/metabolismoRESUMO
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.
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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 , RatosRESUMO
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/metabolismoRESUMO
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 CimaRESUMO
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.
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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 VascularRESUMO
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-LikeRESUMO
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-DawleyRESUMO
Pulmonary arterial hypertension (PAH) encompasses a group of clinical entities characterized by sustained vasoconstriction and progressive vascular remodeling that act in concert to elevate pulmonary vascular resistance. The current treatments for PAH are mainly dedicated to target the process of vasoconstriction and do not offer a cure. There is now accumulating evidence that expansion of pulmonary artery cells due to increased proliferation and apoptotic evasion is a key pathological component of vascular remodeling that occurs in PAH. Thus, vascular lesions seen in advanced PAH patients present some cancer-like characteristics offering important avenues for exploration and expanding treatment options. In this review article, we will discuss recent advances into mechanisms underlying disease progression, with a focus on pulmonary artery smooth muscle cells.
Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Terapia de Alvo Molecular/métodos , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/imunologiaRESUMO
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 , RatosRESUMO
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 VascularRESUMO
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/metabolismoRESUMO
BACKGROUND: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). METHODS AND RESULTS: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. CONCLUSIONS: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.
Assuntos
Hipertensão Pulmonar/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Túnica Adventícia/patologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Divisão Celular , Endotélio Vascular/patologia , Fibroblastos/patologia , Predisposição Genética para Doença , Hemodinâmica , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/genética , Hipertrofia Ventricular Direita/etiologia , Inflamação , Masculino , Potenciais da Membrana , Monocrotalina/toxicidade , Mutação , Miócitos de Músculo Liso/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/biossíntese , Canais de Potássio de Domínios Poros em Tandem/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sulfonamidas/farmacologia , Resistência Vascular , ortoaminobenzoatos/farmacologiaRESUMO
RATIONALE: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Decreased expression of microRNA-204 has been associated to this phenotype. By a still elusive mechanism, microRNA-204 downregulation promotes the expression of oncogenes, including nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. In cancer, increased expression of the epigenetic reader bromodomain-containing protein 4 (BRD4) sustains cell survival and proliferation. Interestingly, BRD4 is a predicted target of microRNA-204 and has binding sites on the nuclear factor of activated T cells promoter region. OBJECTIVE: To investigate the role of BRD4 in PAH pathogenesis. METHODS AND RESULTS: BRD4 is upregulated in lungs, distal PAs, and PASMCs of patients with PAH compared with controls. With mechanistic in vitro experiments, we demonstrated that BRD4 expression in PAH is microRNA-204 dependent. We further studied the molecular downstream targets of BRD4 by inhibiting its activity in PAH-PASMCs using a clinically available inhibitor JQ1. JQ1 treatment in PAH-PASMCs increased p21 expression, thus triggering cell cycle arrest. Furthermore, BRD4 inhibition, by JQ1 or siBRD4, decreased the expression of 3 major oncogenes, which are overexpressed in PAH: nuclear factor of activated T cells, B-cell lymphoma 2, and Survivin. Blocking this oncogenic signature led to decreased PAH-PASMC proliferation and increased apoptosis in a BRD4-dependent manner. Indeed, pharmacological JQ1 or molecular (siRNA) inhibition of BRD4 reversed this pathological phenotype in addition to restoring mitochondrial membrane potential and to increasing cells spare respiratory capacity. Moreover, BRD4 inhibition in vivo reversed established PAH in the Sugen/hypoxia rat model. CONCLUSIONS: BRD4 plays a key role in the pathological phenotype in PAH, which could offer new therapeutic perspectives for patients with PAH.