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Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.
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Hipertensão Pulmonar , Quinase I-kappa B , Hipertensão Arterial Pulmonar , Remodelação Vascular , Animais , Humanos , Ratos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Quinase I-kappa B/metabolismo , Miócitos de Músculo Liso , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar , Proteínas de Sinalização YAP/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismoRESUMO
Increased proliferation and survival of cells in small pulmonary arteries (PAs) drive pulmonary arterial hypertension (PAH). Because cell growth mediated by the mTOR-containing mTORC1 complex is inhibited by tuberous sclerosis complex 2 (TSC2), we investigated the role of this GTPase-activating protein in PAH pathology. TSC2 abundance was decreased in remodeled small PAs and PA vascular smooth muscle cells (PAVSMCs) from patients with PAH or from rodent pulmonary hypertension (PH) models, as well as PAVSMCs maintained on substrates that reproduced pathology-induced stiffness. Accordingly, mice with smooth muscle-specific reduction in TSC2 developed PH. At the molecular level, decreased TSC2 abundance led to stiffness-induced PAVSMC proliferation, increased abundance of the mechanosensitive transcriptional coactivators YAP/TAZ, and enhanced mTOR kinase activity. Moreover, extracellular matrix (ECM) produced by TSC2-deficient PAVSMCs stimulated the proliferation of nondiseased PA adventitial fibroblasts and PAVSMCs through fibronectin and its receptor, the α5ß1 integrin. Reconstituting TSC2 in PAVSMCs from patients with PAH through overexpression or treatment with the SIRT1 activator SRT2104 decreased YAP/TAZ abundance, mTOR activity, and ECM production, as well as inhibited proliferation and induced apoptosis. In two rodent models of PH, SRT2104 treatment restored TSC2 abundance, attenuated pulmonary vascular remodeling, and ameliorated PH. Thus, TSC2 in PAVSMCs integrates ECM composition and stiffness with pro-proliferative and survival signaling, and restoring TSC2 abundance could be an attractive therapeutic option to treat PH.
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Hipertensão Pulmonar , Esclerose Tuberosa , Animais , Camundongos , Proliferação de Células , Matriz Extracelular , Hipertensão Pulmonar/genética , HumanosRESUMO
Aging is accompanied by declining lung function and increasing susceptibility to lung diseases. The role of endothelial dysfunction and vascular remodeling in these changes is supported by growing evidence, but underlying mechanisms remain elusive. In this review we summarize functional, structural, and molecular changes in the aging pulmonary vasculature and explore how interacting aging and mechanobiological cues may drive progressive vascular remodeling in the lungs.
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Doenças Vasculares , Remodelação Vascular , Envelhecimento , Biofísica , Humanos , PulmãoRESUMO
Rationale: Mechanical signaling through cell-matrix interactions plays a major role in progressive vascular remodeling in pulmonary arterial hypertension (PAH). MMP-8 (matrix metalloproteinase-8) is an interstitial collagenase involved in regulating inflammation and fibrosis of the lung and systemic vasculature, but its role in PAH pathogenesis remains unexplored. Objectives: To evaluate MMP-8 as a modulator of pathogenic mechanical signaling in PAH. Methods: MMP-8 levels were measured in plasma from patients with pulmonary hypertension (PH) and controls by ELISA. MMP-8 vascular expression was examined in lung tissue from patients with PAH and rodent models of PH. MMP-8-/- and MMP-8+/+ mice were exposed to normobaric hypoxia or normoxia for 4-8 weeks. PH severity was evaluated by right ventricular systolic pressure, echocardiography, pulmonary artery morphometry, and immunostaining. Proliferation, migration, matrix component expression, and mechanical signaling were assessed in MMP-8-/- and MMP-8+/+ pulmonary artery smooth muscle cells (PASMCs). Measurements and Main Results: MMP-8 expression was significantly increased in plasma and pulmonary arteries of patients with PH compared with controls and induced in the pulmonary vasculature in rodent PH models. Hypoxia-exposed MMP-8-/- mice had significant mortality, increased right ventricular systolic pressure, severe right ventricular dysfunction, and exaggerated vascular remodeling compared with MMP-8+/+ mice. MMP-8-/- PASMCs demonstrated exaggerated proliferation and migration mediated by altered matrix protein expression, elevated integrin-ß3 levels, and induction of FAK (focal adhesion kinase) and downstream YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) activity. Conclusions: MMP-8 is a novel protective factor upregulated in the pulmonary vasculature during PAH pathogenesis. MMP-8 opposes pathologic mechanobiological feedback by altering matrix composition and disrupting integrin-ß3/FAK and YAP/TAZ-dependent mechanical signaling in PASMCs.
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Metaloproteinase 8 da Matriz/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Metaloproteinase 8 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/prevenção & controle , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima , Remodelação VascularRESUMO
Low-dose inhaled carbon monoxide is a novel therapeutic under investigation in acute respiratory distress syndrome. The Coburn-Forster-Kane equation is a well-validated model of carbon monoxide uptake that can accurately predict carboxyhemoglobin levels to ensure safe administration of low-dose inhaled carbon monoxide in patients with acute respiratory distress syndrome. Using data from a Phase I trial of low-dose inhaled carbon monoxide, we performed a post hoc analysis to determine if the Coburn-Forster-Kane equation could be used to assess the diffusing capacity of the lung for carbon monoxide and endogenous carbon monoxide production in patients with sepsis-induced acute respiratory distress syndrome. Diffusing capacity of the lung for carbon monoxide was substantially reduced and correlated with Pao2/Fio2 and Sequential Organ Failure Assessment score. Endogenous carbon monoxide production was markedly elevated and was significantly associated with Lung Injury Score in sepsis-induced acute respiratory distress syndrome patients. Our data suggest that the Coburn-Forster-Kane equation can be used to estimate diffusing capacity of the lung for carbon monoxide and endogenous carbon monoxide production in mechanically ventilated patients. We found that increased endogenous carbon monoxide production and reduced diffusing capacity of the lung for carbon monoxide correlate with clinical endpoints associated with outcomes in patients with sepsis-induced acute respiratory distress syndrome.
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Airway epithelial homeostasis is under constant threat due to continuous exposure to the external environment, and abnormally robust sensitivity to external stimuli is critical to the development of airway diseases, including asthma. Ku is a key nonhomologous end-joining DNA repair protein with diverse cellular functions such as VDJ recombination and telomere length maintenance. Here, we show a novel function of Ku in alleviating features of allergic airway inflammation via the regulation of mitochondrial and endoplasmic reticulum (ER) stress. We first determined that airway epithelial cells derived from both asthmatic lungs and murine asthma models demonstrate increased expression of 8-hydroxy-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage. Ku protein expression was dramatically reduced in the bronchial epithelium of patients with asthma as well as in human bronchial epithelial cells exposed to oxidative stress. Knockdown of Ku70 or Ku80 in naïve mice elicited mitochondrial collapse or ER stress, leading to bronchial epithelial cell apoptosis and spontaneous development of asthma-like features, including airway hyperresponsiveness, airway inflammation, and subepithelial fibrosis. These findings demonstrate an essential noncanonical role for Ku proteins in asthma pathogenesis, likely via maintenance of organelle homeostasis. This novel function of Ku proteins may also be important in other disease processes associated with organelle stress.
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Células Epiteliais/metabolismo , Homeostase/fisiologia , Inflamação/prevenção & controle , Autoantígeno Ku/metabolismo , Animais , Asma/patologia , Asma/prevenção & controle , Estresse do Retículo Endoplasmático/fisiologia , Células Epiteliais/patologia , Humanos , Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Estresse Oxidativo/fisiologia , Hipersensibilidade Respiratória/patologiaRESUMO
BACKGROUND: Respiratory pathology is a major driver of mortality in the intensive care unit (ICU), even in the absence of a primary respiratory diagnosis. Prior work has demonstrated that a visual scoring system applied to chest radiographs (CXR) is associated with adverse outcomes in ICU patients with Acute Respiratory Distress Syndrome (ARDS). We hypothesized that a simple, semi-quantitative CXR score would be associated with clinical outcomes for the general ICU population, regardless of underlying diagnosis. METHODS: All individuals enrolled in the Registry of Critical Illness at Brigham and Women's Hospital between June 2008 and August 2018 who had a CXR within 24 h of admission were included. Each patient's CXR was assigned an opacification score of 0-4 in each of four quadrants with the total score being the sum of all four quadrants. Multivariable negative binomial, logistic, and Cox regression, adjusted for age, sex, race, immunosuppression, a history of chronic obstructive pulmonary disease, a history of congestive heart failure, and APACHE II scores, were used to assess the total score's association with ICU length of stay (LOS), duration of mechanical ventilation, in-hospital mortality, 60-day mortality, and overall mortality, respectively. RESULTS: A total of 560 patients were included. Higher CXR scores were associated with increased mortality; for every one-point increase in score, in-hospital mortality increased 10% (OR 1.10, CI 1.05-1.16, p < 0.001) and 60-day mortality increased by 12% (OR 1.12, CI 1.07-1.17, p < 0.001). CXR scores were also independently associated with both ICU length of stay (rate ratio 1.06, CI 1.04-1.07, p < 0.001) and duration of mechanical ventilation (rate ratio 1.05, CI 1.02-1.07, p < 0.001). CONCLUSIONS: Higher values on a simple visual score of a patient's CXR on admission to the medical ICU are associated with increased in-hospital mortality, 60-day mortality, overall mortality, length of ICU stay, and duration of mechanical ventilation.
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Estado Terminal , Síndrome do Desconforto Respiratório/diagnóstico por imagem , Tórax/diagnóstico por imagem , APACHE , Adulto , Idoso , Biomarcadores/sangue , Feminino , Mortalidade Hospitalar , Humanos , Tempo de Internação , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Respiração Artificial , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/terapia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are causally linked to pulmonary arterial hypertension (PAH) pathogenesis. Carbonic anhydrase inhibition induces mild metabolic acidosis and exerts protective effects in hypoxic pulmonary hypertension. Carbonic anhydrases and metabolic acidosis are further known to modulate immune cell activation. To evaluate if carbonic anhydrase inhibition modulates macrophage activation, inflammation, and VSMC phenotypic switching in severe experimental pulmonary hypertension, pulmonary hypertension was assessed in Sugen 5416/hypoxia (SU/Hx) rats after treatment with acetazolamide or ammonium chloride (NH4Cl). We evaluated pulmonary and systemic inflammation and characterized the effect of carbonic anhydrase inhibition and metabolic acidosis in alveolar macrophages and bone marrow-derived macrophages (BMDMs). We further evaluated the treatment effects on VSMC phenotypic switching in pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) and corroborated some of our findings in lungs and pulmonary arteries of patients with PAH. Both patients with idiopathic PAH and SU/Hx rats had increased expression of lung inflammatory markers and signs of PASMC dedifferentiation in pulmonary arteries. Acetazolamide and NH4Cl ameliorated SU/Hx-induced pulmonary hypertension and blunted pulmonary and systemic inflammation. Expression of carbonic anhydrase isoform 2 was increased in alveolar macrophages from SU/Hx animals, classically (M1) and alternatively (M2) activated BMDMs, and lungs of patients with PAH. Carbonic anhydrase inhibition and acidosis had distinct effects on M1 and M2 markers in BMDMs. Inflammatory cytokines drove PASMC dedifferentiation, and this was inhibited by acetazolamide and acidosis. The protective antiinflammatory effect of acetazolamide in pulmonary hypertension is mediated by a dual mechanism of macrophage carbonic anhydrase inhibition and systemic metabolic acidosis.
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Acetazolamida/uso terapêutico , Cloreto de Amônio/uso terapêutico , Inibidores da Anidrase Carbônica/uso terapêutico , Anidrases Carbônicas/fisiologia , Hipertensão Pulmonar/tratamento farmacológico , Acidose/induzido quimicamente , Acidose/complicações , Acidose/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proteínas Contráteis/biossíntese , Proteínas Contráteis/genética , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/enzimologia , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Isoformas de Proteínas/antagonistas & inibidores , Artéria Pulmonar/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The acute respiratory distress syndrome (ARDS) is characterized by the acute onset of hypoxemia and bilateral lung infiltrates in response to an inciting event, and is associated with high morbidity and mortality. Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for ARDS. We hypothesized that HSCT patients with ARDS would have a unique transcriptomic profile identifiable in peripheral blood compared to those that did not undergo HSCT. METHODS: We isolated RNA from banked peripheral blood samples from a biorepository of critically ill ICU patients. RNA-Seq was performed on 11 patients with ARDS (5 that had undergone HSCT and 6 that had not) and 12 patients with sepsis without ARDS (5 that that had undergone HCST and 7 that had not). RESULTS: We identified 687 differentially expressed genes between ARDS and ARDS-HSCT (adjusted p-value < 0.01), including IFI44L, OAS3, LY6E, and SPATS2L that had increased expression in ARDS vs. ARDS-HSCT; these genes were not differentially expressed in sepsis vs sepsis-HSCT. Gene ontology enrichment analysis revealed that many differentially expressed genes were related to response to type I interferon. CONCLUSIONS: Our findings reveal significant differences in whole blood transcriptomic profiles of patients with non-HSCT ARDS compared to ARDS-HSCT patients and point toward different immune responses underlying ARDS and ARDS-HSCT that contribute to lung injury.
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Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/terapia , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Adulto , Feminino , Transplante de Células-Tronco Hematopoéticas/tendências , Humanos , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Síndrome do Desconforto Respiratório/sangue , Análise de Sequência de RNA/tendênciasRESUMO
Cardiac tamponade is a rare cause of shock in the medical intensive care unit. This paper describes the case of a focal cardiac tamponade caused by compression of the left atrium due to an esophageal stent. Echocardiography yielded a diagnosis when other diagnostic methods did not. (Level of Difficulty: Advanced.).
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BACKGROUND: Acute respiratory distress syndrome (ARDS) is a prevalent disease with significant mortality for which no effective pharmacologic therapy exists. Low-dose inhaled carbon monoxide (iCO) confers cytoprotection in preclinical models of sepsis and ARDS. METHODS: We conducted a phase I dose escalation trial to assess feasibility and safety of low-dose iCO administration in patients with sepsis-induced ARDS. Twelve participants were randomized to iCO or placebo air 2:1 in two cohorts. Four subjects each were administered iCO (100 ppm in cohort 1 or 200 ppm in cohort 2) or placebo for 90 minutes for up to 5 consecutive days. Primary outcomes included the incidence of carboxyhemoglobin (COHb) level ≥10%, prespecified administration-associated adverse events (AEs), and severe adverse events (SAEs). Secondary endpoints included the accuracy of the Coburn-Forster-Kane (CFK) equation to predict COHb levels, biomarker levels, and clinical outcomes. RESULTS: No participants exceeded a COHb level of 10%, and there were no administration-associated AEs or study-related SAEs. CO-treated participants had a significant increase in COHb (3.48% ± 0.7% [cohort 1]; 4.9% ± 0.28% [cohort 2]) compared with placebo-treated subjects (1.97% ± 0.39%). The CFK equation was highly accurate at predicting COHb levels, particularly in cohort 2 (R2 = 0.9205; P < 0.0001). Circulating mitochondrial DNA levels were reduced in iCO-treated participants compared with placebo-treated subjects. CONCLUSION: Precise administration of low-dose iCO is feasible, well-tolerated, and appears to be safe in patients with sepsis-induced ARDS. Excellent agreement between predicted and observed COHb should ensure that COHb levels remain in the target range during future efficacy trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02425579. FUNDING: NIH grants P01HL108801, KL2TR002385, K08HL130557, and K08GM102695.
Assuntos
Administração por Inalação , Monóxido de Carbono/administração & dosagem , Síndrome do Desconforto Respiratório/tratamento farmacológico , Terapia Respiratória/métodos , Sepse/tratamento farmacológico , Adulto , Idoso , Biomarcadores/sangue , Gasometria , Carboxihemoglobina , DNA Mitocondrial , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Vascular stiffening in the pulmonary arterial bed is increasingly recognized as an early disease marker and contributor to right ventricular workload in pulmonary hypertension. Changes in pulmonary artery stiffness throughout the pulmonary vascular tree lead to physiologic alterations in pressure and flow characteristics that may contribute to disease progression. These findings have led to a greater focus on the potential contributions of extracellular matrix remodeling and mechanical signaling to pulmonary hypertension pathogenesis. Several recent studies have demonstrated that the cellular response to vascular stiffness includes upregulation of signaling pathways that precipitate further vascular remodeling, a process known as mechanobiological feedback. The extracellular matrix modifiers, mechanosensors, and mechanotransducers responsible for this process have become increasingly well-recognized. In this review, we discuss the impact of vascular stiffening on pulmonary hypertension morbidity and mortality, evidence in favor of mechanobiological feedback in pulmonary hypertension pathogenesis, and the major contributors to mechanical signaling in the pulmonary vasculature.
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Germline mutations involving small mothers against decapentaplegic-transforming growth factor-ß (SMAD-TGF-ß) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-ß ligand trap and, thus, was not contingent on TGF-ß signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-ß-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colágeno Tipo III/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Pulmão/metabolismo , Pulmão/patologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Colágeno Tipo III/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Pulmão/fisiopatologia , Masculino , Fosfoproteínas/genética , Ligação Proteica , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Proteína Smad3/genética , Proteína Smad3/metabolismo , Biologia de Sistemas/métodosRESUMO
Pulmonary arterial stiffness is an independent risk factor for mortality in pulmonary hypertension (PH) and plays a critical role in PH pathophysiology. Our laboratory has recently demonstrated arterial stiffening early in experimental PH, along with evidence for a mechanobiological feedback loop by which arterial stiffening promotes further cellular remodeling behaviors (Liu F, Haeger CM, Dieffenbach PB, Sicard D, Chrobak I, Coronata AM, Suárez Velandia MM, Vitali S, Colas RA, Norris PC, Marinkovic A, Liu X, Ma J, Rose CD, Lee SJ, Comhair SA, Erzurum SC, McDonald JD, Serhan CN, Walsh SR, Tschumperlin DJ, Fredenburgh LE. JCI Insight 1: e86987, 2016). Cyclooxygenase-2 (COX-2) and prostaglandin signaling have been implicated in stiffness-mediated regulation, with prostaglandin activity inversely correlated to matrix stiffness and remodeling behaviors in vitro, as well as to disease progression in rodent PH models. The mechanism by which mechanical signaling translates to reduced COX-2 activity in pulmonary vascular cells is unknown. The present work investigated the transcriptional regulators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1, a.k.a., TAZ), which are known drivers of downstream mechanical signaling, in mediating stiffness-induced changes in COX-2 and prostaglandin activity in pulmonary artery smooth muscle cells (PASMCs). We found that YAP/TAZ activity is increased in PAH PASMCs and experimental PH and is necessary for the development of stiffness-dependent remodeling phenotypes. Knockdown of YAP and TAZ markedly induces COX-2 expression and downstream prostaglandin production by approximately threefold, whereas overexpression of YAP or TAZ reduces COX-2 expression and prostaglandin production to near undetectable levels. Together, our findings demonstrate a stiffness-dependent YAP/TAZ-mediated positive feedback loop that drives remodeling phenotypes in PASMCs via reduced COX-2 and prostaglandin activity. The ability to interrupt this critical mechanobiological feedback loop and enhance local prostaglandin activity via manipulation of YAP/TAZ signaling presents a highly attractive novel strategy for the treatment of PH.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Remodelação das Vias Aéreas/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo-Oxigenase 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Rigidez Vascular/fisiologia , Adulto , Animais , Movimento Celular , Proliferação de Células , Demografia , Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hipertensão Pulmonar , Masculino , Pessoa de Meia-Idade , Fenótipo , Artéria Pulmonar/citologia , Ratos Sprague-Dawley , Transdução de Sinais , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2-derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I2 analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.
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Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.
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Matriz Extracelular/metabolismo , Retroalimentação Fisiológica , Hipertensão Pulmonar/metabolismo , Mecanotransdução Celular , MicroRNAs/genética , Fatores de Transcrição/metabolismo , Animais , Apolipoproteínas E/metabolismo , Matriz Extracelular/patologia , Humanos , Concentração de Íons de Hidrogênio , Hipertensão Pulmonar/patologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genéticaRESUMO
Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4DeltaLZ. Primary neurons from Par-4DeltaLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4DeltaLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.
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Depressão/metabolismo , Dopamina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Receptores de Dopamina D2/fisiologia , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Cálcio/metabolismo , Calmodulina/metabolismo , Células Cultivadas , Corpo Estriado/metabolismo , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Depressão/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Receptores de Dopamina D2/metabolismo , Regulação para CimaRESUMO
DNA microarray analyses were used to investigate the effect of cell-incorporated 35S-methionine on human colorectal carcinoma cells. This beta-radiation-induced gene expression profile was compared with that induced by external gamma-radiation. The extent of DNA fragmentation was used as a biomarker to determine the external gamma dose that was bioequivalent to that received by cells incubated in medium containing 35S-methionine. Studies showed that 35S-methionine at 100 microCi/mL induced a much more robust transcriptional response than gamma-radiation (2000 cGy) when evaluated 2 h after the labeling or irradiation period. The cellular response to internal beta-radiation was greater not only with respect to the number of genes induced, but also with respect to the level of gene induction. Not surprisingly, the induced genes overlapped with the set of gamma-responsive genes. However, a distinct beta-gene induction profile that included a large number of cell adhesion proteins was also observed. Taken together, these studies demonstrate that metabolic incorporation of a low energy beta-emitter, such as 35S-methionine, can globally influence a diverse set of cellular activities that can, in turn, affect the outcome of many experiments by altering the cell cycle, metabolic, signaling, or redox status (set point) of the cell. Additional studies of the mechanism of beta-induced proliferation arrest and cell death and of the significance of its differential gene induction/repression profile in comparison to pulsed gamma-irradiation may lead to new insights into the ways in which ionizing radiation can interact with cells.