RESUMEN
Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. cAbl (non-receptor tyrosine kinase c-Abelson) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is downactivated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from patients with PAH (PAH-ECs). We found cAbl and phosphorylated cAbl concentrations to be lower in the endothelium of remodeled pulmonary vessels in the lungs of patients with PAH than in control subjects. Similar observations were obtained for the lungs of Sugen + hypoxia and monocrotaline rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA interference in control ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with 5-(1,3-diaryl-1H-pyrazol-4-yl) hydantoin reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of cross-talk between cAbl and bone morphogenetic protein receptor type II. This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.
Asunto(s)
Células Endoteliales , Hipertensión Arterial Pulmonar , Animales , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Humanos , Monocrotalina , Proteínas Tirosina Quinasas/metabolismo , Arteria Pulmonar/metabolismo , RatasRESUMEN
Previous studies have suggested an association between uric acid (UA) and the severity of pulmonary arterial hypertension (PAH), but it is unknown whether UA contributes to disease pathogenesis.The aim of this study was to determine the prognostic value of circulating UA in the era of current management of PAH and to investigate the role of UA in pulmonary vascular remodelling.Serum UA levels were determined in idiopathic, heritable or anorexigen PAH at baseline and first re-evaluation in the French Pulmonary Hypertension Network. We studied protein levels of xanthine oxidase (XO) and the voltage-driven urate transporter 1 (URATv1) in lungs of control and PAH patients and of monocrotaline (MCT) and Sugen/hypoxia (SuHx) rats. Functional studies were performed using human pulmonary artery smooth muscle cells (PA-SMCs) and two animal models of pulmonary hypertension (PH).High serum UA levels at first follow-up, but not at baseline, were associated with a poor prognosis. Both the generating enzyme XO and URATv1 were upregulated in the wall of remodelled pulmonary arteries in idiopathic PAH patients and MCT and SuHx rats. High UA concentrations promoted a mild increase in cell growth in idiopathic PAH PA-SMCs, but not in control PA-SMCs. Consistent with these observations, oxonic acid-induced hyperuricaemia did not aggravate MCT-induced PH in rats. Finally, chronic treatment of MCT and SuHx rats with benzbromarone mildly attenuated pulmonary vascular remodelling.UA levels in idiopathic PAH patients were associated with an impaired clinical and haemodynamic profile and might be used as a non-invasive indicator of clinical prognosis during follow-up. Our findings also indicate that UA metabolism is disturbed in remodelled pulmonary vascular walls in both experimental and human PAH.
Asunto(s)
Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Animales , Modelos Animales de Enfermedad , Humanos , Pulmón , Monocrotalina , Arteria Pulmonar , Ratas , Ácido ÚricoRESUMEN
RATIONALE: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. OBJECTIVE: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. METHOD AND RESULTS: The absence of BMP9 in Bmp9-/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9-/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates. CONCLUSIONS: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.
Asunto(s)
Factor 2 de Diferenciación de Crecimiento/antagonistas & inhibidores , Hipertensión Pulmonar/prevención & control , Receptores de Activinas Tipo II/farmacología , Animales , Células Cultivadas , Endotelina-1/genética , Factor 2 de Diferenciación de Crecimiento/fisiología , Humanos , Hipoxia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas WistarRESUMEN
The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43+/-) and wild-type littermate (Cx43+/+) mice at 12â weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3â weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43+/- mice were partially protected against CH-PH when compared to Cx43+/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43+/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43+/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy.
Asunto(s)
Conexina 43 , Hipertensión Pulmonar , Animales , Conexina 43/genética , Conexinas , Uniones Comunicantes , Humanos , Hipoxia/complicaciones , RatonesRESUMEN
Heightened pulmonary artery smooth muscle cell (PA-SMC) proliferation and migration and dynamic remodeling of the extracellular matrix are hallmark pathogenic features of pulmonary arterial hypertension (PAH). Pirfenidone (PFD) is an orally bioavailable pyridone derivative with antifibrotic, antiinflammatory, and antioxidative properties currently used in the treatment of idiopathic pulmonary fibrosis. We therefore evaluated the efficacy of curative treatments with PFD in the sugen/hypoxia (SuHx) rat model of severe pulmonary hypertension. Treatment with PFD (30 mg/kg per day by mouth 3 times a day for 3 wk) started 5 wk after sugen injection partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and remodeling. Consistent with these observations, we found that continued PFD treatment decreases PA-SMC proliferation and levels of extracellular matrix deposition in lungs and right ventricles in SuHx rats. Importantly, PFD attenuated the proproliferative and promigratory potentials of cultured PA-SMCs from patients with idiopathic PAH and their capacity to produce extracellular matrix components. Finally, we found that PFD dose dependently enhanced forkhead box O1 protein levels and its nuclear translocation in cultured idiopathic PAH PA-SMCs and in PFD-treated SuHx rats. PFD appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.-Poble, P.-B., Phan, C., Quatremare, T., Bordenave, J., Thuillet, R., Cumont, A., Huertas, A., Tu, L., Dorfmüller, P., Humbert, M., Ghigna, M.-R., Savale, L., Guignabert, C. Therapeutic effect of pirfenidone in the sugen/hypoxia rat model of severe pulmonary hypertension.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/fisiopatología , Piridonas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Humanos , Pulmón/efectos de los fármacos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Wistar , Remodelación Vascular/efectos de los fármacosRESUMEN
In this study, we explored the complex interactions between platelet-derived growth factor (PDGF) and N-methyl-d-aspartate receptor (NMDAR) and their effect on the excessive proliferation and migration of smooth muscle cells leading to obstructed arteries in pulmonary arterial hypertension (PAH). We report lower expression of glutamate receptor NMDA-type subunit 2B (GluN2B), a subunit composing NMDARs expected to affect cell survival/proliferation of pulmonary artery smooth muscle cells (PASMCs), in PAH patient lungs. PASMC exposure to PDGF-BB stimulated immediate increased levels of phosphorylated Src family kinases (SFKs) together with increased phosphorylated GluN2B (its active form) and cell surface relocalization, suggesting a cross talk between PDGFR-recruited SFKs and NMDAR. Selective inhibition of PDGFR-ß or SFKs with imatinib or A-419259, respectively, on one hand, or with specific small-interfering RNAs (siRNAs) on the other hand, aborted PDGF-induced phosphorylation of GluN2B, thus validating the pathway. Selective inhibition of GluN2B using Rö25-6981 and silencing with specific siRNA, in the presence of PDGF-BB, significantly increased both migration and proliferation of PASMCs, thus strengthening the functional importance of the pathway. Together, these results indicate that GluN2B-type NMDAR activation may confer to PASMCs antiproliferative and antimigratory properties. The decreased levels of GluN2B observed in PAH pulmonary arteries could mediate the excessive proliferation of PASMCs, thus contributing to medial hyperplasia and PAH development.
Asunto(s)
Miocitos del Músculo Liso/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Adulto , Anciano , Hipertensión Pulmonar Primaria Familiar/metabolismo , Humanos , Hipertensión Pulmonar/metabolismo , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Arteria Pulmonar/metabolismoRESUMEN
Pleural effusion is a frequent side-effect of dasatinib, a second-generation tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia. However, the underlying mechanisms remain unknown. We hypothesised that dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion.To test this, we established the first animal model of dasatinib-related pleural effusion, by treating rats with a daily regimen of high doses of dasatinib (10â mg·kg-1·day-1 for 8â weeks).Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5â weeks. Consistent with these in vivo observations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of vascular endothelial cadherin and zonula occludens-1 from cell-cell junctions, and the development of actin stress fibres. These results were replicated using human umbilical vein endothelial cells and confirmed by decreased endothelial resistance. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo using a cotreatment with an antioxidant agent, N-acetylcysteine.This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.
Asunto(s)
Dasatinib/efectos adversos , Células Endoteliales/patología , Endotelio Vascular/fisiopatología , Permeabilidad/efectos de los fármacos , Derrame Pleural/fisiopatología , Animales , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Pulmón/patología , Masculino , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , UltrasonografíaRESUMEN
RATIONALE: Inflammation and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). CD74 is a receptor for the proinflammatory cytokine macrophage migration inhibitory factor (MIF). This ligand/receptor complex initiates survival pathways and cell proliferation, and it triggers the synthesis and secretion of major proinflammatory factors and cell adhesion molecules. OBJECTIVES: We hypothesized that the MIF/CD74 signaling pathway is overexpressed in idiopathic PAH (iPAH) and contributes to a proinflammatory endothelial cell (EC) phenotype. METHODS: Primary early passage cultures of human ECs isolated from lung tissues obtained from patients with iPAH and controls were examined for their ability to secrete proinflammatory mediators and bind inflammatory cells with or without modulation of the functional activities of the MIF/CD74 complex. In addition, we tested the efficacies of curative treatments with either the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies on the aberrant proinflammatory EC phenotype in vitro and in vivo and on the progression of monocrotaline-induced pulmonary hypertension. MEASUREMENTS AND MAIN RESULTS: In human lung tissues, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions are markedly up-regulated in the endothelium of distal iPAH pulmonary arteries. Circulating MIF levels are increased in the serum of patients with PAH compared with control subjects, and T-cell lymphocytes represent a source of this overabundance. In addition, CD74 is highly expressed in the endothelium of muscularized pulmonary arterioles and in cultured pulmonary ECs from iPAH, contributing to an exaggerated recruitment of peripheral blood mononuclear cells to pulmonary iPAH ECs. Finally, we found that curative treatments with the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies partially reversed development of pulmonary hypertension in rats and substantially reduced inflammatory cell infiltration. CONCLUSIONS: We report here that CD74 and MIF are markedly increased and activated in patients with iPAH, contributing to the abnormal proinflammatory phenotype of pulmonary ECs in iPAH.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Selectina E/inmunología , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Hipertensión Pulmonar Primaria Familiar/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Adulto , Animales , Antígenos de Diferenciación de Linfocitos B/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Selectina E/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Hipertensión Pulmonar Primaria Familiar/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Técnicas In Vitro , Inflamación , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Arteria Pulmonar/inmunología , Arteria Pulmonar/metabolismo , Ratas , Transducción de Señal , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
RATIONALE: Pulmonary hypertension (PH) is characterized by a progressive elevation in mean pulmonary arterial pressure, often leading to right ventricular failure and death. Growth factors play significant roles in the pathogenesis of PH, and their targeting may therefore offer novel therapeutic strategies in this disease. OBJECTIVES: To evaluate the nerve growth factor (NGF) as a potential new target in PH. METHODS: Expression and/or activation of NGF and its receptors were evaluated in rat experimental PH induced by chronic hypoxia or monocrotaline and in human PH (idiopathic or associated with chronic obstructive pulmonary disease). Effects of exogenous NGF were evaluated ex vivo on pulmonary arterial inflammation and contraction, and in vitro on pulmonary vascular cell proliferation, migration, and cytokine secretion. Effects of NGF inhibition were evaluated in vivo with anti-NGF blocking antibodies administered both in rat chronic hypoxia- and monocrotaline-induced PH. MEASUREMENTS AND MAIN RESULTS: Our results show increased expression of NGF and/or increased expression/activation of its receptors in experimental and human PH. Ex vivo/in vitro, we found out that NGF promotes pulmonary vascular cell proliferation and migration, pulmonary arterial hyperreactivity, and secretion of proinflammatory cytokines. In vivo, we demonstrated that anti-NGF blocking antibodies prevent and reverse PH in rats through significant reduction of pulmonary arterial inflammation, hyperreactivity, and remodeling. CONCLUSIONS: This study highlights the critical role of NGF in PH. Because of the recent development of anti-NGF blocking antibodies as a possible new pain treatment, such a therapeutic strategy of NGF inhibition may be of interest in PH.
Asunto(s)
Hipertensión Pulmonar/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Pericytes and their crosstalk with endothelial cells are critical for the development of a functional microvasculature and vascular remodeling. It is also known that pulmonary endothelial dysfunction is intertwined with the initiation and progression of pulmonary arterial hypertension (PAH). We hypothesized that pulmonary endothelial dysfunction, characterized by abnormal fibroblast growth factor-2 and interleukin-6 signaling, leads to abnormal microvascular pericyte coverage causing pulmonary arterial medial thickening. METHODS AND RESULTS: In human lung tissues, numbers of pericytes are substantially increased (up to 2-fold) in distal PAH pulmonary arteries compared with controls. Interestingly, human pulmonary pericytes exhibit, in vitro, an accentuated proliferative and migratory response to conditioned media from human idiopathic PAH endothelial cells compared with conditioned media from control cells. Importantly, by using an anti-fibroblast growth factor-2 neutralizing antibody, we attenuated these proliferative and migratory responses, whereas by using an anti-interleukin-6 neutralizing antibody, we decreased the migratory response without affecting the proliferative response. Furthermore, in our murine retinal angiogenesis model, both fibroblast growth factor-2 and interleukin-6 administration increased pericyte coverage. Finally, using idiopathic PAH human and NG2DsRedBAC mouse lung tissues, we demonstrated that this increased pericyte coverage contributes to pulmonary vascular remodeling as a source of smooth muscle-like cells. Furthermore, we found that transforming growth factor-ß, in contrast to fibroblast growth factor-2 and interleukin-6, promotes human pulmonary pericyte differentiation into contractile smooth muscle-like cells. CONCLUSIONS: To the best of our knowledge, this is the first report of excessive pericyte coverage in distal pulmonary arteries in human PAH. We also show that this phenomenon is directly linked with pulmonary endothelial dysfunction.
Asunto(s)
Células Endoteliales/citología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Hipertensión Pulmonar/patología , Interleucina-6/fisiología , Músculo Liso Vascular/citología , Pericitos/citología , Adulto , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Hipertensión Pulmonar/fisiopatología , Interleucina-6/farmacología , Masculino , Ratones , Ratones Transgénicos , Microcirculación/fisiología , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Neovascularización Fisiológica/fisiología , Pericitos/efectos de los fármacos , Pericitos/fisiología , Circulación Pulmonar/fisiología , Ratas , Ratas Wistar , Vasos Retinianos/citología , Vasos Retinianos/fisiologíaRESUMEN
Excessive proliferation of pulmonary arterial smooth muscle cells (PA-SMCs) and perivascular inflammation lead to pulmonary arterial hypertension (PAH) progression, but they are not specifically targeted by the current therapies. Since leptin (Ob) and its main receptor ObR-b contribute to systemic vascular cell proliferation and inflammation, we questioned whether targeting Ob/ObR-b axis would be an effective antiproliferative and anti-inflammatory strategy against PAH. In idiopathic PAH (iPAH), using human lung tissues and primary cell cultures (early passages ≤5), we demonstrate that pulmonary endothelial cells (P-ECs) over produce Ob and that PA-SMCs overexpress ObR-b. Furthermore, we obtain evidence that Ob enhances proliferation of human PA-SMCs in vitro and increases right ventricular systolic pressure in Ob-treated mice in the chronic hypoxia-induced pulmonary hypertension (PH) model. Using human cells, we also show that Ob leads to monocyte activation and increases cell adhesion molecule expression levels in P-ECs. We also find that Ob/ObR-b axis contributes to PH susceptibility by using ObR-deficient rats, which display less severe hypoxia-induced PH (pulmonary haemodynamics, arterial muscularisation, PA-SMC proliferation and perivascular inflammation). Importantly, we demonstrate the efficacy of two curative strategies using a soluble Ob neutraliser and dichloroacetate in hypoxia-induced PH. We demonstrate here that Ob/ObR-b axis may represent anti-proliferative and anti-inflammatory targets in PAH.
Asunto(s)
Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/terapia , Hipoxia/fisiopatología , Leptina/genética , Remodelación Vascular/genética , Adulto , Animales , Western Blotting , Estudios de Casos y Controles , Proliferación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Femenino , Hemodinámica/fisiología , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Terapia Molecular Dirigida , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transducción de Señal , Regulación hacia ArribaRESUMEN
AIMS: The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models. METHODS AND RESULTS: Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats. CONCLUSION: We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.
Asunto(s)
Chalconas/farmacología , Quimiocina CXCL2/antagonistas & inhibidores , Hipertensión Pulmonar/tratamiento farmacológico , Arteria Pulmonar/efectos de los fármacos , Pirimidinonas/farmacología , Remodelación Vascular/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Animales , Bencilaminas , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL2/metabolismo , Ciclamas , Modelos Animales de Enfermedad , Compuestos Heterocíclicos/farmacología , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/fisiopatología , Hipertrofia Ventricular Derecha/prevención & control , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Ratas Wistar , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Transducción de SeñalRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by a progressive accumulation of pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterioles leading to the narrowing of the lumen, right heart failure, and death. Although most studies have supported the notion of a role for IL-6/glycoprotein 130 (gp130) signaling in PAH, it remains unclear how this signaling pathway determines the progression of the disease. Here, we identify ectopic upregulation of membrane-bound IL-6 receptor (IL6R) on PA-SMCs in PAH patients and in rodent models of pulmonary hypertension (PH) and demonstrate its key role for PA-SMC accumulation in vitro and in vivo. Using Sm22a-Cre Il6rfl/fl, which lack Il6r in SM22A-expressing cells, we found that these animals are protected against chronic hypoxia-induced PH with reduced PA-SMC accumulation, revealing the potent pro-survival potential of membrane-bound IL6R. Moreover, we determine that treatment with IL6R-specific antagonist reverses experimental PH in two rat models. This therapeutic strategy holds promise for future clinical studies in PAH.
Asunto(s)
Receptor gp130 de Citocinas/biosíntesis , Hipertensión Pulmonar Primaria Familiar/metabolismo , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Regulación hacia Arriba , Remodelación Vascular , Animales , Arteriolas/metabolismo , Arteriolas/patología , Arteriolas/fisiopatología , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/patología , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Humanos , Ratones , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Ratas , Transducción de Señal/genéticaRESUMEN
Macrophage migration inhibitory factor (MIF) is a key pleiotropic mediator and a promising therapeutic target in cancer as well as in several inflammatory and cardiovascular diseases including pulmonary arterial hypertension (PAH). Here, a novel series of N-(phenylmethyl)-benzoxazol-2-thiones 5-32 designed to target the MIF tautomerase active site was synthesized and evaluated for its effects on cell survival. Investigation of structure-activity relationship (SAR) particularly at the 5-position of the benzoxazole core led to the identification of 31 that potently inhibits cell survival in DU-145 prostate cancer cells and pulmonary endothelial cells derived from patients with idiopathic PAH (iPAH-ECs), two cell lines for which survival is MIF-dependent. Molecular docking studies helped to interpret initial SAR related to MIF tautomerase inhibition and propose preferred binding mode for 31 within the MIF tautomerase active site. Interestingly, daily treatment with 31 started 2 weeks after a subcutaneous monocrotaline injection regressed established pulmonary hypertension in rats.
Asunto(s)
Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Benzoxazoles/síntesis química , Benzoxazoles/farmacología , Hipertensión Pulmonar/tratamiento farmacológico , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Tionas/síntesis química , Tionas/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Fibrosis/inducido químicamente , Fibrosis/prevención & control , Hipertensión Pulmonar/inducido químicamente , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Modelos Moleculares , Simulación del Acoplamiento Molecular , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Background: Pulmonary hypertension (PH) is a devastating and progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and remodeling of the lung vasculature. Adenosine signaling through the ADORA2B receptor has previously been implicated in disease progression and tissue remodeling in chronic lung disease. In experimental models of PH associated with chronic lung injury, pharmacological or genetic inhibition of ADORA2B improved markers of chronic lung injury and hallmarks of PH. However, the contribution of ADORA2B expression in the PASMC was not fully evaluated. Hypothesis: We hypothesized that adenosine signaling through the ADORA2B receptor in PASMC mediates the development of PH. Methods: PASMCs from controls and patients with idiopathic pulmonary arterial hypertension (iPAH) were characterized for expression levels of all adenosine receptors. Next, we evaluated the development of PH in ADORA2Bf/f-Transgelin (Tagln)cre mice. These mice or adequate controls were exposed to a combination of SUGEN (SU5416, 20 mg/kg/b.w. IP) and hypoxia (10% O2) for 28 days (HX-SU) or to chronic low doses of bleomycin (BLM, 0.035U/kg/b.w. IP). Cardiovascular readouts including right ventricle systolic pressures (RVSPs), Fulton indices and vascular remodeling were determined. Using PASMCs we identified ADORA2B-dependent mediators involved in vascular remodeling. These mediators: IL-6, hyaluronan synthase 2 (HAS2) and tissue transglutaminase (Tgm2) were determined by RT-PCR and validated in our HX-SU and BLM models. Results: Increased levels of ADORA2B were observed in PASMC from iPAH patients. ADORA2Bf/f-Taglncre mice were protected from the development of PH following HX-SU or BLM exposure. In the BLM model of PH, ADORA2Bf/f- Taglncre mice were not protected from the development of fibrosis. Increased expression of IL-6, HAS2 and Tgm2 was observed in PASMC in an ADORA2B-dependent manner. These mediators were also reduced in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Conclusions: Our studies revealed ADORA2B-dependent increased levels of IL-6, hyaluronan and Tgm2 in PASMC, consistent with reduced levels in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Taken together, our data indicates that ADORA2B on PASMC mediates the development of PH through the induction of IL-6, hyaluronan and Tgm2. These studies point at ADORA2B as a therapeutic target to treat PH.
RESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) encompasses a group of conditions with distinct causes. Immunologic disorders are common features of all forms of PAH and contributes to both disease susceptibility and progression. Regulatory T lymphocytes (Treg) are dysfunctional in patients with idiopathic PAH (iPAH) in a leptin-dependent manner. However, it is not known whether these abnormalities are specific to iPAH. Hence, we hypothesized that (1) Treg dysfunction is also present in heritable (hPAH) and connective tissue disease-associated PAH (CTD-PAH); (2) defective leptin-dependent signaling is present in hPAH and CTD-PAH and could contribute to Treg dysfunction; (3) modulating the leptin axis in vivo could protect against Treg dysfunction; and (4) restoration of Treg activity could limit or reverse experimental chronic hypoxia-induced pulmonary hypertension in vivo. METHODS: We analyzed 62 patients with PAH (30 with iPAH, 18 with hPAH, and 14 with CTD-PAH), 7 patients with CTD without PAH, and 20 healthy control subjects. RESULTS: Our results indicate that Treg are dysfunctional in all PAH forms tested, as well as in patients with CTD without PAH. Importantly, the leptin axis is crucial in Treg dysfunction in patients with iPAH and those with CTD (with or without PAH), whereas in patients with hPAH, Treg are altered in a leptin-independent manner. We found that leptin receptor-deficient rats, which develop less severe hypoxia-induced pulmonary hypertension, are protected against decreased Treg function after hypoxic exposure. CONCLUSIONS: Taken together, our results suggest that Treg dysfunction is common to all forms of PAH and may contribute to the development and the progression of the disease.
Asunto(s)
Enfermedades del Tejido Conjuntivo , Hipertensión Pulmonar Primaria Familiar , Hipoxia , Leptina/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Enfermedades del Tejido Conjuntivo/complicaciones , Enfermedades del Tejido Conjuntivo/inmunología , Enfermedades del Tejido Conjuntivo/patología , Progresión de la Enfermedad , Hipertensión Pulmonar Primaria Familiar/etiología , Hipertensión Pulmonar Primaria Familiar/inmunología , Hipertensión Pulmonar Primaria Familiar/patología , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Femenino , Humanos , Hipoxia/complicaciones , Hipoxia/metabolismo , Hipoxia/fisiopatología , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Pruebas de Función Respiratoria/métodosRESUMEN
Pulmonary arterial hypertension (PAH) is a disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in mean pulmonary arterial pressure (mPAP), resulting in a progressive functional decline despite current available therapeutic options. There are multiple mechanisms predisposing to and/or promoting the aberrant pulmonary vascular remodeling in PAH, and these involve not only altered crosstalk between cells within the vascular wall but also sustained inflammation and dysimmunity, cell accumulation in the vascular wall and excessive activation of some growth factor-stimulated signaling pathways, in addition to the interaction of systemic hormones, local growth factors, cytokines, and transcription factors. Heterozygous germline mutations in the bone morphogenetic protein receptor, type-2 (BMPR2) gene, a gene encoding a receptor for the transforming growth factor (TGF)-ß superfamily, can predispose to the disease. Although the spectrum of therapeutic options for PAH has expanded in the last 20 years, available therapies remain essentially palliative. Over the past decade, however, a better understanding of key regulators of this irreversible remodeling of the pulmonary vasculature has been obtained. New and more effective approaches are likely to emerge. The present article profiles the innovative research into novel pathways and therapeutic targets that may lead to the development of targeted agents in PAH.
Asunto(s)
Comunicación Celular/fisiología , Hipertensión Pulmonar/terapia , Terapia Molecular Dirigida/tendencias , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Humanos , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Transducción de Señal/fisiologíaRESUMEN
Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, a dual Src and BCR-ABL tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). Today, key questions remain regarding the mechanisms involved in the long-term development of dasatinib-induced PAH. Here, we demonstrated that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents. We found that dasatinib treatment attenuated hypoxic pulmonary vasoconstriction responses and increased susceptibility to experimental pulmonary hypertension (PH) in rats, but these effects were absent in rats treated with imatinib, another BCR-ABL tyrosine kinase inhibitor. Furthermore, dasatinib treatment induced pulmonary endothelial cell apoptosis in a dose-dependent manner, while imatinib did not. Dasatinib treatment mediated endothelial cell dysfunction via increased production of ROS that was independent of Src family kinases. Consistent with these findings, we observed elevations in markers of endothelial dysfunction and vascular damage in the serum of CML patients who were treated with dasatinib, compared with CML patients treated with imatinib. Taken together, our findings indicate that dasatinib causes pulmonary vascular damage, induction of ER stress, and mitochondrial ROS production, which leads to increased susceptibility to PH development.