RESUMO
Pulmonary arterial adventitial fibroblasts (PAF), the most abundant cellular constituent of adventitia, act as a key regulator of pulmonary vascular wall structure and function from the outside-in. Previous studies indicate that transient receptor potential vanilloid 4 (TRPV4) channel plays an important role in the development of pulmonary hypertension (PH), but no attention has been given so far to its role in adventitial remodeling. In this study, we thus investigated TRPV4 implication in PAF activation occurring in PH. First, we isolated and cultured PAF from rat adventitial intrapulmonary artery. RT-PCR, Western blot, immunostaining, and calcium imaging (fluo-4/AM) showed that PAF express functional TRPV4 channels. In extension of these results, using pharmacological and siRNA approaches, we demonstrated TRPV4 involvement in PAF proliferation (BrdU incorporation) and migration (wound-healing assay). Then, Western blot experiments revealed that TRPV4 activation upregulates the expression of extracellular matrix protein synthesis (collagen type I and fibronectin). Finally, we explored the role of TRPV4 in the adventitial remodeling occurring in PH. By means of Western blot, we determined that TRPV4 protein expression was upregulated in adventitia from chronically hypoxic and monocrotaline rats, two animal models of PH. Furthermore, morphometric analysis indicated that adventitial remodeling is attenuated in PH-induced trpv4-/- mice. These data support the concept that PAF play an essential role in hypertensive pulmonary vascular remodeling and point out the participation of TRPV4 channel activity in PAF activation leading to excessive adventitial remodeling.
Assuntos
Túnica Adventícia/metabolismo , Fibroblastos/metabolismo , Hipertensão Pulmonar/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monocrotalina/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Regulação para Cima/fisiologiaRESUMO
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.
Assuntos
Conexina 43 , Hipertensão Pulmonar , Animais , Conexina 43/genética , Conexinas , Junções Comunicantes , Humanos , Hipóxia/complicações , CamundongosRESUMO
In intrapulmonary arteries (IPA), endothelial cells (EC) respond to mechanical stimuli by releasing vasoactive factors to set the vascular tone. Piezo1, a stretch-activated, calcium-permeable channel, is a sensor of mechanical stress in EC. The present study was undertaken to investigate the implication of Piezo1 in the endothelium-dependent regulation of IPA tone and potential involvement of Piezo1 in pulmonary hypertension, the main disease of this circulation. IPA tone was quantified by means of a myograph in control Piezo1+/+ mice and in mice lacking endothelial Piezo1 (EC-Piezo1-/-). Endothelial intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) production were measured, in mouse or human EC, with Fluo-4 or DAF-FM probe, respectively. Immunofluorescent labeling and patch-clamp experiments revealed the presence of Piezo1 channels in EC. Yoda1, a Piezo1 agonist, induced an endothelium-dependent relaxation that was significantly reduced in pulmonary arteries in EC-Piezo1-/- compared with Piezo1+/+ mice. Yoda1 as well as mechanical stimulation (by osmotic stress) increased [Ca2+]i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca2+]i increases were reduced in EC from Piezo1-/- mice or in the presence of Piezo1 inhibitors. Furthermore, deletion of Piezo1 increased α-adrenergic agonist-mediated contraction. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, Piezo1 still mediated arterial relaxation, and deletion of this channel did not impair the development of the disease. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i and NO production and that this effect is still present in pulmonary hypertension.
Assuntos
Células Endoteliais/metabolismo , Canais Iônicos/metabolismo , Artéria Pulmonar/metabolismo , Animais , Cálcio/metabolismo , Doença Crônica , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Canais Iônicos/agonistas , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Artéria Pulmonar/patologia , Vasoconstrição , VasodilataçãoRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative, and apoptotic-resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well-established pre-clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi-ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT-treated rats, using an iTRAQ-based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti-proliferative effect of TMP seems to be explained, at least in part, by the down-regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH.
Assuntos
Anti-Hipertensivos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína HMGB1/metabolismo , Hipertensão/tratamento farmacológico , Masoprocol/análogos & derivados , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Hemodinâmica/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Masoprocol/farmacologia , Monocrotalina , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Mapas de Interação de Proteínas , Proteômica/métodos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos Wistar , Recuperação de Função Fisiológica , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Pulmonary arterial hypertension (PAH) is a severe form of pulmonary hypertension that combines multiple alterations of pulmonary arteries, including, in particular, thrombotic and plexiform lesions. Multiple-pathological-insult animal models, developed to more closely mimic this human severe PAH form, often require complex and/or long experimental procedures while not displaying the entire panel of characteristic lesions observed in the human disease. In this study, we further characterized a rat model of severe PAH generated by combining a single injection of monocrotaline with 4 weeks exposure to chronic hypoxia. This model displays increased pulmonary arterial pressure, right heart altered function and remodeling, pulmonary arterial inflammation, hyperresponsiveness and remodeling. In particular, severe pulmonary arteriopathy was observed, with thrombotic, neointimal and plexiform-like lesions similar to those observed in human severe PAH. This model, based on the combination of two conventional procedures, may therefore be valuable to further understand the pathophysiology of severe PAH and identify new potential therapeutic targets in this disease.
Assuntos
Modelos Animais de Doenças , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/complicações , Hipóxia/fisiopatologia , Artéria Pulmonar/fisiopatologia , Animais , Pressão Arterial , Doença Crônica , Humanos , Masculino , Monocrotalina , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Resistência Vascular/efeitos dos fármacosRESUMO
Transient receptor potential (TRP) channels of the vanilloid subfamily, mainly TRPV1 and TRPV4, are expressed in pulmonary artery smooth muscle cells (PASMC) and implicated in the remodeling of pulmonary artery, a landmark of pulmonary hypertension (PH). Among a variety of PH subtypes, PH of group 3 are mostly related to a prolonged hypoxia exposure occurring in a variety of chronic lung diseases. In the present study, we thus investigated the role of hypoxia on TRPV1 and TRPV4 channels independently of the increased pulmonary arterial pressure that occurs during PH. We isolated PASMC from normoxic rat and cultured these cells under in vitro hypoxia. Using microspectrofluorimetry and the patch-clamp technique, we showed that hypoxia (1 % O2 for 48 h) significantly increased stretch- and TRPV4-induced calcium responses. qRT-PCR, Western blotting, and immunostaining experiments revealed that the expression of TRPV1 and TRPV4 was not enhanced under hypoxic conditions, but we observed a membrane translocation of TRPV1. Furthermore, hypoxia induced a reorganization of the F-actin cytoskeleton, the tubulin, and intermediate filament networks (immunostaining experiments), associated with an enhanced TRPV1- and TRPV4-induced migratory response (wound-healing assay). Finally, as assessed by immunostaining, exposure to in vitro hypoxia elicited a significant increase in NFATc4 nuclear localization. Cyclosporin A and BAPTA-AM inhibited NFATc4 translocation, indicating the activation of the Ca(2+)/calcineurin/NFAT pathway. In conclusion, these data point out the effect of hypoxia on TRPV1 and TRPV4 channels in rat PASMC, suggesting that these channels can act as direct signal transducers in the pathophysiology of PH.
Assuntos
Hipóxia/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Oxigênio/metabolismo , Artéria Pulmonar/metabolismo , Canais de Cátion TRPV/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Masculino , Músculo Liso Vascular/citologia , Fatores de Transcrição NFATC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Artéria Pulmonar/citologia , Ratos , Ratos WistarRESUMO
Caveolae are stiff plasma membrane microdomains implicated in various cell response mechanisms like Ca(2+) signaling and mechanotransduction. Pulmonary arterial smooth muscle cells (PASMC) transduce mechanical stimuli into Ca(2+) increase via plasma membrane stretch-activated channels (SAC). This mechanotransduction process is modified in pulmonary hypertension (PH) during which stretch forces are increased by the increase in arterial blood pressure. We propose to investigate how caveolae are involved in the pathophysiology of PH and particularly in mechanotransduction. PASMC were freshly isolated from control rats (Ctrl rats) and rats suffering from PH induced by 3 wk of chronic hypoxia (CH rats). Using a caveolae disrupter (methyl-ß-cyclodextrin), we showed that SAC activity measured by patch-clamp, stretch-induced Ca(2+) increase measured with indo-1 probe and pulmonary arterial ring contraction to osmotic shock are enhanced in Ctrl rats when caveolae are disrupted. In CH rats, SAC activity, Ca(2+), and contraction responses to stretch are all higher compared with Ctrl rats. However, in contrast to Ctrl rats, caveolae disruption in CH-PASMC, reduces SAC activity, Ca(2+) responses to stretch and arterial contractions. Furthermore, by means of immunostainings and transmission electron microscopy, we observed that caveolae and caveolin-1 are expressed in PASMC from both Ctrl and CH rats and localize close to subplasmalemmal sarcoplasmic reticulum (ryanodine receptors) and mitochondria, thus facilitating Ca(2+) exchanges, particularly in CH. In conclusion, caveolae are implicated in mechanotransduction in Ctrl PASMC by buffering mechanical forces. In PH-PASMC, caveolae form a distinct Ca(2+) store facilitating Ca(2+) coupling between SAC and sarcoplasmic reticulum.
Assuntos
Cavéolas/fisiologia , Hipertensão Pulmonar/metabolismo , Mecanotransdução Celular , Animais , Sinalização do Cálcio , Caveolina 1/metabolismo , Células Cultivadas , Hipertensão Pulmonar/patologia , Masculino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos WistarRESUMO
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.
Assuntos
Hipertensão Pulmonar/metabolismo , Fator de Crescimento Neural/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Humanos , Masculino , Ratos , Ratos WistarRESUMO
The development of right heart failure (RHF) is characterized by alterations of right ventricle (RV) structure and function, but the mechanisms of RHF remain still unknown. Thus, understanding the RHF is essential for improved therapies. Therefore, identification by quantitative proteomics of targets specific to RHF may have therapeutic benefits to identify novel potential therapeutic targets. The objective of this study was to analyze the molecular mechanisms changing RV function in the diseased RHF and thus, to identify novel potential therapeutic targets. For this, we have performed differential proteomic analysis of whole RV proteins using two experimental rat models of RHF. Differential protein expression was observed for hundred twenty six RV proteins including proteins involved in structural constituent of cytoskeleton, motor activity, structural molecule activity, cytoskeleton protein binding and microtubule binding. Interestingly, further analysis of down-regulated proteins, reveals that both protein and gene expressions of proteasome subunits were drastically decreased in RHF, which was accompanied by an increase of ubiquitinated proteins. Interestingly, the proteasomal activities chymotrypsin and caspase-like were decreased whereas trypsin-like activity was maintained. In conclusion, this study revealed the involvement of ubiquitin-proteasome system (UPS) in RHF. Three deregulated mechanisms were discovered: (1) decreased gene and protein expressions of proteasome subunits, (2) decreased specific activity of proteasome; and (3) a specific accumulation of ubiquitinated proteins. This modulation of UPS of RV may provide a novel therapeutic avenue for restoration of cardiac function in the diseased RHF.
Assuntos
Insuficiência Cardíaca/genética , Ventrículos do Coração/metabolismo , Hipóxia/genética , Complexo de Endopeptidases do Proteassoma/química , Proteoma/genética , Disfunção Ventricular Direita/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Hipóxia/metabolismo , Hipóxia/patologia , Masculino , Monocrotalina , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Ubiquitinação , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/metabolismo , Disfunção Ventricular Direita/patologiaRESUMO
We have previously shown that exposure to urban particulate matter (UPM) impairs endothelial nitric oxide (NO) bioactivity in intrapulmonary arteries. As UPM is composed of heterogeneous constituents, the aim of this study was to clarify the class of pollutants responsible for such effect. Extracts (aqueous, acidic or organic) were prepared from SRM1648, an UPM sample collected in St. Louis (MO, USA). The metal composition of extracts as well as endotoxin content was determined. The effects of each extract, metal mixture and endotoxin were evaluated on endothelium-dependent relaxation to acetylcholine (reflecting endothelial NO production) in rat isolated intrapulmonary arteries. Aqueous or organic SRM1648 pretreatment altered acetylcholine-induced relaxation, similar to that induced by native SRM1648. Organic extract induced similar attenuation of acetylcholine relaxation than organic-treated SRM1648, whereas aqueous extract had no effect. Acidic pretreatment, which impoverished metal and endotoxin content of SRM1648, prevented the impairment of acetylcholine-induced relaxation. However, neither the acidic extract enriched in metals, nor a metal mixture representative of SRM1648 content, modified acetylcholine relaxation, while endotoxin impaired it. Polymyxin B, which chelates endotoxin, prevented SRM1648-induced decrease in relaxation to acetylcholine. It is concluded that SRM1648-induced impairment of endothelial NO-dependent relaxation in intrapulmonary arteries unlikely involved a soluble factor released by vascular cells during UPM exposure, but rather an organic extractible and acidic-sensitive constituents of UPM. Endotoxin, but not metals, may be responsible for UPM-induced impairment of endothelial NO-dependent relaxation.
Assuntos
Endotoxinas/toxicidade , Metais/toxicidade , Óxido Nítrico/metabolismo , Material Particulado/toxicidade , Artéria Pulmonar/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Endotoxinas/análise , Masculino , Metais/análise , Material Particulado/análise , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Wistar , Medição de Risco , Técnicas de Cultura de Tecidos , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Pulmonary hypertension (PH) is a disease that affects the adult or infant population. Dehydroepiandrosterone (DHEA), a steroid hormone, has been previously shown to prevent and to reverse PH in an adult rat model. We thus investigated its effect in a rat-pup model of chronic hypoxic PH. METHODS: Animals were maintained for 3 wk in a hypobaric chamber to induce PH, with or without concomitant treatment with DHEA (30 mg/kg every alternate day). RESULTS: DHEA significantly reduced mean pulmonary artery pressure (measured by right cardiac catheterization), pulmonary artery remodeling (evaluated by histology), and right-ventricular hypertrophy (measured by echography and by the Fulton index). At the level of the pulmonary artery smooth muscle cell (PASMC), DHEA increased activity and expression of the large-conductance Ca2+-activated potassium channel (BKCa) (assessed by means of the patch clamp technique). DHEA also inhibited both serotonin- and KCl-induced contraction and smooth muscle cell proliferation. CONCLUSION: Collectively, these results indicate that DHEA prevents PH in infant rats and may therefore be clinically relevant for the management of PH in human infants.
Assuntos
Desidroepiandrosterona/farmacologia , Hipertensão Pulmonar/prevenção & controle , Miócitos de Músculo Liso/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Pressão Arterial/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desidroepiandrosterona/administração & dosagem , Técnicas Histológicas , Hipertrofia Ventricular Direita/prevenção & controle , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Contração Muscular/efeitos dos fármacos , Técnicas de Patch-Clamp , Artéria Pulmonar/efeitos dos fármacos , RatosRESUMO
Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.
Assuntos
Movimento Celular , Miócitos de Músculo Liso/fisiologia , Artéria Pulmonar/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Citoesqueleto/fisiologia , Morfolinas/farmacologia , Ésteres de Forbol/farmacologia , Artéria Pulmonar/citologia , Pirróis/farmacologia , RNA Mensageiro/biossíntese , Ratos , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 µM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 µM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 µM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 µM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.
Assuntos
Sinalização do Cálcio , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cafeína/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Hipóxia Celular , Células Cultivadas , Dantroleno/farmacologia , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Técnicas In Vitro , Compostos Macrocíclicos/farmacologia , Masculino , Morfolinas/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxantes Musculares Centrais/farmacologia , Oxazóis/farmacologia , Técnicas de Patch-Clamp , Forbóis/farmacologia , Artéria Pulmonar/fisiopatologia , Pirróis/farmacologia , Ratos , Ratos Wistar , Rianodina/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Dehydroepiandrosterone (DHEA) prevents chronic hypoxia-induced pulmonary hypertension and associated right ventricle dysfunction in rats. In this animal model, reoxygenation following hypoxia reverses pulmonary hypertension but not right ventricle dysfunction. We thus studied the effect of DHEA on the right ventricle after reoxygenation, i.e. after a normoxic recovery phase secondary to chronic hypoxia in rats. Right ventricle function was assessed in vivo by Doppler echocardiography and in vitro by the isolated perfused heart technique in three groups of animals: control, recovery (21 days of hypoxia followed by 21 days of normoxia) and recovery DHEA (30 mg · kg(-1) every 2 days during the recovery phase). Right ventricle tissue was assessed by optical and electron microscopy. DHEA abolished right ventricle diastolic dysfunction, as the echographic E wave remained close to that of controls (mean ± SD 76.5 ± 2.4 and 79.7 ± 1.7 cm · s(-1), respectively), whereas it was diminished to 40.3 ± 3.7 in the recovery group. DHEA also abolished right ventricle systolic dysfunction, as shown by the inhibition of the increase in the slope of the pressure-volume curve in isolated heart. The DHEA effect was related to cardiac myocytes proliferation. In conclusion, DHEA prevents right ventricle dysfunction in this animal model by preventing cardiomyocyte alteration.
Assuntos
Desidroepiandrosterona/farmacologia , Hipóxia/terapia , Oxigênio/metabolismo , Disfunção Ventricular Direita/terapia , Animais , Apoptose , Proteína de Ligação a CREB/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Ecocardiografia Doppler/métodos , Masculino , Microscopia/métodos , Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Perfusão , Ratos , Ratos WistarRESUMO
BACKGROUND: Pulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH. METHODS: Intrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine. RESULTS: Cx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with 37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with 40Gap27 (a specific blocker of Cx 40) or 37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by 40Gap27 or 37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following 37-43Gap27 incubation. CONCLUSION: Altogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.
Assuntos
Endotelina-1/farmacologia , Junções Comunicantes/efeitos dos fármacos , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Fenilefrina/farmacologia , Serotonina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imunofluorescência , Junções Comunicantes/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/complicações , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Monocrotalina , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/metabolismo , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.
Assuntos
Metabolismo Energético/fisiologia , Ventrículos do Coração/metabolismo , Hipóxia/metabolismo , Mitocôndrias Cardíacas/metabolismo , Animais , Doença Crônica , Ventrículos do Coração/patologia , Hipóxia/patologia , Masculino , Mitocôndrias Cardíacas/patologia , Ratos , Ratos WistarRESUMO
RATIONALE: Pulmonary arterial hypertension (PAH) is a severe disease characterized by an increase of pulmonary vascular resistance, which is accompanied by functional and structural changes in pulmonary arteries. Microparticles (MPs) have been described as biological vector of endothelial dysfunction in other pathologies. OBJECTIVES: The purpose of this work was to characterize circulating MPs during hypoxic PAH and to study their effects on endothelial function. METHODS: Male Wistar rats were exposed or not to chronic hypoxia, and normoxic or hypoxic MPs from blood were characterized by flow cytometry. Endothelial cells (ECs) from rat aorta or pulmonary arteries were incubated with MPs, and then expression and phosphorylation of enzymes involved in nitric oxide (NO) and reactive oxygen species productions were analyzed. Hypoxic MPs were injected into rats, and endothelium-dependent relaxation was assessed. MEASUREMENTS AND MAIN RESULTS: Circulating levels of MPs from hypoxic rats were twofold higher than those present in normoxic rats. In vitro treatment of ECs with hypoxic MPs reduced NO production in aortas and pulmonary arteries by enhancing phosphorylation of endothelial NO synthase at the inhibitory site. Hypoxic MPs increased oxidative stress only in pulmonary ECs via xanthine oxidase and mitochondrial implication. In vivo injection of hypoxic MPs into rat impaired endothelium-dependent relaxation both in aorta and pulmonary arteries. CONCLUSIONS: These data provide evidence that hypoxic circulating MPs induce endothelial dysfunction in rat aorta and pulmonary arteries by decreasing NO production. Moreover, MPs display tissue specificity with respect to increased oxidative stress, which occurs only in pulmonary ECs.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Células Endoteliais/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão Pulmonar/patologia , Óxido Nítrico Sintase/metabolismo , Superóxidos/metabolismo , Animais , Aorta/metabolismo , Células Cultivadas , Citometria de Fluxo , Hipóxia/fisiopatologia , Masculino , Estresse Oxidativo/fisiologia , Fosforilação , Artéria Pulmonar , Ratos , Ratos Wistar , Vasodilatação/fisiologiaRESUMO
Smooth muscles are widely distributed in mammal body through various systems such as circulatory, respiratory, gastro-intestinal and urogenital systems. The smooth muscle cell (SMC) is not only a contractile cell but is able to perform other important functions such as migration, proliferation, production of cytokines, chemokines, extracellular matrix proteins, growth factors and cell surface adhesion molecules. Thus, SMC appears today as a fascinating cell with remarkable plasticity that contributes to its roles in physiology and disease. Most of the SMC functions are dependent on a key event: the increase in intracellular calcium concentration ([Ca(2+)](i)). Calcium entry from the extracellular space is a major step in the elevation of [Ca(2+)](i) in SMC and involves a variety of plasmalemmal calcium channels, among them is the superfamily of transient receptor potential (TRP) proteins. TRPC (canonical), TRPM (melastatin), TRPV (vanilloid) and TRPP (polycystin), are widely expressed in both visceral (airways, gastrointestinal tract, uterus) and vascular (systemic and pulmonary circulation) smooth muscles. Mainly, TRPC, TRPV and TRPM are implicated in a variety of physiological and pathophysiological processes such as: SMC contraction, relaxation, growth, migration and proliferation; control of blood pressure, arterial myogenic tone, pulmonary hypertension, intestinal motility, gastric acidity, uterine activity during parturition and labor. Thus it is becoming evident that TRP are major element of SMC calcium homeostasis and, thus, appear as novel drug targets for a better management of diseases originating from SMC dysfunction.
Assuntos
Músculo Liso Vascular/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Divisão Celular/fisiologia , Humanos , Hiperplasia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Canais Iônicos/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Biofísica/métodos , Eletrofisiologia/métodos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ativação do Canal Iônico , Ligantes , Potenciais da Membrana , Modelos Biológicos , Retículo Sarcoplasmático/metabolismoRESUMO
In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.