RESUMO
INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.
Assuntos
Pressão Arterial/efeitos dos fármacos , Cistamina/farmacologia , Inibidores Enzimáticos/farmacologia , Hipertensão Pulmonar/prevenção & controle , Hipóxia/tratamento farmacológico , Proteína 2 Glutamina gama-Glutamiltransferase/antagonistas & inibidores , Artéria Pulmonar/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Hipóxia/complicações , Hipóxia/enzimologia , Hipóxia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Artéria Pulmonar/enzimologia , Artéria Pulmonar/fisiopatologia , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/fisiopatologia , Fibrose Pulmonar/prevenção & controle , Ratos Wistar , Remodelação Vascular/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H2O2 concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hipertrofia Ventricular Direita/enzimologia , Hipóxia/enzimologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , NADPH Oxidase 2/biossíntese , Regulação para Cima , Animais , Doença Crônica , Modelos Animais de Doenças , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/patologia , Hipóxia/complicações , Hipóxia/patologia , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans. METHODS: PAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured. RESULTS: MCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration-findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls. CONCLUSIONS: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.
Assuntos
Hipertensão Pulmonar/enzimologia , Pulmão/enzimologia , Niacinamida/análogos & derivados , Nicotinamida N-Metiltransferase/metabolismo , Transdução de Sinais , 6-Cetoprostaglandina F1 alfa/sangue , Adulto , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Endotelina-1/sangue , Epoprostenol/metabolismo , Feminino , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/fisiopatologia , Fígado/enzimologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Monocrotalina , Niacinamida/sangue , Niacinamida/metabolismo , Óxido Nítrico/metabolismo , Ratos Wistar , Fatores de Tempo , Disfunção Ventricular Direita/enzimologia , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular DireitaRESUMO
AIMS: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. METHODS AND RESULTS: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. CONCLUSION: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.
Assuntos
Hipertensão Pulmonar/enzimologia , Hipertrofia Ventricular Direita/enzimologia , Miócitos de Músculo Liso/enzimologia , Artéria Pulmonar/enzimologia , Piruvato Quinase/genética , Espécies Reativas de Oxigênio/metabolismo , Acetofenonas/farmacologia , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/genética , Hipertrofia Ventricular Direita/patologia , Masculino , Metaloporfirinas/farmacologia , Monocrotalina , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Técnicas de Cultura de Órgãos , Via de Pentose Fosfato , Fosforilação , Cultura Primária de Células , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Piruvato Quinase/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/farmacologiaRESUMO
OBJECTIVE: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a persistent elevation of pulmonary artery pressure and ventricular hypertrophy. Tetrandrine is a bisbenzylisoquinoline alkaloid that can decrease blood pressure, inhibit the proliferation of vascular smooth muscle cells, and block cardiac hypertrophy, but whether it has a therapeutic effect on PAH remains poorly defined. This study was undertaken to investigate the efficacy of tetrandrine on PAH. METHODS: Forty-eight male Sprague-Dawley rats were randomly and equally divided into four groups. The control group was injected with normal saline; the others were injected with monocrotaline (MCT) to induce PAH, then treated with saline, tetrandrine, and vardenafil, respectively, from day 21 to day 42. On day 43, we measured the mean pulmonary artery pressure under general anesthesia, dissected the rat, and calculated the right ventricular hypertrophy index [right ventricle/(left ventricle plus septum)]. Later we observed the changes in the pulmonary vascular wall; measured the expression of cyclic guanosine monophosphate-dependent protein kinase type 1 and inducible nitric oxide synthase; measured the levels of superoxide dismutase, glutathione, malondialdehyde, and catalase; and then compared the results among groups. RESULTS: Compared with the MCT group, rats treated with tetrandrine had attenuated mean pulmonary artery pressure (20.48 ± 1.49 vs 30.07 ± 1.51; P < .01) and right ventricular hypertrophy index (49.19 ± 2.45 vs 68.50 ± 1.95; P < .01), inhibited proliferation of pulmonary artery smooth muscle cells, and improved endothelial function. Tetrandrine also upregulated the expression of protein kinase type 1 (90.86 ± 1.95 vs 67.34 ± 1.50; P < .01); downregulated the expression of inducible nitric oxide synthase (74.76 ± 1.48 vs 80.19 ± 0.28; P < .01); increased levels of superoxide dismutase (245.54 ± 12.98 vs 166.16 ± 21.42; P < .01), glutathione (0.699 ± 0.032 vs 0.514 ± 0.056; P < .01), and catalase (32.13 ± 2.33 vs 27.19 ± 2.72; P < .01); and decreased malondialdehyde (1.027 ± 0.039 vs 1.462 ± 0.055; P < .01). CONCLUSIONS: Tetrandrine alleviated MCT-induced PAH through regulation of nitric oxide signaling pathway and antioxidant and antiproliferation effects.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Arterial/efeitos dos fármacos , Benzilisoquinolinas/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Hipertensão Pulmonar/prevenção & controle , Monocrotalina , Óxido Nítrico Sintase Tipo II/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Glutationa/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Masculino , Malondialdeído/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Tempo , Remodelação Vascular/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
BACKGROUND: The Chinese herbal Bufei Jianpi formula (BJF) provides an effective treatment option for chronic obstructive pulmonary disease (COPD). However, the systems-level mechanism underlying the clinical effects of BJF on COPD remains unknown. METHODS: In this study, a systems pharmacology model based on absorption filtering, network targeting, and systems analyses was applied specifically to clarify the active compounds and therapeutic mechanisms of BJF. Then, a rat model of cigarette smoke- and bacterial infection-induced COPD was used to investigate the therapeutic mechanisms of BJF on COPD and its comorbidity. RESULTS: The pharmacological system successfully identified 145 bioactive ingredients from BJF and revealed 175 potential targets. There was a significant target overlap between the herbal constituents of BJF. These results suggested that each herb of BJF connected with similar multitargets, indicating potential synergistic effects among them. The integrated target-disease network showed that BJF probably was efficient for the treatment of not only respiratory tract diseases but also other diseases, such as nervous system and cardiovascular diseases. The possible mechanisms of action of BJF were related to activation of inflammatory response, immune responses, and matrix metalloproteinases, among others. Furthermore, we demonstrated that BJF treatment could effectively prevent COPD and its comorbidities, such as ventricular hypertrophy, by inhibition of inflammatory cytokine production, matrix metalloproteinases expression, and other cytokine production in vivo. CONCLUSION: This study using the systems pharmacology method, in combination with in vivo experiments, helped us successfully dissect the molecular mechanism of BJF for the treatment of COPD and predict the potential targets of the multicomponent BJF, which provides a new approach to illustrate the synergetic mechanism of the complex prescription and discover more effective drugs against COPD.
Assuntos
Anti-Inflamatórios/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Pulmão/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Medicina Tradicional Chinesa , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Biologia de Sistemas/métodos , Animais , Anti-Inflamatórios/análise , Citocinas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/análise , Feminino , Hipertrofia Ventricular Direita/tratamento farmacológico , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/patologia , Mediadores da Inflamação/metabolismo , Pulmão/enzimologia , Pulmão/microbiologia , Pulmão/fisiopatologia , Masculino , Inibidores de Metaloproteinases de Matriz/análise , Fitoterapia , Plantas Medicinais , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Ratos Sprague-Dawley , Fumar/efeitos adversos , Fatores de TempoRESUMO
BACKGROUND: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway. METHODS: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4. RESULTS: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression. CONCLUSION: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.
Assuntos
Bleomicina , Displasia Broncopulmonar/enzimologia , Células Endoteliais/enzimologia , Hipertensão Pulmonar/enzimologia , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/enzimologia , Artéria Pulmonar/enzimologia , Superóxido Dismutase/deficiência , Remodelação Vascular , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/induzido quimicamente , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Células Endoteliais/patologia , Predisposição Genética para Doença , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/genética , Hipertrofia Ventricular Direita/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Fenótipo , Fosforilação , Alvéolos Pulmonares/patologia , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transdução de Sinais , Superóxido Dismutase/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Disfunção Ventricular Direita/induzido quimicamente , Disfunção Ventricular Direita/enzimologia , Disfunção Ventricular Direita/genética , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular Direita , Pressão VentricularRESUMO
Animal models demonstrate that exposure to supraphysiological oxygen during the neonatal period compromises both lung and pulmonary vascular development, resulting in a phenotype comparable to bronchopulmonary dysplasia (BPD). Our prior work in murine models identified postnatal maturation of antioxidant enzyme capacities as well as developmental regulation of mitochondrial oxidative stress in hyperoxia. We hypothesize that consequences of hyperoxia may also be developmentally regulated and mitochondrial reactive oxygen species (ROS) dependent. To determine whether age of exposure impacts the effect of hyperoxia, neonatal mice were placed in 75% oxygen for 72 h at either postnatal day 0 (early postnatal) or day 4 (late postnatal). Mice exposed to early, but not late, postnatal hyperoxia demonstrated decreased alveolarization and septation, increased muscularization of resistance pulmonary arteries, and right ventricular hypertrophy (RVH) compared with normoxic controls. Treatment with a mitochondria-specific antioxidant, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), during early postnatal hyperoxia protected against compromised alveolarization and RVH. In addition, early, but not late, postnatal hyperoxia resulted in induction of NOX1 expression that was mitochondrial ROS dependent. Because early, but not late, exposure resulted in compromised lung and cardiovascular development, we conclude that the consequences of hyperoxia are developmentally regulated and decrease with age. Attenuated disease in mitoTEMPO-treated mice implicates mitochondrial ROS in the pathophysiology of neonatal hyperoxic lung injury, with potential for amplification of ROS signaling through NOX1 induction. Furthermore, it suggests a potential role for targeted antioxidant therapy in the prevention or treatment of BPD.
Assuntos
Displasia Broncopulmonar/enzimologia , Hiperóxia/enzimologia , Animais , Indução Enzimática , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Pulmão/enzimologia , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Calorie restriction (CR) is one of the most effective nonpharmacological interventions protecting against cardiovascular disease, such as hypertension in the systemic circulation. However, whether CR could attenuate pulmonary arterial hypertension (PAH) is largely unknown. The PAH model was developed by subjecting the rats to a single subcutaneous injection of monocrotaline. CR lowered mean pulmonary arterial pressure (mPAP) and reduced vascular remodeling and right ventricular hypertrophy in PAH rats. Meanwhile, CR attenuated endothelial dysfunction as evidenced by increased relaxation in response to acetylcholine. The beneficial effects of CR were associated with restored sirtuin-1 (SIRT1) expression and endothelial nitric oxide synthase (eNOS) phosphorylation and reduced eNOS acetylation in pulmonary arteries of PAH rats. To further clarify the role of SIRT1 in the protective effects of CR, adenoviral vectors for overexpression of SIRT1 were administered intratracheally at 1 day before monocrotaline injection. Overexpression of SIRT1 exhibited similar beneficial effects on mPAP and endothelial function, and increased eNOS phosphorylation and reduced eNOS acetylation in the absence of CR. Moreover, SIRT1 overexpression attenuated the increase in mPAP in hypoxia-induced PAH animals. Overall, the present data demonstrate that CR may serve as an effective treatment of PAH, and targeting the SIRT1/eNOS pathway may improve treatment of PAH.
Assuntos
Pressão Arterial , Restrição Calórica , Hipertensão Pulmonar/prevenção & controle , Monocrotalina , Artéria Pulmonar/fisiopatologia , Remodelação Vascular , Acetilação , Adenoviridae/genética , Animais , Pressão Arterial/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Vetores Genéticos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de Tempo , Transdução Genética , Vasodilatação , Vasodilatadores/farmacologiaRESUMO
Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.
Assuntos
Remodelação das Vias Aéreas , Arginase/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Animais , Arginase/antagonistas & inibidores , Fibrose , Cobaias , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/imunologia , Hipertrofia Ventricular Direita/patologia , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Pulmão/patologia , Mucina-5AC/metabolismo , Neutrófilos/patologia , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
PURPOSE: Pulmonary hypertension (pHTN), a main determinant of survival in congenital diaphragmatic hernia (CDH), results from in utero vascular remodeling. Phosphodiesterase type 5 (PDE5) inhibitors have never been used antenatally to treat pHTN. The purpose of this study is to determine if antenatal PDE5 inhibitors can prevent pHTN in the fetal lamb model of CDH. METHODS: CDH was created in pregnant ewes. Postoperatively, pregnant ewes received oral placebo or tadalafil, a PDE5 inhibitor, until delivery. Near term gestation, lambs underwent resuscitations, and lung tissue was snap frozen for protein analysis. RESULTS: Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs (p=0.002). Normalized expression of eNOS was 82%±12% in Normal-Placebo, 61%±5% in CDH-Placebo, 116%±6% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Normalized expression of ß-sGC was 105%±15% in Normal-Placebo, 82%±3% in CDH-Placebo, 158%±16% in Normal-Tadalafil, and 86%±8% in CDH-Tadalafil lambs. Endothelial NOS and ß-sGC were significantly decreased in CDH (p=0.0007 and 0.01 for eNOS and ß-sGC, respectively), and tadalafil significantly increased eNOS expression (p=0.0002). CONCLUSIONS: PDE5 inhibitors can cross the placental barrier. ß-sGC and eNOS are downregulated in fetal lambs with CDH. Antenatal PDE5 inhibitors normalize eNOS and may prevent in utero vascular remodeling in CDH.
Assuntos
Carbolinas/uso terapêutico , Doenças Fetais/tratamento farmacológico , Terapias Fetais , Hérnias Diafragmáticas Congênitas , Óxido Nítrico Sintase Tipo III/biossíntese , Inibidores da Fosfodiesterase 5/uso terapêutico , Animais , Carbolinas/administração & dosagem , Carbolinas/farmacologia , GMP Cíclico/análise , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Feminino , Hérnia Diafragmática/complicações , Hérnia Diafragmática/embriologia , Hérnia Diafragmática/enzimologia , Hérnia Diafragmática/prevenção & controle , Hipertensão Pulmonar/embriologia , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/prevenção & controle , Hipertrofia Ventricular Direita/embriologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/embriologia , Pulmão/patologia , Troca Materno-Fetal , Óxido Nítrico Sintase Tipo III/genética , Tamanho do Órgão/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/administração & dosagem , Inibidores da Fosfodiesterase 5/farmacologia , Gravidez , Distribuição Aleatória , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Ovinos , TadalafilaRESUMO
OBJETIVO: O aumento da atividade miocárdica da Glicose 6-Fosfato Desidrogenase tem sido demonstrado na insuficiência cardíaca. Este estudo avalia a atividade miocárdica da Glicose 6-Fosfato Desidrogenase no treinamento do ventrículo subpulmonar de cabras adultas. MÉTODOS: Foram utilizadas 18 cabras adultas, divididas em três grupos: convencional (bandagem fixa), sham e intermitente (bandagem ajustável; 12 horas diárias de sobrecarga). A sobrecarga sistólica (70% da pressão sistêmica) foi mantida durante quatro semanas. As avaliações hemodinâmica e ecocardiográfica foram realizadas durante todo o estudo. Depois de cumprido o protocolo, os animais foram mortos para avaliação morfológica e da atividade da Glicose 6-Fosfato Desidrogenase dos ventrículos. RESULTADOS: Apesar de haver sobrecarga sistólica proporcionalmente menor no ventrículo subpulmonar do grupo intermitente (P=0,001), ambos os grupos de estudo apresentaram aumento da massa muscular de magnitude similar. Os grupos intermitente e convencional apresentaram aumento da massa de 55,7% e 36,7% (P<0,05), respectivamente, em comparação ao grupo sham. O conteúdo de água do miocárdio não variou entre os grupos estudados (P=0,27). O ecocardiograma demonstrou maior aumento (37,2%) na espessura do ventrículo subpulmonar do grupo intermitente, em relação aos grupos sham e convencional (P<0,05). Foi observada maior atividade da Glicose 6-Fosfato Desidrogenase na hipertrofia miocárdica do ventrículo subpulmonar do grupo convencional, comparada aos grupos sham e intermitente (P=0,05). CONCLUSÃO: Ambos os grupos de treinamento ventricular desenvolveram hipertrofia ventricular, a despeito do menor tempo de sobrecarga sistólica no grupo intermitente. A maior atividade de Glicose 6-Fosfato Desidrogenase observada no grupo convencional pode refletir um desequilíbrio redox, com maior produção de fosfato de dinucleotídeo de nicotinamida e adenina e glutationa reduzida, um mecanismo importante da fisiopatologia da insuficiência cardíaca.
OBJECTIVE: Increased glucose 6-phosphate dehydrogenase activity has been demonstrated in heart failure. This study sought to assess myocardial glucose 6-phosphate dehydrogenase activity in retraining of the subpulmonary ventricle of adult goats. METHODS: Eighteen adult goats were divided into three groups: traditional (fixed banding), sham, and intermittent (adjustable banding, daily 12-hour systolic overload). Systolic overload (70% of systemic pressure) was maintained during a 4-week period. Right ventricle, pulmonary artery and aortic pressures were measured throughout the study. All animals were submitted to echocardiographic and hemodynamic evaluations throughout the protocol. After the study period, the animals were killed for morphological and glucose 6-phosphate dehydrogenase activity assessment. RESULTS: A 55.7% and 36.7% increase occurred in the intermittent and traditional right ventricle masses, respectively, when compared with the sham group (P<0.05), despite less exposure of intermittent group to systolic overload. No significant changes were observed in myocardial water content in the 3 groups (P=0.27). A 37.2% increase was found in right ventricle wall thickness of intermittent group, compared to sham and traditional groups (P<0.05). Right ventricle glucose 6-phosphate dehydrogenase activity was elevated in the traditional group, when compared to sham and intermittent groups (P=0.05). CONCLUSION: Both study groups have developed similar right ventricle hypertrophy, regardless less systolic overload exposure of intermittent group. Traditional systolic overload for adult subpulmonary ventricle retraining causes upregulation of myocardial glucose 6-phosphate dehydrogenase activity. It may suggest that the undesirable "pathologic systolic overload" is influenced by activation of penthose pathway and cytosolic Nicotinamide adenine dinucleotide phosphate availability. This altered energy substrate metabolism can elevate levels of free radicals by Nicotinamide adenine dinucleotide phosphate oxidase, an important mechanism in the pathophysiology of heart failure.
Assuntos
Animais , Glucosefosfato Desidrogenase/metabolismo , Hipertrofia Ventricular Direita/enzimologia , Miocárdio/enzimologia , Artéria Pulmonar/cirurgia , Transposição dos Grandes Vasos/cirurgia , Pressão Sanguínea , Biomarcadores/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Cabras , Hemodinâmica , Ventrículos do Coração/enzimologia , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Direita/fisiopatologia , Fatores de Tempo , Disfunção Ventricular Direita/enzimologia , Disfunção Ventricular Direita/fisiopatologiaRESUMO
Chronic exposure to cold caused pulmonary arterial hypertension (cold-induced pulmonary hypertension [CIPH]) and increased phosphodiesterase-1C (PDE-1C) expression in pulmonary arteries (PAs) in rats. The purpose of this study is to investigate a hypothesis that inhibition of PDE-1 would decrease inflammatory infiltrates and superoxide production leading to attenuation of CIPH. Three groups of male rats were exposed to moderate cold (5±1°C) continuously, whereas 3 groups were maintained at room temperature (23.5±1°C, warm; 6 rats/group). After 8-week exposure to cold, 3 groups in each temperature condition received continuous intravenous infusion of 8-isobutyl-methylxanthine (8-IBMX) (PDE-1 inhibitor), apocynin (NADPH oxidase inhibitor) or vehicle, respectively, for 1 week. Cold exposure significantly increased right-ventricular systolic pressure compared with warm groups (33.8±3.2 versus 18.6±0.3 mm Hg), indicating that animals developed CIPH. Notably, treatment with 8-IBMX significantly attenuated the cold-induced increase in right ventricular pressure (23.5±1.8 mm Hg). Cold exposure also caused right-ventricular hypertrophy, whereas 8-IBMX reversed cold-induced right ventricular hypertrophy. Cold exposure increased PDE-1C protein expression, macrophage infiltration, NADPH oxidase activity, and superoxide production in PAs and resulted in PA remodeling. 8-IBMX abolished cold-induced upregulation of PDE-1C in PAs. Interestingly, inhibition of PDE-1 eliminated cold-induced macrophage infiltration, NADPH oxidase activation, and superoxide production in PAs and reversed PA remodeling. Inhibition of NADPH oxidase by apocynin abolished cold-induced superoxide production and attenuated CIPH and PA remodeling. In conclusion, inhibition of PDE-1 attenuated CIPH and reversed cold-induced PA remodeling by suppressing macrophage infiltration and superoxide production, suggesting that upregulation of PDE-1C expression may be involved in the pathogenesis of CIPH.
Assuntos
Temperatura Baixa/efeitos adversos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/fisiologia , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , 1-Metil-3-Isobutilxantina/uso terapêutico , Acetofenonas/uso terapêutico , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/biossíntese , Inibidores Enzimáticos/uso terapêutico , Hipertensão Pulmonar Primária Familiar , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/tratamento farmacológico , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/fisiopatologia , Pneumopatias/tratamento farmacológico , Pneumopatias/enzimologia , Pneumopatias/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , NADPH Oxidases/antagonistas & inibidores , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismoRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. METHODS AND RESULTS: Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcription-polymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxic+SU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1(-/-) mice, and the potent CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg · kg(-1) · d(-1) IP) significantly attenuated hypoxic PAH and hypoxic+SU5416 PAH in vivo. TMS also abolished estrogen-induced proliferation in human pulmonary artery smooth muscle cells and PAH-pulmonary artery smooth muscle cells. The estrogen metabolite 16α-hydroxyestrone provoked human pulmonary artery smooth muscle cell proliferation, and this mitogenic effect was greatly pronounced in PAH-pulmonary artery smooth muscle cells. ELISA analysis revealed that 16α-hydroxyestrone concentration was elevated in PAH, consistent with CYP1B1 overexpression and activity. Finally, administration of the CYP1B1 metabolite 16α-hydroxyestrone (1.5 mg · kg(-1) · d(-1) IP) caused the development of PAH in mice. CONCLUSIONS: Increased CYP1B1-mediated estrogen metabolism promotes the development of PAH, likely via the formation of mitogens, including 16α-hydroxyestrone. Collectively, this study reveals a possible novel therapeutic target in clinical PAH.
Assuntos
Hidrocarboneto de Aril Hidroxilases/fisiologia , Estrogênios/metabolismo , Hipertensão Pulmonar/enzimologia , Artéria Pulmonar/enzimologia , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/deficiência , Hidrocarboneto de Aril Hidroxilases/genética , Hipóxia Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Doença Crônica , Citocromo P-450 CYP1B1 , Indução Enzimática , Estradiol/farmacologia , Feminino , Humanos , Hidroxiestronas/metabolismo , Hidroxiestronas/farmacologia , Hidroxiestronas/toxicidade , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/enzimologia , Hipóxia/complicações , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estilbenos/farmacologia , Regulação para CimaRESUMO
OBJECTIVE: Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such as platelet-derived growth factor (PDGF) are closely involved in the pathogenesis of pulmonary arterial hypertension. We investigated the effects of 2 novel PDGF inhibitors, nilotinib/AMN107 (Abl kinases/PDGF receptor inhibitor) and dasatinib/BMS-354825 (Abl kinases/PDGF receptor/Src inhibitor), on the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and on the hemodynamics and pulmonary vascular remodeling in experimental pulmonary hypertension, and determined the expression and regulation of Src family kinases. METHODS AND RESULTS: Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 µmol/L), nilotinib (0.3 µmol/L), and imatinib (1 µmol/L) potently inhibited PDGF-induced signal transducer and activator of transcription 3 and Akt phosphorylation. All 3 inhibitors decreased PDGF-induced proliferation, cell cycle gene regulation, and migration. In contrast, only dasatinib inhibited multiple growth factor-induced PASMC proliferation, and this was associated with the inhibition of Src phosphorylation. Combination of specific Src inhibitors (phosphoprotein phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor-induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per body weight) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per body weight) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. CONCLUSIONS: We suggest that dual inhibition of PDGF receptor and Src kinases potently inhibits mitogenic and motogenic responses to growth factors in PASMCs and pulmonary vascular remodeling in vivo so that dual inhibition may represent an alternative therapeutic approach for pulmonary arterial hypertension.
Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Becaplermina , Benzamidas , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Dasatinibe , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hemodinâmica , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/prevenção & controle , Hipóxia/complicações , Mesilato de Imatinib , Monocrotalina , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Fosforilação , Piperazinas/farmacologia , Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Pirimidinas/farmacologia , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Fatores de Tempo , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: Increased myocardial glucose-6-phosphate dehydrogenase (G6PD) activity occurs in heart failure. This study compared G6PD activity in 2 protocols of right ventricle (RV) systolic overload in young goats. METHODS: Twenty-seven goats were separated into 3 groups: sham (no overload), continuous (continuous systolic overload), and intermittent (four 12-hour periods of systolic overload paired with a 12-hour resting period). During a 96-hour protocol, systolic overload was adjusted to achieve a 0.7 RV/aortic pressure ratio. Echocardiographic and hemodynamic evaluations were performed before and after systolic overload every day postoperatively. After the study period, the animals were humanely killed for morphologic and G6PD tissue activity assessment. RESULTS: A 92.1% and 46.5% increase occurred in RV and septal mass, respectively, in the intermittent group compared with the sham group; continuous systolic overload resulted in a 37.2% increase in septal mass. A worsening RV myocardial performance index occurred in the continuous group at 72 hours and 96 hours, compared with the sham (P < .039) and intermittent groups at the end of the protocol (P < .001). Compared with the sham group, RV G6PD activity was elevated 130.1% in the continuous group (P = .012) and 39.8% in the intermittent group (P = .764). CONCLUSIONS: Continuous systolic overload for ventricle retraining causes RV dysfunction and upregulation of myocardial G6PD activity, which can elevate levels of free radicals by NADPH oxidase, an important mechanism in the pathophysiology of heart failure. Intermittent systolic overload promotes a more efficient RV hypertrophy, with better preservation of myocardial performance and and less exposure to hypertrophic triggers.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Hipertrofia Ventricular Direita/enzimologia , Miocárdio/enzimologia , Artéria Pulmonar/cirurgia , Disfunção Ventricular Direita/enzimologia , Fatores Etários , Animais , Aorta/fisiopatologia , Pressão Sanguínea , Modelos Animais de Doenças , Metabolismo Energético , Cabras , Hipertrofia Ventricular Direita/diagnóstico por imagem , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/fisiopatologia , Ligadura , Miocárdio/patologia , Fatores de Tempo , Ultrassonografia , Disfunção Ventricular Direita/diagnóstico por imagem , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/fisiopatologia , Função Ventricular Direita , Pressão Ventricular , Remodelação VentricularRESUMO
BACKGROUND: Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH. METHODS: The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB. RESULTS: HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB. CONCLUSION: In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.
Assuntos
Células Endoteliais/virologia , Infecções por HIV/virologia , HIV-1/genética , Hipertensão Pulmonar/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/irrigação sanguínea , Estresse Oxidativo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Antioxidantes/farmacologia , Becaplermina , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Hipertensão Pulmonar Primária Familiar , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/complicações , Infecções por HIV/enzimologia , Infecções por HIV/genética , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/genética , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Microvasos/enzimologia , Microvasos/virologia , Estresse Oxidativo/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-sis , Artéria Pulmonar/enzimologia , Artéria Pulmonar/virologia , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Regulação para Cima , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Rho-kinase (ROCK) inhibitors prevent pulmonary hypertension (PHT) in adult rodents, but little is known about their effects on the neonatal lung. Our objective was to examine the effects of ROCK inhibition on chronic hypoxia (CH)-induced PHT and abnormal lung structure in the neonatal rat. Pups were exposed to air or CH from postnatal d 1-14 while receiving Y-27632 (5 or 10 mg x kg(-1) x d(-1)), fasudil (20 mg x kg(-1) x d(-1)), or saline intraperitoneally. Relative to air, CH-exposed pups had increased pulmonary vascular resistance, right ventricular hypertrophy, arterial medial wall thickening, and abnormal distal airway morphology characterized by septal thinning and decreased secondary septation. Treatment with 10 mg/kg Y-27632 or fasudil attenuated the structural and hemodynamic changes of PHT while having no effect on septal thinning or inhibited secondary septation. In addition, Y-27632 (10 mg/kg) and fasudil augmented CH-induced somatic growth restriction. Pulmonary arteries of CH-exposed pups had increased ROCK activity, up-regulated expression of PDGF-BB and increased smooth muscle DNA synthesis, all of which were attenuated by treatment with 10 mg/kg Y-27632. Systemically administered ROCK inhibitors prevented PHT in the CH-exposed neonatal rat but at the cost of inhibited somatic growth. Limiting effects on vascular remodeling likely resulted, in major part, from attenuated vascular PDGF-BB/beta-receptor signaling.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Anti-Hipertensivos/farmacologia , Hiperóxia/tratamento farmacológico , Hipertensão Pulmonar/prevenção & controle , Pulmão/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Becaplermina , Peso Corporal , Doença Crônica , Replicação do DNA , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hiperóxia/complicações , Hiperóxia/enzimologia , Hiperóxia/patologia , Hiperóxia/fisiopatologia , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/prevenção & controle , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Pulmão/patologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Tamanho do Órgão , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Resistência Vascular/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays an important role in integrin-mediated signal transduction. To explore the role and mechanisms of FAK in cardiac development, we inactivated FAK in embryonic cardiomyocytes by crossing the floxed FAK mice with myosin light chain-2a (MLC2a) Cre mice, which expressed Cre as early as embryonic day 9.5 in the heart. The majority of conditional FAK knockout mice generated from MLC2a-Cre (CFKO-2a) died in the embryonic stage with thin ventricular wall and ventricular septal defects. A small fraction of CFKO-2a mice survived to adulthood with spontaneous eccentric right ventricle hypertrophy. Transmission electron microscopy analysis displayed swelling in the rough endoplasmic reticulum in CFKO-2a embryonic cardiomyocytes. We found that decreased cell proliferation, but not increased cell apoptosis or differentiation, is the reason for the thin ventricular wall in CFKO-2a mice. Microarray analysis suggests that myocyte enhancer factor 2a (MEF2a) can be regulated by FAK and that inactivation of FAK in the embryonic heart compromised MEF2a expression. Last, we found that Src, but not PI3K, is important in mediating signal transduction for the regulation of MEF2a by FAK. Together, these results identified the role and mechanisms of FAK in embryonic cardiac development.
Assuntos
Anormalidades Cardiovasculares/embriologia , Anormalidades Cardiovasculares/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/deficiência , Hipertrofia Ventricular Direita/enzimologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Animais , Proliferação de Células , Embrião de Mamíferos/enzimologia , Desenvolvimento Embrionário , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Deleção de Genes , Integrases/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Camundongos Knockout , Miocárdio/enzimologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Fatores de Regulação Miogênica/metabolismo , Cadeias Leves de Miosina/metabolismo , Especificidade de Órgãos , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1-3, PDGF receptor (PDGFR)-beta, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O(2) fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.