RESUMO
Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1-/-) and ROCK2-deficient (cROCK2-/-) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1-/- mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2-/- mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1-/- hearts and down-regulated in cROCK2-/- hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1-/- mice, whereas their expressions were significantly lower in cROCK2-/- mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.
Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Hipertensão Pulmonar/metabolismo , Pulmão/metabolismo , Miocárdio/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Basigina/biossíntese , Basigina/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Ciclofilina A/biossíntese , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Miocárdio/patologia , Quinases Associadas a rho/genéticaRESUMO
OBJECTIVE: Excessive proliferation and apoptosis resistance are special characteristics of pulmonary artery smooth muscle cells (PASMCs) in pulmonary arterial hypertension (PAH). However, the drugs in clinical use for PAH target vascular dilatation, which do not exert adequate effects in patients with advanced PAH. Here, we report a novel therapeutic effect of emetine, a principal alkaloid extracted from the root of ipecac clinically used as an emetic and antiprotozoal drug. Approach and Results: We performed stepwise screenings for 5562 compounds from original library. First, we performed high-throughput screening with PASMCs from patients with PAH (PAH-PASMCs) and found 80 compounds that effectively inhibited proliferation. Second, we performed the repeatability and counter assay. Finally, we performed a concentration-dependent assay and found that emetine inhibits PAH-PASMC proliferation. Interestingly, emetine significantly reduced protein levels of HIFs (hypoxia-inducible factors; HIF-1α and HIF-2α) and downstream PDK1 (pyruvate dehydrogenase kinase 1). Moreover, emetine significantly reduced the protein levels of RhoA (Ras homolog gene family, member A), Rho-kinases (ROCK1 and ROCK2 [rho-associated coiled-coil containing protein kinases 1 and 2]), and their downstream CyPA (cyclophilin A), and Bsg (basigin) in PAH-PASMCs. Consistently, emetine treatment significantly reduced the secretion of cytokines/chemokines and growth factors from PAH-PASMCs. Interestingly, emetine reduced protein levels of BRD4 (bromodomain-containing protein 4) and downstream survivin, both of which are involved in many cellular functions, such as cell cycle, apoptosis, and inflammation. Finally, emetine treatment ameliorated pulmonary hypertension in 2 experimental rat models, accompanied by reduced inflammatory changes in the lungs and recovered right ventricular functions. CONCLUSIONS: Emetine is an old but novel drug for PAH that reduces excessive proliferation of PAH-PASMCs and improves right ventricular functions.
Assuntos
Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Emetina/farmacologia , Emetina/uso terapêutico , Hipertensão Pulmonar/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Animais , Basigina/metabolismo , Proteínas Sanguíneas/metabolismo , Ciclofilina A/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão Pulmonar/fisiopatologia , Masculino , Mitocôndrias Musculares/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: Excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) are key mechanisms of pulmonary arterial hypertension (PAH). Despite the multiple combination therapy, a considerable number of patients develop severe pulmonary hypertension (PH) because of the lack of diagnostic biomarker and antiproliferative therapies for PASMCs. METHODS: Microarray analyses were used to identify a novel therapeutic target for PAH. In vitro experiments, including lung and serum samples from patients with PAH, cultured PAH-PASMCs, and high-throughput screening of 3336 low-molecular-weight compounds, were used for mechanistic study and exploring a novel therapeutic agent. Five genetically modified mouse strains, including PASMC-specific selenoprotein P (SeP) knockout mice and PH model rats, were used to study the role of SeP and therapeutic capacity of the compounds for the development of PH in vivo. RESULTS: Microarray analysis revealed a 32-fold increase in SeP in PAH-PASMCs compared with control PASMCs. SeP is a widely expressed extracellular protein maintaining cellular metabolism. Immunoreactivity of SeP was enhanced in the thickened media of pulmonary arteries in PAH. Serum SeP levels were also elevated in patients with PH compared with controls, and high serum SeP predicted poor outcome. SeP-knockout mice ( SeP-/-) exposed to chronic hypoxia showed significantly reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling compared with controls. In contrast, systemic SeP-overexpressing mice showed exacerbation of hypoxia-induced PH. Furthermore, PASMC-specific SeP-/- mice showed reduced hypoxia-induced PH compared with controls, whereas neither liver-specific SeP knockout nor liver-specific SeP-overexpressing mice showed significant differences with controls. Altogether, protein levels of SeP in the lungs were associated with the development of PH. Mechanistic experiments demonstrated that SeP promotes PASMC proliferation and resistance to apoptosis through increased oxidative stress and mitochondrial dysfunction, which were associated with activated hypoxia-inducible factor-1α and dysregulated glutathione metabolism. It is important to note that the high-throughput screening of 3336 compounds identified that sanguinarine, a plant alkaloid with antiproliferative effects, reduced SeP expression and proliferation in PASMCs and ameliorated PH in mice and rats. CONCLUSIONS: These results indicate that SeP promotes the development of PH, suggesting that it is a novel biomarker and therapeutic target of the disorder.
Assuntos
Pressão Arterial , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Selenoproteína P/metabolismo , Remodelação Vascular , Animais , Anti-Hipertensivos/farmacologia , Apoptose , Pressão Arterial/efeitos dos fármacos , Benzofenantridinas/farmacologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/farmacologia , Masculino , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Estresse Oxidativo , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Transdução de Sinais , Remodelação Vascular/efeitos dos fármacosRESUMO
BACKGROUND: Thoracic aortic aneurysm (TAA) and dissection are fatal diseases that cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in aortic smooth muscle cells (AoSMCs) causes TAA and thoracic aortic dissection. METHODS: To examine the role of SmgGDS in TAA formation, we used an angiotensin II (1000 ng·min-1·kg-1, 4 weeks)-induced TAA model. RESULTS: We found that 33% of Apoe-/- SmgGDS+/- mice died suddenly as a result of TAA rupture, whereas there was no TAA rupture in Apoe-/- control mice. In contrast, there was no significant difference in the ratio of abdominal aortic aneurysm rupture between the 2 genotypes. We performed ultrasound imaging every week to follow up the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe-/- SmgGDS+/- mice compared with Apoe-/- mice, whereas that of the abdominal aorta remained comparable between the 2 genotypes. Histological analysis of Apoe-/- SmgGDS+/- mice showed dissections of major thoracic aorta in the early phase of angiotensin II infusion (day 3 to 5) and more severe elastin degradation compared with Apoe-/- mice. Mechanistically, Apoe-/- SmgGDS+/- mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe-/- mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After angiotensin II (100 nmol/L) treatment for 24 hours, Apoe-/- SmgGDS+/- AoSMCs showed significantly increased matrix metalloproteinase activity and oxidative stress levels compared with Apoe-/- AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of fibrillin-1 ( FBN1), α-smooth muscle actin ( ACTA2), myosin-11 ( MYH11), MYLLK, and PRKG1, which are force generation genes, were significantly reduced in Apoe-/- SmgGDS+/- AoSMCs compared with Apoe-/- AoSMCs. A similar tendency was noted in AoSMCs from patients with TAA compared with those from control subjects. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe-/- SmgGDS+/- mice. CONCLUSIONS: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.
Assuntos
Aorta/metabolismo , Aneurisma da Aorta Torácica/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Actinas/genética , Actinas/metabolismo , Angiotensina II/administração & dosagem , Angiotensina II/efeitos adversos , Animais , Aorta/citologia , Aorta/patologia , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrilina-1/genética , Fibrilina-1/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary artery (PA) remodeling, elevated PA pressure and right ventricular (RV) failure. It has been previously demonstrated that treatment with a Rho-kinase inhibitor, fasudil, ameliorates PH in animal models. Here, whether combination therapy with fasudil and sildenafil further ameliorates PH in rats was examined. METHODS AND RESULTS: PH was induced in Sprague-Dawley rats by the use of a single subcutaneous monocrotaline (MCT) injection, which caused PA remodeling, elevated RV systolic pressure (RVSP), and RV hypertrophy (RVH). While fasudil and sildenafil monotherapy inhibited the development of MCT-induced PH in the prevention and treatment protocols, their combination therapy further improved RVSP and RVH. Moreover, a histological examination demonstrated significant improvements of PA remodeling in the combination group compared with the monotherapy groups. An echocardiographic examination also revealed significant reduction in RV diameter in the combination group compared with the monotherapy groups. Mechanistic experiments revealed significant inhibition of Rho-kinase activity in PA trunk, lung and RV tissues in the combination group as well as in the monotherapy groups. Finally, the combination therapy markedly improved the long-term survival compared with the monotherapy groups. CONCLUSIONS: These results indicate that the combination therapy with fasudil and sildenafil shows the synergistic effects through the inhibition of Rho-kinase activity for the treatment of PH.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Bloqueadores dos Canais de Cálcio/farmacologia , Hipertensão Pulmonar , Monocrotalina/toxicidade , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Quimioterapia Combinada , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Masculino , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Citrato de SildenafilaRESUMO
BACKGROUND: Pulmonary hypertension (PH) causes elevated right ventricular (RV) systolic pressure, RV remodeling and finally RV failure to death. However, the mechanisms of RV remodeling in PH remain to be fully elucidated. METHODS AND RESULTS: RV autopsy samples from 6 PH patients with RV failure against 3 age- and sex-matched controls were first examined. Next, RV remodeling in 2 mouse models of chronic hypoxia-induced PH with endothelial nitric oxide synthase-deficient (eNOS(-/-)) and collagenase-resistant knock-in (Col(R/R)) mice were examined. In humans, RV failure was associated with RV hypertrophy, interstitial and perivascular fibrosis, decreased RV capillary density and increased macrophage recruitment. Furthermore, immunostaining showed that perivascular matrix metalloproteinase-2 was increased in PH patients with RV failure. In animals, both hypoxic eNOS(-/-) and Col(R/R) mice developed a greater extent of RV hypertrophy, perivascular remodeling and macrophage infiltration compared with wild-type mice. Capillary rarefaction was developed in hypoxic eNOS(-/-) mice, while Col(R/R) mice were able to increase their capillary density in the RV in response to chronic hypoxia. Both mouse models showed increased autophagy even under normoxic condition. CONCLUSIONS: These results indicate that RV remodeling occurs early during PH development through fibrosis, perivascular remodeling, capillary rarefaction and autophagy, in which the eNOS pathway and collagen metabolism might be involved.
Assuntos
Colágeno/metabolismo , Hipertensão Pulmonar/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Remodelação Ventricular , Adulto , Animais , Autofagia/genética , Colágeno/genética , Feminino , Fibrose , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/genéticaRESUMO
The objective of this study was to evaluate the effect of drug loading and the effect of excipients on the release pattern of tamsulosin tydrochloride from in situ PLGA implants formed in vitro in gelatin gel. This system is prepared by dissolving a biodegradable polymer (DL-PLGA 70K) in biocompatible solvent, dimethyl sulfoxide (DMSO). Then either the drug or drug with excipients was added to it. The drug solution was poured into the hollow of gelatin gel, the solvent dissipated into the surrounding gelatin base through diffusion leading phase separation and subsequent coagulation of the polymer. The drug formed a rod like implant in situ. Two types of implants were prepared such as implants containing tamsulosin hydrochloride and implants containing tamsulosin hydrochloride with biocompatible excipients such as Tween 20, Tween 60, Span 20, Span 80, Chremophore EL, or Chremophore RH 40. In vitro dissolution studies were performed in static condition using phosphate buffer (pH 7.4) to observe the release of drugs from these implants for 10 days. Formulation containing only tamsulosin hydrochloride showed that drug loading was 83.54%, 90.23%, 86.72%, 89.17% and 94.08% against the actual drug content of 9.09%, 13.04%, 16.67%, 20% and 23.08% respectively. The release rate of drug was 64.51%, 70.64%, 74.08%, 76.12% and 80.05% accordingly. It can be concluded that the release rate of drug increases with increasing drug concentrations. The other formulation containing tamsulosin with excipients showed that the release rate was 74.70%, 75.14%, 60.03%, 63.83%, 70.82% and 76.43% against same conc. of drug (8.7% of drug) but different excipients such as tween 20, tween 60, span 20, span 80, chremophore EL and chremophore RH 40 respectively. The loading efficiency was 79.33%, 87.34%, 91.91%, 94.19%, 88.48% and 95.34% respectively. It can be concluded that excipient lowers the release rate of the drug and may prolong the activity and overall release kinetics.