RESUMO
Although OPC-28326, 4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl) piperidine hydrochloride monohydrate, was developed as a selective peripheral vasodilator with α2-adrenergic antagonist properties, it also reportedly exhibits angiogenic activity in an ischemic leg model. The purpose of this study was to examine the effect of OPC-28326 on the architectural dynamics and function of the infarcted left ventricle during the chronic stage of myocardial infarction. Myocardial infarction was induced in male C3H/He mice, after which the mice were randomly assigned into two groups: a control group receiving a normal diet and an OPC group whose diet contained 0.05% OPC-28326. The survival rate among the mice (n = 18 in each group) 4 wk postinfarction was significantly greater in the OPC than control group (83 vs. 44%; P < 0.05), and left ventricular remodeling and dysfunction were significantly mitigated. Histologically, infarct wall thickness was significantly greater in the OPC group, due in part to an abundance of nonmyocyte components, including blood vessels and myofibroblasts. Five days postinfarction, Ki-67-positive proliferating cells were more abundant in the granulation tissue in the OPC group, and there were fewer apoptotic cells. These effects were accompanied by activation of myocardial Akt and endothelial nitric oxide synthase. Hypoxia within the infarct issue, assessed using pimonidazole staining, was markedly attenuated in the OPC group. In summary, OPC-28326 increased the nonmyocyte population in infarct tissue by increasing proliferation and reducing apoptosis, thereby altering the tissue dynamics such that wall stress was reduced, which might have contributed to a mitigation of postinfarction cardiac remodeling and dysfunction.
Assuntos
Indutores da Angiogênese/farmacologia , Compostos de Anilina/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Piperidinas/farmacologia , Vasodilatadores/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Antígeno Ki-67/metabolismo , Camundongos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taxa de Sobrevida , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologiaRESUMO
BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.
RESUMO
Although recanalization of the infarct-related artery late after myocardial infarction (MI) is known to reduce both cardiac remodeling and mortality, the mechanisms responsible are not yet fully understood. We compared infarcted rat hearts in which the infarct-related coronary artery was opened 24 hours after infarction (late reperfusion [LR] group) with those having a permanently occluded artery. Left ventricular dilatation and dysfunction were significantly mitigated in the LR group 1, 2, and 4 weeks post-MI. Attributable, in large part, to the greater number of cells present, the infarcted wall was significantly thicker in the LR group, which likely reduced wall stress and mitigated cardiac dysfunction. Granulation tissue cell proliferation was increased to a greater degree in the LR group 4 days post-MI, whereas the incidence of apoptosis was significantly lower throughout the subacute stage (4 days, 1 week, and 2 weeks post-MI), further suggesting preservation of granulation tissue cells contributes to the thick, cell-rich scar. Functionally, myocardial debris was more rapidly removed from the infarcted areas in the LR group during subacute stages, and stouter collagen was more rapidly synthesized in those areas. Direct acceleration of Fas-mediated apoptosis by hypoxia was confirmed in vitro using infarct tissue-derived myofibroblasts. In salvaged cardiomyocytes, degenerative changes, but not apoptosis, were mitigated in the LR group, accompanied by restoration of GATA-4 and sarcomeric protein expression. Along with various mechanisms proposed earlier, the present findings appear to provide an additional pathophysiological basis for the benefits of late reperfusion.
Assuntos
Infarto do Miocárdio/fisiopatologia , Reperfusão Miocárdica , Miócitos Cardíacos , Remodelação Ventricular , Animais , Apoptose , Proteínas de Transporte/metabolismo , Colágeno/biossíntese , Proteínas do Citoesqueleto , Fibroblastos/metabolismo , Fibroblastos/patologia , Fator de Transcrição GATA4/metabolismo , Humanos , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Reperfusão Miocárdica/métodos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
BACKGROUND: In patients with end-stage renal disease, angiotensin II type 1A receptor (AT1) blockade attenuates the associated cardiac dysfunction. We investigated the molecular signaling mediating that effect. METHODS AND RESULTS: We used 5/6 nephrectomy to induce significant renal dysfunction in AT1 knockout (AT1KO) and wild-type mice (WT). Twelve weeks after nephrectomy, WT showed significant left ventricular dilation and dysfunction that were accompanied by cardiomyocyte hypertrophy, fibrosis, and reduced capillary density. All of these effects were significantly mitigated in AT1KO. Nephrectomy led to upregulation of myocardial expression of AT1, transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and phosphorylated Akt (p-Akt), and also led to increased oxidative damage in cardiomyocytes. In AT1KO, TGF-beta1, TIMP-1, oxidative damage levels were lower, whereas MMPs and p-Akt levels were higher. Treating nephrectomized WT mice with valsartan (an AT1 blocker), but not hydralazine, improved cardiac function and altered molecular signaling in a manner similar to that seen in AT1KO mice. Notably, AT1 expression was downregulated in valsartan-treated but not hydralazine-treated hearts. CONCLUSIONS: These findings provide novel insight into the mechanism underlying the beneficial effects of AT1 blockade on cardiac function in a model of renal dysfunction-associated heart failure.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Insuficiência Cardíaca/etiologia , Falência Renal Crônica/prevenção & controle , Receptor Tipo 1 de Angiotensina/sangue , Transdução de Sinais/fisiologia , Tetrazóis/uso terapêutico , Valina/análogos & derivados , Animais , Nitrogênio da Ureia Sanguínea , Western Blotting , Creatinina/sangue , DNA/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Imuno-Histoquímica , Falência Renal Crônica/complicações , Falência Renal Crônica/metabolismo , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Nefrectomia/efeitos adversos , Estresse Oxidativo/genética , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Valina/uso terapêutico , Valsartana , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
In order to examine the ability of the umu test detecting system, 18 antitumor drugs were tested using the Salmonella umu test. The tested antitumor drugs were selected so as to produce different biochemical actions, and they were classified into three categories; five agents of group I (antimetabolites), eight agents of group II (alkylating agents), and five agents of group III (antibiotics). The results showed that all antimetabolites, all alkylating agents, and three of the antibiotics had positive responses, but the antibiotics aclarubicin (ACR) and chromomycin A3 (CHR) had negative responses. Both antibiotics that gave negative responses were anthracyclines, but daunomycin (DNR), which was one of the anthracyclines, had a positive result in the umu test. These results suggest that it is possible for the umu test to detect genotoxicity of chemicals regardless of the types of DNA damage (inhibition of DNA synthesis relative enzyme, DNA base alkylating, DNA strand-break, and DNA adduct), but difficult for it to detect genotoxicity of any anthracyclines.
Assuntos
Antineoplásicos/toxicidade , Dano ao DNA , Testes de Mutagenicidade/métodos , Salmonella typhimurium/efeitos dos fármacosRESUMO
The liver micronucleus (MN) assay is useful for predicting genotoxic rodent hepatocarcinogenicity. We have recently established the repeated-dose liver MN (RDLMN) assay in rats for integration into general toxicity studies. To investigate the effectiveness of the RDLMN assay, the genotoxic rodent hepatocarcinogen, monocrotaline (MCT), was administered by oral gavage to 6-week old male rats once daily for 14 days at 0.5 and 1.5mg/kg/day, and for 28 days at 0.15, 0.5, 1.5, 3.75, 7.5 and 15mg/kg/day. Then, MN induction was measured in the liver and bone marrow (BM), and histopathological hepatotoxicity was examined. Additionally, in order to evaluate the effects of repeated dosing periods on MN inducibility, a double-dose examination of MCT at doses of 15, 30 and 60mg/kg/day in juvenile (26-days old) and young adult (7-weeks old) rats was also conducted, as an acute dose MN assay. The peripheral blood (PB) and liver were sampled at 48h and 4 days after the second dosing, respectively. In the repeated-dose MN assay, MCT produced a positive result in the liver at a non-hepatotoxic lower dose level, but not in the BM at any dose level. In contrast, in the double-dose MN assay, MCT showed a negative result in the young adult rat livers, although it gave positive responses in the livers of juvenile rats and in the PB with both age groups. The maximum dose used in the repeated-dose assay was considerably lower than that used in the acute dose assay. These results suggest that a repeated dosing regimen is more suitable for the liver MN assay using young adult rats than an acute dose regimen, and the RDLMN assay might be capable of detecting genotoxic rodent hepatocarcinogens at dose levels that are typically undetectable in BM MN assays.
Assuntos
Carcinógenos/toxicidade , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Testes para Micronúcleos , Monocrotalina/toxicidade , Administração Oral , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Comportamento Cooperativo , Relação Dose-Resposta a Droga , Esquema de Medicação , Hepatócitos/patologia , Humanos , Japão , Leucócitos Mononucleares/efeitos dos fármacos , Fígado/patologia , Masculino , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacos , Sociedades FarmacêuticasRESUMO
As part of a collaborative study by the Collaborative Study Group for Micronucleus Test (CSGMT) of the Mammalian Mutagenicity Study Group (MMS) in the Japanese Environmental Mutagen Society (JEMS), the present study evaluated the effectiveness of the repeated dose liver micronucleus (RDLMN) assay. Two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF), were administered orally to male rats (6 weeks old at the initial dosing) once daily for 14 and 28 days to evaluate the micronucleus (MN) inducibility in the liver. In addition, these chemicals were evaluated for MN inducibility in the bone marrow (BM) and gastrointestinal (GI) tract, i.e. glandular stomach and colon of the same animals used in the RDLMN assay. As a result, both chemicals produced positive results in the liver, although a weak positive response was given by 2-AAF. DMN gave negative results in the tissues other than the liver. 2-AAF produced positive responses in the BM and glandular stomach, and a prominent response was particularly observed in the glandular stomach, which is directly exposed to the test chemicals by gavage. The present results suggest that the RDLMN assay is a useful method for detecting genotoxic hepatocarcinogens, and that it is especially effective for evaluating test chemicals, such as DMN, undetectable by the BM and GI tract MN assay. Moreover, the results in this investigation indicate that the use of multiple tissues in the study integrating the MN tests is more effective than using a single tissue, for detection of the MN induction produced by chemical exposure to rats, and helps to determine the characteristics of the test chemicals.
Assuntos
2-Acetilaminofluoreno/toxicidade , Carcinógenos/toxicidade , Dimetilnitrosamina/toxicidade , Trato Gastrointestinal/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Testes para Micronúcleos , Administração Oral , Animais , Medula Óssea/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Comportamento Cooperativo , Relação Dose-Resposta a Droga , Esquema de Medicação , Humanos , Japão , Masculino , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Sociedades FarmacêuticasRESUMO
The most critical determinant of prognosis in patients with acute myocardial infarction (MI) is infarct magnitude, which can be established within several hours of an attack. The importance of the subsequent healing process is not negligible, however. In fact, much experimental and clinical evidence suggests that late reperfusion of the infarct-related coronary artery--i.e. at times too late to salvage the myocardium within the area at risk-is beneficial for reducing left ventricular remodelling and decreasing mortality ('open artery hypothesis'). For instance, one recent study highlighted the beneficial effects of late reperfusion therapy on the infarct tissue cell dynamics following acute MI. Nonetheless, several recent large, randomized clinical trials have failed to provide evidence of such benefits, refuting the clinical efficacy of late reperfusion. In addition, they also underscore the need for revised clinical studies in which there is less heterogeneity in the timing of reperfusion and in the initial infarct size, as well as the need for sustained patency of the recanalized artery. This review focuses on the effects of late reperfusion on the pathophysiology of MI in the context of the infarct tissue dynamics and clinical outcomes. We also discuss the issues that need to be resolved to improve clinical application.
Assuntos
Angioplastia Coronária com Balão , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Reperfusão Miocárdica/métodos , Miocárdio/patologia , Remodelação Ventricular , Angioplastia Coronária com Balão/efeitos adversos , Animais , Humanos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Reperfusão Miocárdica/efeitos adversos , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
To address whether adult rat ventricular cardiomyocytes (ARVCs) exposed to oxidant stress die via apoptosis (secondarily by necrosis) or primarily by necrosis, we exposed ARVCs to hydrogen peroxide (H2O2; 0.1-100 microM) for up to 24 h and then compared them with isoproterenol-induced apoptotic and Triton X-induced necrotic controls. Cellular shrinkage preceded plasma membrane disruption, reflected by trypan blue uptake in ARVCs exposed to lower concentrations of H2O2 (<1 microM; an apoptotic pattern), but the order was reversed in cells exposed to higher concentrations of H2O2 (>1 microM; a necrotic pattern). DNA fragmentation, caspase-3 activation, mitochondrial membrane potential preservation, and ATP preservation were all apparent in ARVCs treated with low H2O2 (0.5 microM), but not in those treated with high H2O2 (10 microM). In addition, electron microscopy revealed unique morphology in H2O2-treated ARVCs; i.e., the nuclei had a homogeneous ground glass-like appearance that was never accompanied by chromatin condensation. Apparently, high concentrations of H2O2 caused primary necrosis in ARVCs, whereas low concentrations induced biochemically comparable apoptosis, although the latter did not satisfy the morphological criteria of apoptosis. These findings caution against the use of oxidant stress, H2O2 in particular, as an inducer of apoptosis in ARVCs.
Assuntos
Apoptose , Peróxido de Hidrogênio/farmacologia , Miócitos Cardíacos/citologia , Oxidantes/farmacologia , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Fragmentação do DNA , Ventrículos do Coração/citologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/ultraestrutura , Modelos Animais , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Necrose , Ratos , Ratos Sprague-DawleyRESUMO
We hypothesized that therapy, composed of antiapoptotic soluble Fas (sFas) gene transfer, combined with administration of the cardioprotective cytokine granulocyte colony-stimulating factor (G-CSF), would markedly mitigate cardiac remodeling and dysfunction following myocardial infarction (MI). On the 3rd day after MI induced by ligating the left coronary artery in mice, four different treatments were initiated: saline injection (Group C, n = 26); G-CSF administration (Group G, n = 27); adenoviral transfer of sFas gene (Group F, n = 26); and the latter two together (Group G+F, n = 26). Four weeks post-MI, Group G+F showed better survival than Group C (96 vs. 65%, P < 0.05) and the best cardiac function among the four groups. In Group G, the infarct scar was smaller and less fibrotic, whereas in Group F the scar was thicker, without a reduction in area, and contained abundant myofibroblasts and vascular cells; Group G+F showed both phenotypes. G-CSF exerted a beneficial effect on infarct tissue dynamics through antifibrotic and proliferative effects on granulation tissue; however, it also exerts an adverse proapoptotic effect that leads to thinning of the infarct scar. sFas appeared to offset the latter drawback. In vitro study using cultured myofibroblasts derived from the infarct tissue revealed that G-CSF increased proliferating activity of those cells accompanying activation of Akt and signal transducer and activator of transcription 3, while accelerating Fas-mediated apoptosis with increasing Bax-to-Bcl-2 ratio. The results suggest that combined use of G-CSF administration and sFas gene therapy is a potentially powerful tool against post-MI heart failure.
Assuntos
Apoptose , Cardiotônicos/farmacologia , Técnicas de Transferência de Genes , Terapia Genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Insuficiência Cardíaca/terapia , Infarto do Miocárdio/terapia , Miocárdio/patologia , Receptor fas/genética , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Terapia Combinada , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Vetores Genéticos , Tecido de Granulação/efeitos dos fármacos , Tecido de Granulação/patologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Lenograstim , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , Regeneração/genética , Fatores de Tempo , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/terapia , Remodelação Ventricular/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismoRESUMO
Although beneficial effects of granulocyte colony-stimulating factor (G-CSF) have been demonstrated on post-myocardia infarction (MI) process, the mechanisms and feasibility are not fully agreed yet. We investigated effects of a long-term treatment with a low-dose G-CSF started 1 day after the onset of MI, on post-infarction process. One day after being made MI by left coronary ligation, mice were given G-CSF (10 microg/kg/day) for 4 weeks. The G-CSF treatment resulted in a significant mitigation of cardiac remodelling and dysfunction. In the G-CSF-treated hearts, the infarcted scar was smaller with less fibrosis and abundant vessels while in the non-infarcted area, hypertrophic cardiomyocytes with attenuated degenerative changes and reduced fibrosis were apparent. These effects were accompanied by activation of signal transducer and activator of transcription 3 (STAT3) and Akt and also by up-regulation of GATA-4, myosin heavy chain and matrix metalloproteinases-2 and -9. Apoptosis of cardiomyocytes appeared insignificant at any stages. Parthenolide, a STAT3 inhibitor, completely abolished the beneficial effects of G-CSF on cardiac function and remodelling with loss of effect on both anti-cardiomyocyte degeneration and anti-fibrosis. In contrast, wortmannin, an Akt inhibitor, did not affect G-CSF-induced benefits despite cancelling vessel increase. In conclusion, treatment with G-CSF at a small dose but for a long duration beneficially affects the post-infarction process possibly through STAT3-mediated anti-cardiomyocyte degeneration and anti-fibrosis, but not through anti-cardiomyocyte apoptosis or Akt-mediated angio-genesis. The findings may also imply a more feasible way of G-CSF administration in the clinical settings.
Assuntos
Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/patologia , Animais , Apoptose/efeitos dos fármacos , Contagem de Células Sanguíneas , Sobrevivência Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Testes de Função Cardíaca , Humanos , Janus Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/enzimologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Recombinantes , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Abnormal vascular smooth muscle cell (VSMC) proliferation and migration are involved in restenosis following percutaneous transluminal angioplasty (PTCA) as well as in the development and progression of atherosclerosis. We investigated the mechanisms underlying the inhibitory effect of the sesquiterpene 3-oxo-5alphaH,8betaH-eudesma-1,4(15),7(11)-trien-8,12-olide (1) on rat VSMC proliferation and migration. VSMCs were isolated from rat aorta, and then the effect of 1 on cell proliferation and migration was examined using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and chemotaxis assays, respectively. Compound 1 had a potent inhibitory effect on fetal calf serum-induced VSMC proliferation. This effect correlated with reduced expression of cyclin D(1). In addition, 1 also inhibited platelet derived growth factor (PDGF)-induced migration of VSMCs. These results indicate that 1 is a promising candidate for additional biological evaluation to further define its potential as an inhibitory modulator of VSMC responses that contribute to restenosis following PTCA and to the development and progression of atherosclerosis.
Assuntos
Ciclo Celular/efeitos dos fármacos , Músculo Liso Vascular/citologia , Sesquiterpenos/farmacologia , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Ciclina D1/biossíntese , Depressão Química , Fase G1/efeitos dos fármacos , Indicadores e Reagentes , Lactonas/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/biossíntese , Ratos , Ratos Sprague-Dawley , Sais de Tetrazólio , TiazóisRESUMO
Blockade of angiotensin II type 1 receptor (AT1) signaling attenuates heart failure following myocardial infarction (MI), perhaps through reduction of fibrosis in the noninfarcted myocardium. However, its specific effect on the infarct tissue itself has not been fully clarified, which we examined in the present study. After MI induction in mice, treatment with the AT1 blocker olmesartan, beginning on the 3rd day post-MI, significantly improved survival (94%) 4 wk post-MI, compared with saline (53%) and hydralazine (73%). Olmesartan-treated mice also showed significant attenuation of left ventricular dilatation and dysfunction, as well as significantly greater infarct wall thickness, although the absolute size of the infarct scar was unchanged. In addition, significantly greater numbers of nonmyocytes (mainly vascular cells and myofibroblasts) were present within the infarct scar in olmesartan-treated hearts. Ten days post-MI, apoptosis among granulation tissue cells was significantly suppressed in the olmesartan-treated hearts, where expression of Fas, Bax, procaspase-3, and Daxx and activation of caspase-3, c-Jun NH(2)-terminal kinase, and c-Jun were all significantly attenuated. By contrast, expression of Fas ligand, Bcl-2, and Fas-associated death domain and activation of caspase-8 were unaffected, suggesting olmesartan exerts a negative regulatory effect on the alternate pathway downstream of Fas receptor. In vitro, olmesartan dose-dependently inhibited Fas-mediated apoptosis in granulation tissue-derived myofibroblasts. The present study proposes this antiapoptotic effect as another important mechanism for an AT1 blocker in improving post-MI ventricular remodeling, as well as its antifibrotic effect, and also suggests a significant link between renin-angiotensin and Fas/Fas ligand systems in postinfarction hearts.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Imidazóis/farmacologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Tetrazóis/farmacologia , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação Ventricular/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/complicações , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/patologiaRESUMO
It is not well-known yet how granulocyte colony-stimulating factor (G-CSF) affects nonischemic cardiomyopathy, though its beneficial effects on acute myocardial infarction are well-established. We hypothesize that G-CSF beneficially might affect nonischemic cardiomyopathy through the direct cardioprotective effects. Here, we show that a single injection of doxorubicin (DOX, 15 mg/kg) induced left ventricular dilatation and dysfunction in mice within 2 weeks, and that these effects were significantly attenuated by human recombinant G-CSF (100 microg/kg/day for 5 days). G-CSF also protected hearts against DOX-induced cardiomyocyte atrophy/degeneration, fibrosis, inflammatory cell infiltration and down regulation of GATA-4 and sarcomeric proteins, myosin heavy chain, troponin I and desmin, both in vivo and in vitro. Cardiac cyclooxygenase-2 was upregulated and G-CSF receptor was downregulated in DOX-induced cardiomyopathy, but both of those effects were largely reversed by G-CSF. No DOX-induced apoptotic effects were seen, nor were there any changes in tumor necrosis factor-alpha or transforming growth factor-beta1 levels. Among downstream mediators of G-CSF receptor signaling, DOX-induced cardiomyopathy involved inactivation of extracellular signal-regulated protein kinase (ERK); the ERK inactivation was reversed by G-CSF. Inhibition of ERK activation, but not cyclooxygenase-2 inhibition, completely abolished beneficial effect of G-CSF on cardiac function. G-CSF did not promote differentiation of bone marrow cells into cardiomyocytes according to the experiment using green fluorescent protein-chimeric mice, and inhibition of CXCR4+ cell homing using AMD3100 did not diminish the effect of G-CSF. Finally, G-CSF was also effective when administered after cardiomyopathy was established. In conclusion, these findings imply the therapeutic usefulness of G-CSF mainly through restoring ERK activation against DOX-induced nonischemic cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/prevenção & controle , Cardiotônicos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Disfunção Ventricular Esquerda/prevenção & controle , Animais , Animais Recém-Nascidos , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doxorrubicina/toxicidade , Combinação de Medicamentos , Quimioterapia Combinada , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Proteínas Recombinantes , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/patologiaRESUMO
Blockade of ANG II type 1A receptor (AT(1A)) is known to attenuate postinfarction [postmyocardial infarction (post-MI)] heart failure, accompanying reduction in fibrosis of the noninfarcted area. In the present study, we investigated the influence of AT(1A) blockade on the infarcted tissue itself. Consistent with earlier reports, AT(1A) knockout (AT(1A)KO) mice showed significantly attenuated left ventricular (LV) remodeling (dilatation) and dysfunction compared with wild-type (WT) mice. Morphometry revealed that the infarcted wall was thicker and had a smaller circumferential length in AT(1A)KO than WT hearts. In addition, significantly greater numbers of cells were present within infarcts in AT(1A)KO hearts 4 wk post-MI; most notably, there was an abundance of vessels and myofibroblasts. One week post-MI, the incidence of apoptosis among granulation tissue cells was fewer (3.3 +/- 0.4 vs. 4.4 +/- 0.5% in WT, P < 0.05), whereas vessel proliferation was higher in AT(1A)KO hearts, which likely explains the later abundance of cells within the scar tissue. Insulin-like growth factor receptor-I was upregulated and its downstream signal protein kinase B (Akt) was significantly activated in infarcted AT(1A)KO hearts compared with WT hearts. Inactivation of Akt with wortmannin partially but significantly prevented the benefits observed in AT(1A)KO. Collectively, in AT(1A)KO hearts, Akt-mediated granulation tissue cell proliferation and preservation resulting from antiapoptosis likely contributed to an abundant cell population that altered the infarct scar structure, thereby reducing wall stress and attenuating LV dilatation and dysfunction at the chronic stage. In conclusion, altered structural dynamics of infarct scar and increasing myocardial fibrosis may be responsible for the deleterious effects of AT(1A) signaling following MI.
Assuntos
Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais , Androstadienos/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Apoptose , Pressão Sanguínea/efeitos dos fármacos , Proliferação de Células , Fibrose , Tecido de Granulação/metabolismo , Tecido de Granulação/patologia , Hidralazina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Função Ventricular Esquerda , Remodelação Ventricular , WortmaninaRESUMO
Although amlodipine, a long-acting L-type calcium channel blocker, reportedly prevents left ventricular remodeling and dysfunction after myocardial infarction, the mechanism responsible is not yet well understood. Myocardial infarction was induced in mice by ligating the left coronary artery. Treatment of mice with amlodipine (10 mg x kg(-1) x day(-1)), beginning on the third day postinfarction, significantly improved survival and attenuated left ventricular dilatation and dysfunction 4 wk postinfarction compared with treatment with saline or hydralazine. Although infarct sizes did not differ among the groups, the infarcted wall thickness was greater and the infarct segment length was smaller in the amlodipine-treated group, and cellular components, including vessels and myofibroblasts, were abundant within the infarcted area. Ten days postinfarction (the subacute stage), the proliferation of granulation tissue cells in the infarcted area was similar among the groups, but the incidence of apoptosis was significantly lower in the amlodipine-treated group, where Bad, a proapoptotic Bcl-2 family protein, was significantly phosphorylated (inactivated). Calcineurin, which dephosphorylates (activates) Bad, was upregulated in infarcted hearts, but its levels were significantly reduced by amlodipine treatment. In vitro, Fas stimulation augmented calcineurin activity and induced apoptosis among infarct tissue-derived myofibroblasts; both of those effects were strongly inhibited by amlodipine, two other calcium channel blockers (verapamil or nifedipine), and two calcineurin inhibitors (cyclosporin A or FK-506). Amlodipine inhibits Fas-mediated granulation tissue cell apoptosis in infarcted hearts, possibly by attenuating the activities of calcineurin and Bad. These findings may provide new insight into the mechanism by which calcium channel blockers attenuate postinfarction cardiac remodeling and dysfunction.
Assuntos
Anlodipino/farmacologia , Apoptose/efeitos dos fármacos , Calcineurina/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Tecido de Granulação/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular/efeitos dos fármacos , Anlodipino/uso terapêutico , Animais , Inibidores de Calcineurina , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/metabolismo , Células Cultivadas , Vasos Coronários/cirurgia , Ciclosporina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Tecido de Granulação/metabolismo , Tecido de Granulação/patologia , Tecido de Granulação/fisiopatologia , Hidralazina/farmacologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Fosforilação , Projetos de Pesquisa , Tacrolimo/farmacologia , Fatores de Tempo , Vasodilatadores/farmacologia , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda/efeitos dos fármacos , Proteína de Morte Celular Associada a bcl/metabolismo , Receptor fas/metabolismoRESUMO
We examined effects of a physiologic concentration of pitavastatin (0.01 micromol/L) on oxidant-induced apoptosis in cultured human vascular smooth muscle cells (VSMCs). Apoptosis was induced in VSMCs by hydrogen peroxide (H2O2, 300 micromol/L), as evidenced by in situ nick end-labeling and scanning electron microscopy. This apoptotic response was accompanied by increased activation of mitogen-activated protein kinases (MAPKs--ie, increases in the phosphorylated forms of extracellular signal-regulated kinase (p-ERK), c-Jun N-terminal kinase (p-JNK), and p38 MAPK (p-p38 MAPK). Although pitavastatin alone did not induce VSMC death, pretreatment with pitavastatin significantly enhanced H2O2-induced apoptosis and prolonged activation of JNK and p38 MAPK (for up to 24 h) but not ERK. Expression of MAPK phosphatase-1 (MKP-1) also was upregulated by H2O2, but this was not affected by pitavastatin. The apoptosis accelerating effect was observed also in simvastatin but not in pravastatin. Treating VSMCs with mevalonate, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate completely blocked the statin-induced enhancement of VSMC apoptosis, suggesting that protein prenylation is critically involved. It thus appears that pitavastatin enhances H2O2-induced VSMC apoptosis, at least in part, via increases in MAPK activation and protein prenylation, but independently of MKP-1 expression, which consequently results in reduction of VSMC population.