RESUMO
Anti-apoptotic therapy for cardiomyocytes could be an effective strategy for preventing or treating heart failure. Notably, however, morphological evidence definitively demonstrating cardiomyocyte apoptosis has been very rare in actual heart diseases such as acute myocardial infarction and heart failure. By contrast, within the postinfarction heart, interstitial noncardiomyocytes such as granulation tissue cells do die via apoptosis to form scar tissue. Blockade of this apoptosis improves survival and mitigates ventricular remodeling and dysfunction during the chronic stage. Possible mechanisms to explain this benefit might be preservation of infarcted wall thickness and preservation of myofibroblasts, which could promote infarct shrinkage; both would reduce wall stress through Laplace's law. On the other hand, autophagy is an intracellular degradation mechanism that compensates for energy insufficiency by digesting and recycling intracellular components, and is often observed in cardiomyocytes within failing hearts with various origins including postinfarction. Starvation strongly induces and activates autophagic degeneration within cardiomyocytes. When that activation is inhibited, the starved animals suffer from heart failure. Promoting autophagy through caloric restriction or several reagents not only reduces the acute infarct size but also mitigates postinfarction cardiac remodeling and dysfunction during chronic stages. Moreover, augmenting autophagy by the treatment with resveratrol or exercise can bring about reverse remodeling in failing hearts with a large old myocardial infarction. In conclusion, we propose two strategies for managing postinfarction heart failure through control of cell death/degeneration: (1) anti-apoptosis in granulation tissue noncardiomyocytes; and (2) pro-autophagy in salvaged cardiomyocytes.
Assuntos
Insuficiência Cardíaca/prevenção & controle , Infarto do Miocárdio/complicações , Miocárdio/patologia , Miócitos Cardíacos/patologia , Animais , Apoptose , Autofagia , Progressão da Doença , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Treatment with granulocyte colony-stimulating factor (G-CSF) reportedly mitigates postinfarction cardiac remodeling and dysfunction. We herein examined the effects of G-CSF knockout (G-CSF-KO) on the postinfarction remodeling process in the hearts of mice. Unexpectedly, the acute infarct size 24 hours after ligation was similar in the two groups. At the chronic stage (4 weeks later), there was no difference in the left ventricular dimension, left ventricular function, or histological findings, including vascular density, between the two groups. In addition, expression of vascular endothelial growth factor (VEGF) was markedly up-regulated in hearts from G-CSF-KO mice, compared with wild-type mice. Microarray failed in detecting up-regulation of VEGF mRNA, whereas G-CSF administration significantly decreased myocardial VEGF expression in mice, indicating that G-CSF post-transcriptionally down-regulates VEGF expression. When G-CSF-KO mice were treated with an anti-VEGF antibody (bevacizumab), cardiac remodeling was significantly aggravated, with thinning of the infarct wall and reduction of the cellular component, including blood vessels. In the granulation tissue of bevacizumab-treated hearts 4 days after infarction, vascular development was scarce, with reduced cell proliferation and increased apoptosis, which likely contributed to the infarct wall thinning and the resultant increase in wall stress and cardiac remodeling at the chronic stage. In conclusion, overexpression of VEGF may compensate for the G-CSF deficit through preservation of cellular components, including blood vessels, in the postinfarction heart.
Assuntos
Fator Estimulador de Colônias de Granulócitos/genética , Infarto do Miocárdio/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Remodelação Ventricular/genética , Animais , Apoptose , Proliferação de Células , Tecido de Granulação/metabolismo , Tecido de Granulação/patologia , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/deficiência , Masculino , Camundongos , Camundongos Knockout , Infarto do Miocárdio/induzido quimicamente , Miocárdio/patologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular EsquerdaRESUMO
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
We investigated the effect of restriction of food intake, a potent inducer of autophagy, on postinfarction cardiac remodeling and dysfunction. Myocardial infarction was induced in mice by left coronary artery ligation. At 1 week after infarction, mice were randomly divided into four groups: the control group was fed ad libitum (100%); the food restriction (FR) groups were fed 80%, 60%, or 40% of the mean amount of food consumed by the control mice. After 2 weeks on the respective diets, left ventricular dilatation and hypofunction were apparent in the control group, but both parameters were significantly mitigated in the FR groups, with the 60% FR group showing the strongest therapeutic effect. Cardiomyocyte autophagy was strongly activated in the FR groups, as indicated by up-regulation of microtubule-associated protein 1 light chain 3-II, autophagosome formation, and myocardial ATP content. Chloroquine, an autophagy inhibitor, completely canceled the therapeutic effect of FR. This negative effect was associated with reduced activation of AMP-activated protein kinase and of ULK1 (a homolog of yeast Atg1), both of which were enhanced in hearts from the FR group. In vitro, the AMP-activated protein kinase inhibitor compound C suppressed glucose depletion-induced autophagy in cardiomyocytes, but did not influence activity of chloroquine. Our findings imply that a dietary protocol with FR could be a preventive strategy against postinfarction heart failure.
Assuntos
Autofagia , Privação de Alimentos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/prevenção & controle , Miócitos Cardíacos/patologia , Trifosfato de Adenosina/metabolismo , Animais , Remodelamento Atrial , Western Blotting , Peso Corporal , Cateterismo Cardíaco , Sobrevivência Celular , Células Cultivadas , Densitometria , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Lisossomos/patologia , Lisossomos/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Tamanho do Órgão , Transdução de Sinais , Ultrassonografia , Vacúolos/patologia , Vacúolos/ultraestruturaRESUMO
We investigated the effect of resveratrol, a popular natural polyphenolic compound with antioxidant and proautophagic actions, on postinfarction heart failure. Myocardial infarction was induced in mice by left coronary artery ligation. Four weeks postinfarction, when heart failure was established, the surviving mice were started on 2-week treatments with one of the following: vehicle, low- or high-dose resveratrol (5 or 50 mg/kg/day, respectively), chloroquine (an autophagy inhibitor), or high-dose resveratrol plus chloroquine. High-dose resveratrol partially reversed left ventricular dilation (reverse remodeling) and significantly improved cardiac function. Autophagy was augmented in those hearts, as indicated by up-regulation of myocardial microtubule-associated protein-1 light chain 3-II, ATP content, and autophagic vacuoles. The activities of AMP-activated protein kinase and silent information regulator-1 were enhanced in hearts treated with resveratrol, whereas Akt activity and manganese superoxide dismutase expression were unchanged, and the activities of mammalian target of rapamycin and p70 S6 kinase were suppressed. Chloroquine elicited opposite results, including exacerbation of cardiac remodeling associated with a reduction in autophagic activity. When resveratrol and chloroquine were administered together, the effects offset one another. In vitro, compound C (AMP-activated protein kinase inhibitor) suppressed resveratrol-induced autophagy in cardiomyocytes, but did not affect the events evoked by chloroquine. In conclusion, resveratrol is a beneficial pharmacological tool that augments autophagy to bring about reverse remodeling in the postinfarction heart.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/enzimologia , Transdução de Sinais , Estilbenos/uso terapêutico , Remodelação Ventricular , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células Cultivadas , Densitometria , Metabolismo Energético/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Testes de Função Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resveratrol , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Superóxido Dismutase/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Remodelação Ventricular/efeitos dos fármacosRESUMO
Ischemia is known to potently stimulate autophagy in the heart, which may contribute to cardiomyocyte survival. In vitro, transfection with small interfering RNAs targeting Atg5 or Lamp-2 (an autophagy-related gene necessary, respectively, for the initiation and digestion step of autophagy), which specifically inhibited autophagy, diminished survival among cultured cardiomyocytes subjected to anoxia and significantly reduced their ATP content, confirming an autophagy-mediated protective effect against anoxia. We next examined the dynamics of cardiomyocyte autophagy and the effects of manipulating autophagy during acute myocardial infarction in vivo. Myocardial infarction was induced by permanent ligation of the left coronary artery in green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) transgenic mice in which GFP-LC3 aggregates to be visible in the cytoplasm when autophagy is activated. Autophagy was rapidly (within 30 min after coronary ligation) activated in cardiomyocytes, and autophagic activity was particularly strong in salvaged cardiomyocytes bordering the infarcted area. Treatment with bafilomycin A1, an autophagy inhibitor, significantly increased infarct size (31% expansion) 24 h postinfarction. Interestingly, acute infarct size was significantly reduced (23% reduction) in starved mice showing prominent autophagy before infarction. Treatment with bafilomycin A1 reduced postinfarction myocardial ATP content, whereas starvation increased myocardial levels of amino acids and ATP, and the combined effects of bafilomycin A1 and starvation on acute infarct size offset one another. The present findings suggest that autophagy is an innate and potent process that protects cardiomyocytes from ischemic death during acute myocardial infarction.
Assuntos
Autofagia/fisiologia , Oclusão Coronária/complicações , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/antagonistas & inibidores , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Macrolídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Infarto do Miocárdio/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Interferente Pequeno/farmacologiaRESUMO
Activation of Fas signaling is a key mediator of doxorubicin cardiotoxicity, which involves both cardiomyocyte apoptosis and myocardial inflammation. In this study, acute cardiotoxicity was induced in mice by doxorubicin, and some mice simultaneously received an intramuscular injection of adenoviral vector encoding mouse soluble Fas (sFas) gene (Ad.CAG-sFas), an inhibitor of Fas/Fas ligand interaction. Two weeks later, left ventricular dilatation and dysfunction were apparent in the LacZ-treated control group, but both were significantly mitigated in the sFas-treated group. The in situ nick-end labeling-positive rate were similar in the two groups, and although electron microscopy revealed cardiomyocyte degeneration, no apoptotic structural features and no activation of caspases were detected, suggesting an insignificant role of apoptosis in this model. Instead, sFas treatment reversed doxorubicin-induced down-regulation of GATA-4 and attenuated ubiquitination of myosin heavy chain and troponin I to preserve these sarcomeric proteins. In addition, doxorubicin-induced significant leukocyte infiltration, fibrosis, and oxidative damage to the myocardium, all of which were largely reversed by sFas treatment. sFas treatment also suppressed doxorubicin-induced p53 overexpression, phosphorylation of c-Jun N-terminal kinase, c-Jun, and inhibitor of nuclear factor-kappaB, as well as production of cyclooxygenase-2 and monocyte chemoattractant protein-1, and it restored extracellular signal-regulated kinase activation. Therefore, sFas gene therapy prevents the progression of doxorubicin-induced acute cardiotoxicity, with accompanying attenuation of the cardiomyocyte degeneration, inflammation, fibrosis, and oxidative damage caused by Fas signaling.
Assuntos
Apoptose/fisiologia , Doxorrubicina , Terapia Genética/métodos , Cardiopatias/induzido quimicamente , Cardiopatias/terapia , Receptor fas/genética , Animais , Apoptose/genética , Dano ao DNA/genética , Ecocardiografia , Fibrose Endomiocárdica/genética , Fibrose Endomiocárdica/patologia , Fibrose Endomiocárdica/prevenção & controle , Cardiopatias/genética , Cardiopatias/patologia , Inflamação/genética , Inflamação/patologia , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/patologia , Miocárdio/ultraestrutura , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Solubilidade , Receptor fas/antagonistas & inibidoresRESUMO
Granulocyte colony-stimulating factor (G-CSF) is a potent angiogenic factor. We hypothesized that G-CSF-immersed gelatin hydrogel microspheres (G-CSF-GHMs) injected into the ischemic legs might continuously release a small amount of G-CSF to locally stimulate angiogenesis without unfavorable systemic effects. Just after ligation of the right femoral artery of BALB/c mice, recombinant human G-CSF (100-µg/kg)-immersed GHM was injected into the right hindlimb muscles; the controls included a saline-injected group, an intramuscularly injected G-CSF group, a subcutaneously injected G-CSG group, and an empty GHM-injected group. Eight weeks later, improvement of blood perfusion to the ischemic limb was significantly augmented in the G-CSF-GHM group compared with any of the control groups. Despite there being no increase in the serum concentration of G-CSF, in peripheral granulocytes, or in circulating endothelial progenitor cells, not only capillary but also arteriolar density was significantly increased in this group. Next, we started treatment with G-CSF-GHM 4 weeks after ligation to examine whether the treatment is effective if performed during the chronic stage of ischemia. The late treatment was also found to effectively improve blood flow in the ischemic leg. In conclusion, G-CSF-GHM administration is suggested to be a promising and readily usable approach to treating peripheral artery disease, applicable even during the chronic stage.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Isquemia/tratamento farmacológico , Microesferas , Doença Arterial Periférica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Gelatina/química , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Humanos , Hidrogéis , Injeções Intramusculares , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Doença Arterial Periférica/patologia , Proteínas Recombinantes , Fatores de TempoRESUMO
Anemia may accelerate angiogenesis in ischemic organs through its ability to augment tissue hypoxia-induced generation of several known angiogenic factors and to increase erythropoietin levels, which are also potently angiogenic. We examined the effect of controlled phlebotomy (bloodletting) on blood flow in a mouse ischemic leg model. We ligated the right femoral artery of BALB/c mice. In the phlebotomy group, 200 microl of blood were drawn from the tail vein once a week. After 4 wk, blood flow in the ischemic leg was significantly better in the phlebotomy group (flow ratio of the ischemic to nonischemic leg, 0.87 + or - 0.04) than the control group (0.59 + or - 0.05, P < 0.05), and capillary density was significantly higher. Repeated phlebotomy increased serum erythropoietin levels as well as the expression of hypoxia-inducible transcription factor-1alpha and vascular endothelial growth factor and both the expression and activity of Akt and endothelial nitric oxide synthase (eNOS) in ischemic legs. Treatment with wortmannin or N(omega)-nitro-l-arginine methyl ester significantly attenuated the phlebotomy-induced improvement of blood flow. In addition, fluorescence-activated cell sorting analysis revealed an increase in circulating peripheral endothelial progenitor cells in the phlebotomy group, and treatment with AMD3100, a specific inhibitor of the chemokine receptor CXCR4, blocked the beneficial effect of phlebotomy. These findings suggest that repeated phlebotomy improves blood flow in ischemic legs through an angiogenic action that involves the Akt/eNOS pathway, endothelial progenitor cell mobilization, and their complicated cross talk. An adequately controlled phlebotomy might be one method by which to induce therapeutic angiogenesis.
Assuntos
Capilares/fisiopatologia , Isquemia/terapia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Flebotomia , Androstadienos/farmacologia , Animais , Benzilaminas , Capilares/efeitos dos fármacos , Capilares/metabolismo , Capilares/patologia , Ciclamos , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Inibidores Enzimáticos/farmacologia , Eritropoetina/sangue , Artéria Femoral/cirurgia , Compostos Heterocíclicos/farmacologia , Membro Posterior , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Isquemia/fisiopatologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NG-Nitroarginina Metil Éster/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Fluxo Sanguíneo Regional , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , WortmaninaRESUMO
To examine the functional significance and morphological characteristics of starvation-induced autophagy in the adult heart, we made green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) transgenic mice starve for up to 3 days. Electron microscopy revealed round, homogenous, electron-dense lipid droplet-like vacuoles that initially appeared in cardiomyocytes as early as 12 hours after starvation; these vacuoles were identified as lysosomes based on cathepsin D-immunopositive reactivity and acid phosphatase activity. The increase in the number of lysosomes depended on the starvation interval; typical autophagolysosomes with intracellular organelles also appeared, and their numbers increased at the later phases of starvation. Myocardial expression of autophagy-related proteins, LC3-II, cathepsin D, and ubiquitin, increased, whereas both myocardial ATP content and starvation integral decreased. Treatment with bafilomycin A1, an autophagy inhibitor, did not affect cardiac function in normally fed mice but significantly depressed cardiac function and caused significant left ventricular dilatation in mice starved for 3 days. The cardiomyocytes were occupied with markedly accumulated lysosomes in starved mice treated with bafilomycin A1, and both the myocardial amino acid content, which was increased during starvation, and the myocardial ATP content were severely decreased, potentially contributing to cardiac dysfunction. The present findings suggest a critical role of autophagy in the maintenance of cardiac function during starvation in the adult.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Peso Corporal/efeitos dos fármacos , Catepsina D/metabolismo , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Testes de Função Cardíaca , Técnicas Imunoenzimáticas , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Macrolídeos/farmacologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ratos , Ubiquitina/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Disfunção Ventricular Esquerda/metabolismoRESUMO
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
The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-beta (TGF-beta) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorin's chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-beta1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-beta signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.
Assuntos
Adenoviridae/genética , Proteínas da Matriz Extracelular/biossíntese , Terapia Genética , Vetores Genéticos , Hipertrofia Ventricular Esquerda/prevenção & controle , Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Proteoglicanas/biossíntese , Disfunção Ventricular Esquerda/prevenção & controle , Remodelação Ventricular , Animais , Apoptose , Proliferação de Células , Doença Crônica , Decorina , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fibrose , Células HeLa , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Proteoglicanas/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Tempo , Transdução Genética , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
1. In the present study, we examined the effects of inhibiting transforming growth factor (TGF)-beta in a mouse model of diabetic nephropathy. 2. An adenovirus harbouring the gene encoding soluble TGF-beta type II receptor (Ad.CAG-sTbetaRII), a competitive inhibitor of TGF-beta, was injected into hindlimb muscles (systemic delivery) of mice 5 weeks after the induction of diabetes with streptozotocin. The control group was injected with an adenovirus encoding the LacZ gene (Ad-LacZ). 3. Five weeks after administration, anti-TGF-beta gene therapy was found to have had no effect on renal function, albuminuria or glucose metabolism in mice with diabetic nephropathy. Nonetheless, this gene therapy did significantly reduce fibrosis in both glomeruli and renal tubules. These effects were accompanied by attenuation of the increased expression of alpha-smooth muscle actin normally seen in kidneys of diabetic mice and better preservation of glomerular cell numbers, although the thickness of the glomerular capillary basement membrane was unchanged. The plasma concentration of soluble TGF-beta type II receptor peaked on Day 7 after treatment, but was undetectable by Day 14. Moreover, a second treatment with Ad.CAG-sTbetaRII failed to prolong the interval of gene product expression in the blood. 4. The present anti-TGF-beta gene therapy showed a significant antifibrotic effect in a model of diabetic nephropathy, but failed to improve renal function. The inadequacy of the observed effect is likely due to the relatively short interval of gene product expression. This problem will have to be overcome if gene therapies for slowly progressing diseases, like diabetic nephropathy, are to be realised.
Assuntos
Adenoviridae/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/terapia , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Proteínas Serina-Treonina Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagem , Receptores de Fatores de Crescimento Transformadores beta/genética , Animais , Galinhas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Nefropatias Diabéticas/patologia , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Receptor do Fator de Crescimento Transformador beta Tipo II , Solubilidade , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
PURPOSE: Occlusal trauma, resulting in the destruction of alveolar bone, is a form of periodontal disease caused by excessive mechanical stress (MS) during hyperocclusion. Previously, we showed that CC chemokine ligand (CCL) 2/CCR2 receptor axis plays a crucial role in MS-dependent osteoclastogenesis. However, in the previous work, we were unable to precisely measure changes in alveolar bone profiles. In the present study, we sought to establish a precise method for evaluating alveolar bone resorption induced by hyperocclusion using micro-computed tomography. METHODS: Under anesthesia, a stainless steel wire was attached to the molars of 5-week-old C57/BL6 wild-type (WT) mice, CCL2-/- mice, and CCR2-/-mice to induce occlusal force overload. At days 0 and 7, hard tissue samples were harvested and analyzed by micro-computed tomography. RESULTS: In the WT mice, bone mineral density of the alveolar bone was significantly decreased at day 7 as compared with day 0, with marked alveolar bone resorption observed. Similarly, significant alveolar bone resorption was observed in the CCL2-/- and CCR2-/- mice at day 7 as compared with day 0. CONCLUSIONS: Micro-computed tomographic images can be used to measure changes in bone mineral density in a mouse model of hyperocclusion. This method may be useful for further investigating bone changes in other periodontal disease research fields.
Assuntos
Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/etiologia , Força de Mordida , Oclusão Dentária Traumática/complicações , Estresse Mecânico , Perda do Osso Alveolar/metabolismo , Animais , Densidade Óssea , Oclusão Dentária Traumática/metabolismo , Oclusão Dentária Traumática/fisiopatologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Alvéolo Dental/metabolismo , Microtomografia por Raio-XRESUMO
In myocardial infarction (MI), granulation tissue cells disappear via apoptosis to complete a final scarring with scanty cells. Blockade of this apoptosis was reported to improve post-MI ventricular remodeling and heart failure. However, the molecular biological mechanisms for the apoptosis are unknown. Fas and Fas ligand were overexpressed in the granulation tissue at the subacute stage of MI (1 week after MI) in mice, where apoptosis frequently occurred. In mice lacking functioning Fas (lpr strain) and in those lacking Fas ligand (gld strain), apoptotic rate of granulation tissue cells was significantly fewer compared with that of genetically controlled mice, and post-MI ventricular remodeling and dysfunction were greatly attenuated. Mice were transfected with adenovirus encoding soluble Fas (sFas), a competitive inhibitor of Fas ligand, on the third day of MI. The treatment resulted in suppression of granulation tissue cell apoptosis and produced a thick, cell-rich infarct scar containing rich vessels and bundles of smooth muscle cells with a contractile phenotype at the chronic stage (4 weeks after MI). This accompanied not only alleviation of heart failure but also survival improvement. However, the sFas gene delivery during scar tissue phase was ineffective, suggesting that beneficial effects of the sFas gene therapy owes to inhibition of granulation tissue cell apoptosis. The Fas/Fas ligand interaction plays a critical role for granulation tissue cell apoptosis after MI. Blockade of this apoptosis by interfering with the Fas/Fas ligand interaction may become one of the therapeutic strategies against chronic heart failure after large MI.
Assuntos
Apoptose/fisiologia , Tecido de Granulação/patologia , Insuficiência Cardíaca/fisiopatologia , Glicoproteínas de Membrana/fisiologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Remodelação Ventricular/fisiologia , Receptor fas/fisiologia , Adenoviridae/genética , Animais , Caspases/análise , Cicatriz/patologia , Vasos Coronários , Vírus Defeituosos/genética , Proteína Ligante Fas , Genes Sintéticos , Terapia Genética , Vetores Genéticos/uso terapêutico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/prevenção & controle , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Imunoglobulina G/genética , Ligadura , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Infarto do Miocárdio/complicações , Infarto do Miocárdio/terapia , Solubilidade , Fatores de Tempo , Receptor fas/genética , Receptor fas/uso terapêuticoRESUMO
OBJECTIVES: It is known that excessive mechanical force exerted by hyperocclusion induces occlusal trauma. However, the mechanism of the process remains unclear. In the present study, we employed an in vivo hyperocclusion rodent model to examine morphological and biological mechanisms of occlusal trauma in periodontal ligament tissue. DESIGN: To investigate alveolar bone resorption, tooth sections were stained to detect osteoclasts. To investigate the relationship between hyperocclusion and the regeneration of the cell matrix, we examined the effect of hyperocclusal force on the expression of collagens using immunohistochemistry and quantitative PCR methods. RESULTS: The arrangement of collagen fibers in the furcation area of the teeth was undisturbed before hyperocclusion (control). Type I collagen was localized in the extracellular area at the furcation and there was faint expression and localization of type XII collagen in the periodontal ligament. The number of osteoclasts significantly increased in the furcation and lingual cervical regions on day 4 after hyperocclusion was induced. Type XII collagens were gradually up-regulated following the induction of hyperocclusion, in a time-dependent manner. Although type I collagen mRNA expression was stable before and after hyperocclusion, type XII collagen mRNA was significantly up-regulated on day 2 and day 4 after hyperocclusion treatment. CONCLUSIONS: Our findings indicate that hyperocclusal force predominantly up-regulates the expression of type XII collagen in periodontal tissue, but not type I collagen, suggesting that there is a mechanism for regeneration of periodontal tissues as a response to occlusal trauma.
Assuntos
Colágeno Tipo XII/biossíntese , Oclusão Dentária , Ligamento Periodontal/metabolismo , Perda do Osso Alveolar/patologia , Animais , Fenômenos Biomecânicos , Força de Mordida , Colágeno/biossíntese , Colágeno Tipo I/metabolismo , Colágenos Associados a Fibrilas/biossíntese , Colágenos Fibrilares/biossíntese , Camundongos , Osteoclastos/metabolismo , Ligamento Periodontal/citologia , Ratos , Ratos Wistar , Regulação para CimaRESUMO
When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin ß1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin ß1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin ß1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin ß1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed in the ischemic leg tissue, even at the chronic stage. Moreover, the cells appeared functional, as evidenced by the improved blood flow. The cell type used (ECFCs), the route of administration (intravenous, not directly injected into the affected area), and the use of ligand-receptor interactions (extracellular matrix and integrins) for homing represent substantial advantages over previously reported cell therapies for the treatment of peripheral artery disease.
Assuntos
Células Progenitoras Endoteliais/transplante , Fibronectinas/agonistas , Membro Posterior/irrigação sanguínea , Integrina beta1/genética , Isquemia/terapia , Neovascularização Fisiológica , Animais , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Artéria Femoral/cirurgia , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior/metabolismo , Membro Posterior/patologia , Humanos , Injeções Intravenosas , Integrina beta1/metabolismo , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Lentivirus/genética , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução Genética , Transgenes , Transplante HeterólogoRESUMO
BACKGROUND: We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony-stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. METHODS AND RESULTS: In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 microg x kg(-1) x d(-1) of human recombinant G-CSF (G) was injected subcutaneously from 1 to 5 days after MI. Smaller left ventricular (LV) dimension, increased LV ejection fraction, and thicker infarct-LV wall were seen in G at 3 months after MI. At 2, 7, and 14 days and 3 months after MI, necrotic tissue areas were 14.2+/-1.5/13.4+/-1.1, 0.4+/-0.1/1.8+/-0.5*, 0/0, and 0/0 mm2 x slice(-1) x kg(-1), granulation areas 0/0, 4.0+/-0.7/8.5+/-1.0*, 3.9+/-0.8/5.7+/-0.7,* and 0/0 mm2 x slice(-1) x kg(-1), and scar areas 0/0, 0/0, 0/0, and 4.2+/-0.5/7.9+/-0.9* mm2 x slice(-1) x kg(-1) in G and S, respectively (*P<0.05, G versus S). Clear increases of macrophages and of matrix metalloproteinases (MMP) 1 and 9 were seen in G at 7 days after MI. This suggests that G accelerates absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues via expression of MMPs. Meanwhile, surviving myocardial tissue areas within the risk areas were significantly increased in G despite there being no difference in LV weight, LV wall area, or cardiomyocyte size between G and S. Confocal microscopy revealed significant increases of cardiomyocytes with positive 3,3,3',3'-tetramethylindocarbocyanine perchlorate and positive troponin I in G, suggesting enhanced myocardial regeneration by G. CONCLUSIONS: The acceleration of the healing process and myocardial regeneration may play an important role for the beneficial effect of post-MI G-CSF treatment.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Coração/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/patologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Tamanho Celular/efeitos dos fármacos , Cicatriz/etiologia , Cicatriz/patologia , Cicatriz/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Ecocardiografia , Tecido de Granulação/patologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Macrófagos/fisiologia , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Microscopia Confocal , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Regeneração/efeitos dos fármacosRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) is implicated in tissue regeneration, angiogenesis, and antiapoptosis. However, its chronic effects are undetermined on postinfarction left ventricular (LV) remodeling and heart failure. METHODS AND RESULTS: In mice, on day 3 after myocardial infarction (MI), adenovirus encoding human HGF (Ad.CAG-HGF) was injected into the hindlimb muscles (n=13). As a control (n=15), LacZ gene was used. A persistent increase in plasma human HGF was confirmed in the treated mice: 1.0+/-0.2 ng/mL 4 weeks later. At 4 weeks after MI, the HGF-treated mice showed improved LV remodeling and dysfunction compared with controls, as indicated by the smaller LV cavity and heart/body weight ratio, greater % fractional shortening and LV +/-dP/dt, and lower LV end-diastolic pressure. The cardiomyocytes near MI, including the papillary muscles and trabeculae, were greatly hypertrophied in the treated mice. The old infarct size was similar between the groups, but the infarct wall was thicker in the treated mice, where the density of noncardiomyocyte cells, including vessels, was greater. Fibrosis of the ventricular wall was significantly reduced in them. Examination of 10-day-old MI revealed no proliferation or apoptosis but showed augmented expression of c-Met/HGF receptor in cardiomyocytes near MI, whereas a greater proliferating activity and smaller apoptotic rate of granulation tissue cells in the HGF-treated hearts was observed compared with controls. CONCLUSIONS: Postinfarction HGF gene therapy improved LV remodeling and dysfunction through hypertrophy of cardiomyocytes, infarct wall thickening, preservation of vessels, and antifibrosis. These findings imply a novel therapeutic approach against postinfarction heart failure.
Assuntos
Adenoviridae , Vetores Genéticos/uso terapêutico , Fator de Crescimento de Hepatócito/biossíntese , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Adenoviridae/genética , Animais , Bromodesoxiuridina , Divisão Celular/efeitos dos fármacos , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Vetores Genéticos/genética , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia , Taxa de Sobrevida , Fatores de Tempo , Transdução Genética/métodos , Resultado do Tratamento , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/genética , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/genéticaRESUMO
Little is known about the association between autophagy and diabetic cardiomyopathy. Also unknown are possible distinguishing features of cardiac autophagy in type 1 and type 2 diabetes. In hearts from streptozotocin-induced type 1 diabetic mice, diastolic function was impaired, though autophagic activity was significantly increased, as evidenced by increases in microtubule-associated protein 1 light chain 3/LC3 and LC3-II/-I ratios, SQSTM1/p62 (sequestosome 1) and CTSD (cathepsin D), and by the abundance of autophagic vacuoles and lysosomes detected electron-microscopically. AMP-activated protein kinase (AMPK) was activated and ATP content was reduced in type 1 diabetic hearts. Treatment with chloroquine, an autophagy inhibitor, worsened cardiac performance in type 1 diabetes. In addition, hearts from db/db type 2 diabetic model mice exhibited poorer diastolic function than control hearts from db/+ mice. However, levels of LC3-II, SQSTM1 and phosphorylated MTOR (mechanistic target of rapamycin) were increased, but CTSD was decreased and very few lysosomes were detected ultrastructurally, despite the abundance of autophagic vacuoles. AMPK activity was suppressed and ATP content was reduced in type 2 diabetic hearts. These findings suggest the autophagic process is suppressed at the final digestion step in type 2 diabetic hearts. Resveratrol, an autophagy enhancer, mitigated diastolic dysfunction, while chloroquine had the opposite effects in type 2 diabetic hearts. Autophagy in the heart is enhanced in type 1 diabetes, but is suppressed in type 2 diabetes. This difference provides important insight into the pathophysiology of diabetic cardiomyopathy, which is essential for the development of new treatment strategies.