RESUMO
In contrast to hepatocyte growth factor (HGF), the therapeutic potential and pathophysiologic roles of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in liver diseases remain relatively unknown. To address the lack of effective pharmacologic treatments for cholestatic liver injuries, as well as to clarify the biologic features of these growth factors, we explored the effects of HB-EGF and HGF in mice with cholestatic liver injury induced by bile duct ligation (BDL). The mice were assessed 3, 5 and/or 14 days after BDL (acute, subacute and/or chronic phases, respectively) and intravenous injection of adenoviral vector expressing LacZ (control), HB-EGF, HGF, or HB-EGF and HGF. HB-EGF, HGF, or a combination of the growth factors exerted potent antioncotic (antinecrotic), antiapoptotic, anticholestatic, and regenerative effects on hepatocytes in vivo, whereas no robust antiapoptotic or regenerative effects were detected in interlobular bile ducts. Based on serum transaminase levels, the acute protective effects of HB-EGF on hepatocytes were greater than those of HGF. On the other hand, liver fibrosis and cholestasis during the chronic phase were more potently inhibited by HGF compared with HB-EGF. Compared with either growth factor alone, combining HB-EGF and HGF produced greater anticholestatic and regenerative effects during the chronic phase. Taken together, these findings suggest that HB-EGF and HGF inhibited BDL-induced cholestatic liver injury, predominantly by exerting acute cytoprotective and chronic antifibrotic effects, respectively; combining the growth factors enhanced the anticholestatic effects and liver regeneration during the chronic phase. Our results contribute to a better understanding of the pathophysiologic roles of HB-EGF and HGF, as well as to the development of novel effective therapies for cholestatic liver injuries.
Assuntos
Colestase Intra-Hepática/metabolismo , Colestase Intra-Hepática/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Adenoviridae/genética , Animais , Apoptose/genética , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/terapia , Modelos Animais de Doenças , Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento de Hepatócito/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Testes de Função Hepática , Regeneração Hepática , Masculino , Camundongos , Necrose , Transdução GenéticaRESUMO
Inflammatory bowel disease (IBD) severely affects the quality of life of patients. At present, there is no clinical solution for this condition; therefore, there is a need for innovative therapies for IBD. Hepatocyte growth factor (HGF) exerts various biological activities in various organs. However, a clinically applicable and effective HGF-based therapy for IBD has yet to be developed. In this study, we examined the therapeutic effect of injecting an adenoviral vector encoding the human HGF gene (Ad.HGF) into the hindlimbs of mice with dextran sodium sulfate (DSS)-induced colitis. Plasma levels of circulating human HGF (hHGF) were measured in injected mice. The results showed that weight loss and colon shortening were significantly lower in Ad.HGF-infected mice as compared to control (Ad.LacZ-infected) colitic mice. Additionally, inflammation and crypt scores were significantly reduced in the entire length of the colon, particularly in the distal section. This therapeutic effect was associated with increased cell proliferation and an antiapoptotic effect, as well as a reduction in the number of CD4+ cells and a decreased CD4/CD8 ratio. The levels of inflammatory, as well as Th1 and Th2 cytokines were higher in Ad.HGF-infected mice as compared to the control colitic mice. Thus, systemically circulating hHGF protein, produced by an adenovirally transduced hHGF gene introduced at distal sites in the limbs, significantly ameliorated DSS-induced colitis by promoting cell proliferation (i.e., regeneration), preventing apoptosis, and immunomodulation. Owing to its clinical feasibility and potent therapeutic effects, this method may be developed into a clinical therapy for treating IBD.
Assuntos
Adenoviridae/genética , Colite/induzido quimicamente , Colite/terapia , Sulfato de Dextrana/toxicidade , Fator de Crescimento de Hepatócito/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Fator de Crescimento de Hepatócito/genética , Humanos , Imunoprecipitação , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.
Assuntos
Neovascularização de Coroide/terapia , Endotélio Vascular/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Neovascularização Retiniana/terapia , Animais , Anticorpos/farmacologia , Antígenos CD/genética , Células Cultivadas , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/administração & dosagem , Ratos , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/genética , Tetraspanina 29 , Transdução GenéticaRESUMO
BACKGROUND/AIMS: It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. METHODS: Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. RESULTS: Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. CONCLUSIONS: HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.
Assuntos
Fator de Crescimento Epidérmico/genética , Terapia Genética , Hepatopatias/terapia , Regeneração Hepática/genética , Adenoviridae/genética , Animais , Anticorpos/farmacologia , Apoptose/genética , Membrana Celular/química , Fator de Crescimento Epidérmico/análise , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Fator de Crescimento de Hepatócito/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/enzimologia , Hepatopatias/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética , Receptor fas/efeitos dos fármacosRESUMO
BACKGROUND: Doxorubicin is a highly effective antineoplastic drug, but its clinical use is limited by its adverse side effects on the heart. We investigated possible protective effects of erythropoietin against doxorubicin-induced cardiomyopathy. METHODS AND RESULTS: Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, human recombinant erythropoietin (5000 U/kg) was started simultaneously. Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by erythropoietin treatment. Erythropoietin also protected hearts against doxorubicin-induced cardiomyocyte atrophy and degeneration, myocardial fibrosis, inflammatory cell infiltration, and downregulation of expression of GATA-4 and 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin. Cyclooxygenase-2 expression was upregulated in doxorubicin-treated hearts, and that, too, was attenuated by erythropoietin. No doxorubicin-induced apoptotic effects were seen, nor were any changes seen in the expression of tumor necrosis factor-alpha or transforming growth factor-beta1. Antiatrophic and GATA-4 restoring effects of erythropoietin were demonstrated in the in vitro experiments with cultured cardiomyocytes exposed to doxorubicin, which indicated the direct cardioprotective effects of erythropoietin beyond erythropoiesis. Cardiac erythropoietin receptor expression was downregulated in doxorubicin-induced cardiomyopathy but was restored by erythropoietin. Among the downstream mediators of erythropoietin receptor signaling, activation of extracellular signal-regulated kinase was reduced by doxorubicin but restored by erythropoietin. By contrast, erythropoietin was ineffective when administered after cardiac dysfunction was established in the chronic stage. CONCLUSIONS: The present study indicates a protective effect of erythropoietin against doxorubicin-induced cardiomyopathy.
Assuntos
Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/prevenção & controle , Eritropoetina/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Animais , Antibióticos Antineoplásicos , Cardiomiopatias/induzido quimicamente , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Doxorrubicina , Contagem de Eritrócitos , Eritropoetina/sangue , Fator de Transcrição GATA4/metabolismo , Insuficiência Cardíaca/induzido quimicamente , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Receptores da Eritropoetina/metabolismo , Sarcômeros/metabolismo , Taxa de Sobrevida , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/prevenção & controleRESUMO
Hepatocyte growth factor (HGF) gene therapy may have potential for treating chronic hepatitis (CH) and liver cirrhosis (LC). However, the lack of an HGF gene therapy study on hepatomas that are often associated with CH or LC, together with the stimulatory effects of HGF on many types of cancer, may hamper its application. This study explored the effects of adenoviral HGF gene transduction and their mechanisms on two types of hepatoma cells (hepatoblastoma and hepatocellular carcinoma) in in vitro experiments. Both types of hepatomas were revealed to have higher adenoviral gene transduction efficiencies and more efficient expressions of the HGF transgene, which successfully activated the HGF receptor/c-Met in an autocrine fashion, than those of other types of cancer. Notably, not only HGF, but also adenoviral infection, inhibited DNA synthesis, whereas only HGF but not adenoviral infection exerted a potent apoptotic effect. Moreover, adenoviral HGF gene transduction additively exerted inhibitory effects on cisplatin-treated hepatomas. In conclusion, inhibitory and apoptotic effects of adenoviral HGF gene transduction in hepatomas in contrast to potent mitogenic and antiapoptotic effects of HGF for hepatocytes are not only of biological interest, but also pose clinical benefits for adenoviral HGF gene therapy for CH and LC.
Assuntos
Adenoviridae/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Terapia Genética/métodos , Fator de Crescimento de Hepatócito/metabolismo , Adenoviridae/metabolismo , Bromodesoxiuridina/farmacologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Cisplatino/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Fosforilação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Sais de Tetrazólio/farmacologia , Fatores de TempoRESUMO
Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.
Assuntos
Fator de Crescimento Epidérmico/biossíntese , Hipertrofia Ventricular Esquerda/patologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Terapia Genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Regulação para Cima , Receptor fas/genética , Receptor fas/imunologia , Receptor fas/metabolismoRESUMO
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.