RESUMEN
Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl(4)-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-ß (TGFß) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFß, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.
Asunto(s)
Cirrosis Hepática Experimental/etiología , Hígado/patología , Células Madre/fisiología , 2-Acetilaminofluoreno/toxicidad , Actinas/análisis , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Tetracloruro de Carbono/toxicidad , Transición Epitelial-Mesenquimal , Queratina-19/análisis , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in ß-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1ß, TNF-α, lymphotoxin-ß, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-ß and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.
Asunto(s)
Hígado Graso/prevención & control , Hepatitis/prevención & control , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Cirrosis Hepática Experimental/prevención & control , Hígado/metabolismo , Animales , Tetracloruro de Carbono , Proliferación Celular , Deficiencia de Colina/complicaciones , Progresión de la Enfermedad , Etionina , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Regulación de la Expresión Génica , Hepatitis/etiología , Hepatitis/genética , Hepatitis/metabolismo , Hepatitis/patología , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Metabolismo de los Lípidos , Hígado/patología , Cirrosis Hepática Experimental/etiología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/metabolismo , Células Madre/patología , Tioacetamida , Factores de Tiempo , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND/AIMS: Resident macrophages and myofibroblasts derived from hepatic stellate cells play a key role in liver wound healing. We previously reported that these sinusoidal cells secrete the growth arrest-specific protein 6 (Gas6) and express Axl, one of its receptors. Here we address the role of Gas6 in the healing process during acute liver injury. METHODS: Toxic hepatitis was induced by a single carbon tetrachloride injection in Gas6 deficient (Gas6(-/-)) mice and liver recovery was compared with wild-type animals. RESULTS: Gas6 deficiency did not cause any change in CCl(4)-induced liver damage. At 72 h, an efficient tissue repair was observed in wild-type animals whereas in Gas6(-/-) mice, we noticed a defective wound healing accounted by reduced Kupffer cell activation revealed by a decrease in the induction of CD14, TNF-alpha, IL6 and MCP-1. Gas6-deficiency, by limiting cytokine/chemokine release, prevents hepatocyte proliferation, recruitment of circulating monocytes and accumulation of myofibroblasts in healing areas. We also report a direct chemotactic effect of Gas6 on circulating monocytes which might explain defective macrophage infiltration in liver necrotic areas of Gas6(-/-) mice. Interestingly in Gas6(-/-) mice, we observed a high and constitutive expression of Axl and an induction of the suppressor of cytokine signaling SOCS1 after CCl(4) treatment. CONCLUSIONS: The lower level of cytokines/chemokines in Gas6(-/-) mice after CCl(4) injury, is the consequence of an inhibitory signal arising from Axl receptor overexpression, leading to delayed liver repair in deficient mice.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Regeneración Hepática , Enfermedad Aguda , Animales , Tetracloruro de Carbono/toxicidad , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Células Estrelladas Hepáticas/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Macrófagos del Hígado/fisiología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis , Proteínas Oncogénicas/fisiología , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal , Tirosina Quinasa del Receptor AxlRESUMEN
The protein product of the growth arrest-specific gene 6 (Gas6) is a secreted ligand for tyrosine kinase receptors, among which Axl is the most widely distributed and displays the highest affinity for Gas6. The Gas6/Axl signaling pathway has been increasingly implicated in growth and survival processes occurring during development and tissue repair. In liver, after an acute or chronic injury, repair involves macrophages and hepatic stellate cells (HSC) activated into myofibroblastic cells (HSC/MFB), which produce cytokines and matrix proteins. We investigated the expression and the role of Gas6 and its receptor Axl in liver repair. Three days after CCl4-induced liver injury in the rat, we detected the expression of Gas6 in ED1-positive macrophages as well as in desmin-positive HSC, which accumulated in injured areas. Axl, the high-affinity receptor for Gas6, was detected in macrophages, HSC, and HSC/MFB. In vitro, expression of gamma-carboxylated Gas6 was strongly induced in HSC along with their transformation into myofibroblasts, and it exerted an anti-apoptotic effect on both HSC and HSC/MFB mediated by the Axl/PI3-kinase/Akt pathway. In conclusion, Gas6 is a survival factor for these cells and we suggest that Gas6, secreted by macrophages and HSC/MFB in vivo after liver injury, promotes HSC and HSC/MFB survival and might support transient HSC/MFB accumulation during liver healing.
Asunto(s)
Apoptosis/fisiología , Expresión Génica , Hepatocitos/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Hepatopatías/patología , ARN/genética , Animales , Tetracloruro de Carbono/toxicidad , Supervivencia Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hepatopatías/metabolismo , Masculino , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: The growth arrest-specific gene 6 (Gas6) protein is a vitamin K-dependent protein that binds to the Axl subfamily of tyrosine kinase receptors and exerts antiapoptotic and proliferative effects. Because Gas6 plays a role in development and tissue remodelling, we studied its expression as well as that of its high-affinity receptor Axl in a well-characterized model of hepatic regeneration from precursor oval cells. METHODS: Hepatic regeneration was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. RESULTS: Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 and 14 after hepatectomy and declined thereafter. Oval cells expressed Gas6 protein and messenger RNA (mRNA). Axl mRNA hepatic levels paralleled the number of oval cells, and immunohistochemistry showed Axl expression in these cells. WB-F344 cells, a hepatocytic precursor cell line, also expressed Gas6 and Axl. Addition of Gas6 significantly increased the number of WB-F344 cells cultured with or without serum. Gas6 did not increase cell entry in the S phase of the cell cycle but inhibited 15-d-prostaglandin J2-induced WB-F344 cell apoptosis. CONCLUSIONS: Our data demonstrate an expression of Gas6 and of its receptor Axl by oval cells during hepatic regeneration. Because the Gas6/Axl couple protects from apoptosis a hepatocytic precursor cell line, these results strongly suggest that the Gas6/Axl couple favors oval cell accumulation in regenerating liver by an autocrine/paracrine mechanism.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/genética , Regeneración Hepática/fisiología , Hígado/citología , Hígado/fisiología , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Apoptosis/fisiología , Comunicación Autocrina/fisiología , Células Cultivadas , Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas Oncogénicas/metabolismo , Comunicación Paracrina/fisiología , Proteínas Proto-Oncogénicas , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/fisiología , Timidina/farmacocinética , Tritio , Tirosina Quinasa del Receptor AxlRESUMEN
Liver fibrosis is potentially reversible after removal of the injurious agent. Fibrosis resolution is characterized by apoptosis of hepatic myofibroblasts and degradation of extracellular matrix components. Matrix metalloproteinase-2 (MMP-2) is involved in matrix remodeling. In the liver, it is synthesized by myofibroblasts, secreted as a proenzyme, and activated by membrane type-MMPs (MT-MMP) such as MT1-MMP. The goal of this work was to determine whether apoptosis induction in human hepatic myofibroblasts modulates the gene expression of MMP-2 and/or its activation by MT1-MMP. Induction of apoptosis by cytochalasin D or C(2)-ceramide did not modulate MMP-2 mRNA expression. In contrast, apoptosis was associated with marked activation of pro-MMP-2, as shown by gelatin zymography, which revealed the presence of the 59-kd active form, whereas untreated cells only expressed the 66-kd proform. SB-203580, a specific inhibitor of p38 (MAPK), selectively abrogated both C(2)-ceramide-induced apoptosis and pro-MMP-2 activation. Apoptosis-induced pro-MMP-2 activation was inhibited by the tissue inhibitors of metalloproteinases (TIMP)-2 but not by TIMP-1, implying involvement of an MT-MMP-mediated process. Induction of apoptosis by cytochalasin D and C(2)-ceramide upregulated MT1-MMP protein expression and MT1-MMP mRNA expression. In conclusion, apoptosis of hepatic myofibroblasts induces pro-MMP-2 activation through increased MT1-MMP expression. HEPATOLOGY 2002;36:615-622.)
Asunto(s)
Apoptosis/fisiología , Fibroblastos/enzimología , Hepatocitos/enzimología , Metaloproteinasa 2 de la Matriz/genética , Esfingosina/análogos & derivados , Apoptosis/efectos de los fármacos , Células Cultivadas , Citocalasina D/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Hepatocitos/citología , Humanos , Imidazoles/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Piridinas/farmacología , ARN Mensajero/análisis , Esfingosina/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.