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1.
J Clin Invest ; 125(3): 981-92, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25621497

RESUMO

Polyploidization is one of the most dramatic changes that can occur in the genome. In the liver, physiological polyploidization events occur during both liver development and throughout adult life. Here, we determined that a pathological polyploidization takes place in nonalcoholic fatty liver disease (NAFLD), a widespread hepatic metabolic disorder that is believed to be a risk factor for hepatocellular carcinoma (HCC). In murine models of NAFLD, the parenchyma of fatty livers displayed alterations of the polyploidization process, including the presence of a large proportion of highly polyploid mononuclear cells, which are rarely observed in normal hepatic parenchyma. Biopsies from patients with nonalcoholic steatohepatitis (NASH) revealed the presence of alterations in hepatocyte ploidy compared with tissue from control individuals. Hepatocytes from NAFLD mice revealed that progression through the S/G2 phases of the cell cycle was inefficient. This alteration was associated with activation of a G2/M DNA damage checkpoint, which prevented activation of the cyclin B1/CDK1 complex. Furthermore, we determined that oxidative stress promotes the appearance of highly polyploid cells, and antioxidant-treated NAFLD hepatocytes resumed normal cell division and returned to a physiological state of polyploidy. Collectively, these findings indicate that oxidative stress promotes pathological polyploidization and suggest that this is an early event in NAFLD that may contribute to HCC development.


Assuntos
Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo , Poliploidia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Dano ao DNA , Dieta Hiperlipídica/efeitos adversos , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Fatores de Risco
2.
Hum Gene Ther ; 24(2): 143-51, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23249390

RESUMO

Lentiviral vectors are efficient gene delivery vehicles for therapeutic and research applications. In contrast to oncoretroviral vectors, they are able to infect most nonproliferating cells. In the liver, induction of cell proliferation dramatically improved hepatocyte transduction using all types of retroviral vectors. However, the precise relationship between hepatocyte division and transduction efficiency has not been determined yet. Here we compared gene transfer efficiency in the liver after in vivo injection of recombinant lentiviral or Moloney murine leukemia viral (MoMuLV) vectors in hepatectomized rats treated or not with retrorsine, an alkaloid that blocks hepatocyte division and induces megalocytosis. Partial hepatectomy alone resulted in a similar increase in hepatocyte transduction using either vector. In retrorsine-treated and partially hepatectomized rats, transduction with MoMuLV vectors dropped dramatically. In contrast, we observed that retrorsine treatment combined with partial hepatectomy increased lentiviral transduction to higher levels than hepatectomy alone. Analysis of nuclear ploidy in single cells showed that a high level of transduction was associated with polyploidization. In conclusion, endoreplication could be exploited to improve the efficiency of liver-directed lentiviral gene therapy.


Assuntos
Vetores Genéticos/metabolismo , Lentivirus/metabolismo , Fígado/virologia , Mitose , Poliploidia , Recombinação Genética , Animais , Núcleo Celular/virologia , Proliferação de Células , Endorreduplicação , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatectomia , Hepatócitos/efeitos dos fármacos , Imuno-Histoquímica , Lentivirus/genética , Fígado/efeitos dos fármacos , Masculino , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Alcaloides de Pirrolizidina/farmacologia , Ratos , Ratos Endogâmicos F344 , Transdução Genética
3.
Cell Cycle ; 9(3): 460-6, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20090410

RESUMO

The formation of polyploid cells is part of the developmental program in several tissues. Polyploidy is a characteristic feature of mammalian hepatocytes and it is emerging that this process is an important mechanism of restricting liver growth. We previously demonstrated that during post-natal development, binucleated tetraploid hepatocytes arise due to a failure in cytokinesis. The genesis of such binucleated tetraploid cells is the crucial step for the establishment of liver polyploidization. Our recent work identified the cellular signaling pathway controlling this process. Rats with low levels of circulating insulin exhibit reduced formation of binucleated tetraploid hepatocytes, whereas rats injected with insulin exhibit increased formation of binucleated tetraploid hepatocytes. Furthermore, modulation of Akt activity clearly controls cytokinesis failure events indicating that the PI3K-Akt pathway, downstream from the insulin signal, is central to tetraploidization process. Here, we discuss these findings in the context of how cells become polyploid during physiological or pathological growth.


Assuntos
Hepatócitos/citologia , Insulina/metabolismo , Fígado/citologia , Poliploidia , Transdução de Sinais , Animais , Animais Recém-Nascidos , Citocinese , Hepatócitos/metabolismo , Humanos , Fígado/crescimento & desenvolvimento , Camundongos , Ratos
4.
Am J Pathol ; 175(5): 1929-37, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19808650

RESUMO

Accumulating evidence indicates that oxidative stress is involved in the physiopathology of liver fibrogenesis. However, amid the global context of hepatic oxidative stress, the specific role of hepatocyte mitochondrial dysfunction in the fibrogenic process is still unknown. The aim of this study was to determine whether a targeted protection of hepatocytes against mitochondrial dysfunction could modulate fibrosis progression. We induced liver fibrogenesis by chronic carbon tetrachloride treatment (3 or 6 weeks of biweekly injections) in transgenic mice expressing Bcl-2 in their hepatocytes or in normal control mice. Analyses of mitochondrial DNA, respiratory chain complexes, and lipid peroxidation showed that Bcl-2 transgenic animals were protected against mitochondrial dysfunction and oxidative stress resulting from carbon tetrachloride injury. Picrosirius red staining, alpha-smooth muscle actin immunohistochemistry, and real-time PCR for transforming growth factor-beta and collagen alpha-I revealed that Bcl-2 transgenic mice presented reduced fibrosis at early stages of fibrogenesis. However, at later stages increased nonmitochondrial/nonhepatocytic oxidative stress eventually overcame the capacity of Bcl-2 overexpression to prevent the fibrotic process. In conclusion, we demonstrate for the first time that specific protection against hepatocyte mitochondrial dysfunction plays a preventive role in early stages of fibrogenesis, delaying its onset. However, with the persistence of the aggression, this protection is no longer sufficient to impede fibrosis progression.


Assuntos
Hepatócitos/citologia , Hepatócitos/patologia , Mitocôndrias Hepáticas , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Tetracloreto de Carbono/toxicidade , Caspases/metabolismo , Progressão da Doença , Fibrose/metabolismo , Fibrose/patologia , Fibrose/fisiopatologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Peroxidação de Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
5.
J Clin Invest ; 119(7): 1880-7, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19603546

RESUMO

The formation of polyploid cells is part of the developmental program of several tissues. During postnatal development, binucleated tetraploid cells arise in the liver, caused by failure in cytokinesis. In this report, we have shown that the initiation of cytokinesis failure events and the subsequent appearance of binucleated tetraploid cells are strictly controlled by the suckling-to-weaning transition in rodents. We found that daily light/dark rhythms and carbohydrate intake did not affect liver tetraploidy. In contrast, impairment of insulin signaling drastically reduced the formation of binucleated tetraploid cells, whereas repeated insulin injections promoted the generation of these liver cells. Furthermore, inhibition of Akt activity decreased the number of cytokinesis failure events, possibly through the mammalian target of rapamycin signaling complex 2 (mTORC2), which indicates that the PI3K/Akt pathway lies downstream of the insulin signal to regulate the tetraploidization process. To our knowledge, these results are the first demonstration in a physiological context that insulin signaling through Akt controls a specific cell division program and leads to the physiologic generation of binucleated tetraploid liver cells.


Assuntos
Divisão Celular , Hepatócitos/fisiologia , Insulina/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Carboidratos da Dieta/administração & dosagem , Camundongos , Camundongos Obesos , Fosfatidilinositol 3-Quinases/fisiologia , Poliploidia , Ratos , Ratos Wistar , Transdução de Sinais
6.
Am J Pathol ; 174(5): 1766-75, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19359521

RESUMO

Inflammation has been shown to induce the progression of fibrosis in response to liver injury. Among inflammatory cells, macrophages and lymphocytes play major roles in both the constitution and resolution of liver fibrosis. The chemokine receptor CCR2 is involved in the recruitment of monocytes to injury sites, and it is known to be induced during the progression of fibrosis in humans. However, its specific role during this process has not yet been unveiled. We first demonstrated that, compared with wild-type mice, CCR2 knockout animals presented a delay in liver injury after acute CCl(4) injection, accompanied by a reduction in infiltrating macrophage populations. We then induced fibrosis using repeated injections of CCl(4) and observed a significantly lower level of fibrotic scars at the peak of fibrosis in mutant animals compared with control mice. This diminished fibrosis was associated with a reduction in F4/80(+)CD11b(+) and CD11c(+) populations at the sites of injury. Subsequent analysis of the kinetics of the resolution of fibrosis showed that fibrosis rapidly regressed in wild-type, but not in CCR2(-/-) mice. The persistence of hepatic injury in mutant animals was correlated with sustained tissue inhibitor of metalloproteinase-1 mRNA expression levels and a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-13 expression levels. In conclusion, these findings underline the role of the CCR2 signaling pathway in both the constitution and resolution of liver fibrotic scars.


Assuntos
Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Receptores CCR2/fisiologia , Animais , Western Blotting , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Tetracloreto de Carbono/toxicidade , Citometria de Fluxo , Técnicas Imunoenzimáticas , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
7.
J Cell Sci ; 120(Pt 20): 3633-9, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17895361

RESUMO

Cytokinesis is precisely controlled in both time and space to ensure equal distribution of the genetic material between daughter cells. Incomplete cytokinesis can be associated with developmental or pathological cell division programs leading to tetraploid progenies. In this study we decipher a new mechanism of incomplete cytokinesis taking place in hepatocytes during post-natal liver growth. This process is initiated in vivo after weaning and is associated with an absence of anaphase cell elongation. In this process, formation of a functional contractile actomyosin ring was never observed; indeed, actin filaments spread out along the cortex were not concentrated to the putative site of furrowing. Recruitment of myosin II to the cortex, controlled by Rho-kinase, was impaired. Astral microtubules failed to contact the equatorial cortex and to deliver their molecular signal, preventing activation of the RhoA pathway. These findings reveal a new developmental cell division program in the liver that prevents cleavage-plane specification.


Assuntos
Citocinese , Hepatócitos/citologia , Poliploidia , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Anáfase , Animais , Ciclo Celular , Forma Celular , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Hepatócitos/metabolismo , Fígado/crescimento & desenvolvimento , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Miosina Tipo II/metabolismo , Ratos , Ratos Wistar , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura
8.
Mol Ther ; 15(9): 1710-5, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17565348

RESUMO

Orthotopic liver transplantation is limited by the shortage of liver donors, leading to elderly patients being enrolled as donors with increasing frequency. Alternative strategies such as cell therapy are therefore needed. Because transplanted hepatocytes do not proliferate into a recipient liver, repopulation strategies have been developed. We have previously published a proof of concept that hepatocytes harboring a survival selective advantage can efficiently repopulate a mouse liver. We develop here an alternative approach by conferring a selective proliferative advantage on transplanted hepatocytes over resident ones. FoxM1B is a transcription factor that, when over-expressed into hepatocytes, accelerates the cell cycle and maintains the hepatocyte in vivo proliferative capacity of aged livers. We now demonstrate that transplanted hepatocytes over-expressing FoxM1B repopulate the liver of mice subjected to continuous injury far more efficiently than control hepatocytes. We show that old hepatocytes that over-express FoxM1B retain their cell division capacity and repopulate liver as well as young ones, in contrast with old non-modified hepatocytes, which lose their proliferative capacity. In conclusion, our results point to the potential use of FoxM1B expression in hepatocyte-based therapy protocols in diseases where host hepatocytes are chronically injured, especially if donor hepatocytes come from old livers.


Assuntos
Fatores de Transcrição Forkhead/genética , Hepatócitos/metabolismo , Hepatócitos/transplante , Fígado/cirurgia , Fatores Etários , Animais , Proliferação de Células , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/fisiologia , Hepatócitos/citologia , Fígado/lesões , Fígado/metabolismo , Masculino , Camundongos , Camundongos SCID , Camundongos Transgênicos , Transplante Homólogo/métodos
9.
Am J Physiol Gastrointest Liver Physiol ; 292(4): G996-G1001, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17170024

RESUMO

Various immediate early genes (IEGs) upregulated during the early process of liver regeneration are transcriptional targets of the serum response factor (SRF). We show here that the expression of SRF is rapidly induced in rodent liver after partial hepatectomy. Because the inactivation of the SRF gene in mice is embryonic lethal, the in vivo role of SRF in liver regeneration after partial hepatectomy was analyzed in mutant mice conditionally deleted for SRF in the liver. We demonstrate that SRF is not an essential factor for liver ontogenesis. However, adult mutant mice show impaired liver regeneration after partial hepatectomy, associated with a blunted upregulation of various SRF target IEGs. In conclusion, our work suggests that SRF is an early response transcription factor that may contribute to the initial phases of liver regeneration through its activation of IEGs.


Assuntos
Regeneração Hepática , Fígado/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , DNA/biossíntese , Hepatectomia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Fígado/citologia , Fígado/fisiologia , Fígado/cirurgia , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/deficiência , Fator de Resposta Sérica/genética , Fatores de Tempo , Ativação Transcricional
10.
Int J Dev Biol ; 50(1): 17-26, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16323074

RESUMO

Recent studies have shown that persistent expression of FGF10 in the developing pancreas of transgenic mice results in enhanced and prolonged proliferation of pancreatic progenitors, pancreatic hyperplasia and impaired pancreatic differentiation. These studies have also suggested that FGF10 prevents the differentiation of pancreatic progenitors by maintaining persistent Notch signalling. Here, we provide experimental evidence sustaining the capacity of FGF10 to induce the proliferation of pancreatic precursors, while preventing their differentiation. Using explant cultures of E10.5 isolated dorsal pancreatic epithelium, we found that FGF10 maintained Notch activation and induced the expansion of pancreatic precursors while blocking their differentiation. In addition, by using a gamma-secretase inhibitor, we were able to down-regulate the expression of Hes1, a target gene of the Notch pathway in explant cultures of pancreatic epithelium treated with FGF10. In such explants, the effect of FGF10 on the proliferation and maintenance of pancreatic progenitors was suppressed. These results demonstrate that activation of the Notch pathway is required as a downstream mediator of FGF10 signalling in pancreatic precursor cells.


Assuntos
Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Fator 10 de Crescimento de Fibroblastos/fisiologia , Pâncreas/citologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Proliferação de Células , Colágeno , Combinação de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fator 10 de Crescimento de Fibroblastos/metabolismo , Glicosiltransferases/metabolismo , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/fisiologia , Laminina , Camundongos , Técnicas de Cultura de Órgãos , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Proteoglicanas , Receptores Notch/metabolismo
11.
J Soc Biol ; 199(3): 243-6, 2005.
Artigo em Francês | MEDLINE | ID: mdl-16471264

RESUMO

In the liver, the importance of apoptosis is not only evident during development and homeostasis of the biliary tree but plays also a prominent role in liver pathogenesis. Ligand binding to cell surface death receptors such as Fas activates the extrinsic pathway. This pathway predominates in autoimmune liver diseases, viral hepatitis, liver allograft rejection. Hepatocyte apoptosis is also significantly increased in patients with alcoholic hepatitis and nonalcoholic steatohepatitis and correlates with disease severity and hepatic fibrosis. We have used this specific susceptibility of the liver to apoptosis to develop two different approaches: 1) a cell therapy strategy based on a survival advantage to an apoptotic stimulus conferred to transplanted hepatocytes and 2) a new model of hepatocyte conditional ablation based on a controlled activation of the cell death program.


Assuntos
Apoptose/fisiologia , Hepatócitos/patologia , Animais , Apolipoproteínas E/deficiência , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/fisiologia , Aterosclerose/etiologia , Aterosclerose/terapia , Efeito Espectador , Caspase 3 , Caspases/química , Caspases/fisiologia , Dimerização , Modelos Animais de Doenças , Ativação Enzimática , Fígado Gorduroso/patologia , Genes bcl-2 , Genes myc , Terapia Genética , Hepatite Alcoólica/patologia , Hepatócitos/transplante , Humanos , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/fisiologia , Proteína bcl-X/genética , Receptor fas/imunologia
12.
Am J Physiol Endocrinol Metab ; 288(4): E715-22, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15585594

RESUMO

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.


Assuntos
Adenilil Ciclases/biossíntese , Fígado/enzimologia , Fígado/metabolismo , Receptores Adrenérgicos beta 2/biossíntese , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Ligação Competitiva/fisiologia , Northern Blotting , Clembuterol/farmacologia , Ingestão de Alimentos/fisiologia , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Propranolol/farmacologia , Piruvato Quinase/biossíntese , Piruvato Quinase/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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