Claudia Mitchell

Dual Role of CCR2 in the Constitution and the Resolution of Liver Fibrosis in Mice

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.

Proinflammatory Cytokine Production in Liver Regeneration Is Myd88-Dependent, but Independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.

The Combination of Ischemic Preconditioning and Liver Bcl-2 Overexpression Is a Suitable Strategy to Prevent Liver and Lung Damage after Hepatic Ischemia-Reperfusion

The present study evaluates the effectiveness of ischemic preconditioning and Bcl-2 overexpression against the liver and lung damage that follow hepatic ischemia-reperfusion and investigates the underlying protective mechanisms. Preconditioning and Bcl-2, respectively, reduced the increased tumor necrosis factor (TNF) and macrophage inflammatory protein-2 (MIP)-2 levels observed after hepatic reperfusion. Bcl-2 overexpression or anti-MIP-2 pretreatment seems to be more effective than preconditioning or anti-TNF pretreatment against inflammatory response, microcirculatory disorders, and subsequent hepatic ischemia-reperfusion injury. Furthermore, each one of these strategies individually was unable to completely inhibit hepatic injury. The combination of preconditioning and Bcl-2 overexpression as well as the combined anti-TNF and anti-MIP-2 pretreatment totally prevented hepatic injury, whereas the benefits of preconditioning and Bcl-2 were abolished by TNF and MIP-2. In contrast to preconditioning, Bcl-2 did not modify lung damage induced by hepatic reperfusion. This could be explained by the differential effect of both treatments on TNF release. Anti-TNF therapy or preconditioning, by reducing TNF release, reduced pulmonary inflammatory response, whereas the benefits of preconditioning on lung damage were abolished by TNF. Thus, the induction of both Bcl-2 overexpression in liver and preconditioning, as well as pharmacological strategies that simulated their benefits, such as anti-TNF and anti-MIP-2 therapies, could be new strategies aimed to reduce lung damage and inhibit the hepatic injury associated with hepatic ischemia-reperfusion.

Protection against hepatocyte mitochondrial dysfunction delays fibrosis progression in mice.

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.

c-Met recruits ICAM-1 as a coreceptor to compensate for the loss of CD44 in Cd44 null mice.

CD44v6 acts as a coreceptor for the receptor tyrosine kinases c-Met and VEGFR-2. It is shown that ICAM-1 can act as a new coreceptor in CD44v6-negative tumor cells. Furthermore, ICAM-1 can substitute for CD44v6 as a coreceptor for c-Met in primary hepatocytes and in liver regeneration in Cd44 null mice.

Liver Repopulation by Bcl-xL Transgenic Hepatocytes

Liver repopulation could constitute a potential therapeutic alternative to liver transplantation in the future. Therefore, the development of liver repopulation strategies is of major interest. We have previously reported that Bcl-2-expressing hepatocytes are resistant to Fas-mediated apoptosis and that these hepatocytes, when transplanted into the spleen, are able to repopulate the liver of normal mice submitted to Fas-mediated apoptosis. We now show that Bcl-x(L)-overexpressing hepatocytes are able to repopulate up to 10% of a normal mouse liver treated with successive injections of anti-Fas antibody. We show that a twofold overexpression of Bcl-x(L) is sufficient to confer a selective advantage to hepatocytes submitted to anti-Fas antibody. Moreover, repopulation percentages obtained here were comparable to those obtained when Bcl-2 hepatocytes were transplanted, suggesting that both proteins are equivalent in conferring a selective advantage to hepatocytes submitted to anti-Fas antibody.

The transforming growth factor-α and cyclin D1 genes are direct targets of β-catenin signaling in hepatocyte proliferation

BACKGROUND & AIMS β-Catenin is an oncogene frequently mutated in hepatocellular carcinoma. In this study, we investigated target genes of β-catenin signaling in hepatocyte proliferation. METHODS We studied transgenic mice displaying either inactivation or activation of the β-catenin pathway, focusing on analysis of liver proliferation due to aberrant β-catenin activation, and on the regeneration process during which β-catenin signaling is transiently activated. We localized in situ the various partners involved in proliferation or identified as targets of β-catenin in these transgenic and regenerating livers. We also performed comparative transcriptome analyses, using microarrays. Finally, we extracted, from deep-sequencing data, both the DNA regulatory elements bound to the β-catenin/Tcf nuclear complex and the expression levels of critical targets identified in microarrays. RESULTS β-Catenin activation during liver regeneration occurred during G1/S cell cycle progression and allowed zonal extension of the normal territory of active β-catenin and panlobular proliferation. We found that β-catenin controlled both cell-autonomous and non-cell-autonomous hepatocyte proliferation, through direct transcriptional and complex control of cyclin D1 gene expression and of the expression of a new target gene, Tgfα. CONCLUSIONS We propose that β-catenin controls panlobular hepatocyte proliferation partly by controlling, together with its Tcf4 nuclear partner, expression of the pro-proliferation cyclin D1 and Tgfα genes. This study constitutes a first step toward understanding the oncogenic properties of this prominent signaling pathway in the liver.

GH Receptor Plays a Major Role in Liver Regeneration through the Control of EGFR and ERK1/2 Activation

GH is a pleiotropic hormone that plays a major role in proliferation, differentiation, and metabolism via its specific receptor. It has been previously suggested that GH signaling pathways are required for normal liver regeneration but the molecular mechanisms involved have yet to be determined. The aim of this study was to identify the mechanisms by which GH controls liver regeneration. We performed two thirds partial hepatectomies in GH receptor (GHR)-deficient mice and wild-type littermates and showed a blunted progression in the G(1)/S transition phase of the mutant hepatocytes. This impaired liver regeneration was not corrected by reestablishing IGF-1 expression. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between mutant and wild-type mice, cell cycle progression was significantly blunted in mutant mice. The main defect in GHR-deficient mice was the deficiency of the epidermal growth factor receptor activation during the process of liver regeneration. Finally, among the pathways activated downstream of GHR during G(1) phase progression, namely Erk1/2, Akt, and signal transducer and activator of transcription 3, we only found a reduced Erk1/2 phosphorylation in mutant mice. In conclusion, our results demonstrate that GH signaling plays a major role in liver regeneration and strongly suggest that it acts through the activation of both epidermal growth factor receptor and Erk1/2 pathways.

Selection of in vivo retrovirally transduced hepatocytes leads to efficient and predictable mouse liver repopulation.

Stable liver gene transfer is generally limited by the low efficiency of commonly used vectors. One way to circumvent this difficulty is to confer a selective advantage on transduced hepatocytes, allowing them to progressively repopulate the liver. We have used a strategy for in vivo selection and controlled amplification of a small percentage of retrovirally engineered hepatocytes. Using a bicistronic retroviral vector encoding a reporter gene and Bcl2, which confers on liver cells a survival advantage against the Fas apoptotic pathway, we demonstrate that 1.5% of initially transduced hepatocytes repopulate up to 85% of the liver after 10 injections of a Fas agonist antibody. Moreover, we show that the kinetics of liver repopulation is highly predictable. This system provides a general means of expanding at will engineered hepatocytes in vivo and offering the possibility to obtain a genetically modified liver expressing a gene of interest in a desired proportion of hepatocytes.