Smad3 signaling in the regenerating liver: implications for the regulation of IL-6 expression.

Liver regeneration is vital for graft survival and adequate organ function. Smad activation regulates hepatocyte proliferation and macrophage function. The aim of the current study was to evaluate the impact of Smad3 signaling during liver regeneration in the mouse. Male C57Bl/6 wild-type (wt) mice or mice deficient in Smad3 (Smad3(-/-) ) were subjected to a 70% partial hepatectomy (pHx) or sham surgery and sacrificed 24, 42, or 48 h later. Tissue was analyzed for TGF-ß signaling, the mitogenic cytokine response [i.e., tumor necrosis factor alpha, TNF-α; interleukin (IL)-6], and liver regeneration. Partial hepatectomy stimulated a strong regenerative response measured by proliferating cell nuclear antigen-positive hepatocytes 42 and 48 h post-pHx in conjunction with an increased expression of IL-6, TNF-α, and Smad2/3 phosphorylation 24 h post-pHx in both hepatocytes and nonparenchymal cells. Surprisingly, Smad3 deficiency led to reduced hepatocyte proliferation 42 h post-pHx which recovered by 48 h, a process that correlated with and was preceded by significant reductions in IL-6 expression and signal transducer and activator of transcription 3 phosphorylation, and cyclin D1 induction 24 h post-pHx. Loss of Smad3 signaling suppresses the expression of key mitogenic cytokines and delays hepatocellular regeneration. Therapies directed at finely regulating Smad3 activation early within the regenerating liver may prove useful in promoting liver cell proliferation and restoration of liver mass.

Angiogenesis is crucial for liver regeneration after partial hepatectomy.

BACKGROUND: Recent studies of hepatic regeneration have mainly focused on the growth of parenchymal cells. However, remodeling of liver vessels seems to be crucial during hepatic regeneration. In this study, we investigated the influence of antiangiogenesis on hepatic regeneration using sFlt-1, a soluble receptor for vascular endothelial growth factor that acts as a dominant negative receptor, and the hepatocyte growth factor antagonist NK4. METHODS: A sFlt-1-expressing adenoviral vector, an NK4-expressing adenoviral vector, or both combined were infected into C57BL6 mice via the tail vein. A 70% partial hepatectomy was performed on all of the mice 48 hours after infection. The remnants of the liver were removed after the partial hepatectomy, and hepatic regeneration was assessed by measuring the remnant liver weight and hepatocyte mitosis, bromodeoxyuridine staining, immunohistochemical staining with anti-platelet endothelial cell adhesion molecule-1 antibodies, and real-time polymerase chain reaction studies for angiogenic factors. RESULTS: The immunohistochemical staining for CD31 showed suppression of sinusoidal endothelial cells growth in sFlt-1-expressing adenoviral vector-and NK4-expressing adenoviral vector-infected mice. Increases in the remnant hepatic weight were significantly lower in the sFlt-1-expressing adenoviral vector-infected mice. The bromodeoxyuridine index and mitotic cell results revealed a significant decrease in hepatic regeneration in the sFlt-1-expressing adenoviral vector-and NK4-expressing adenoviral vector-infected mice. The suppressive effects on hepatic regeneration were significantly enhanced by combined sFlt-1-expressing adenoviral vector and NK4-expressing adenoviral vector infection. Real-time polymerase chain reaction results revealed the significant suppression of angiogenic growth factor receptors Tie-1 and Tie-2. CONCLUSION: The angiogenesis inhibitor significantly suppressed hepatic regeneration. These results suggest that hepatic regeneration after hepatectomy closely correlates with angiogenesis.

Contribution of gut bacteria to liver pathobiology.

Emerging evidence suggests a strong interaction between the gut microbiota and health and disease. The interactions of the gut microbiota and the liver have only recently been investigated in detail. Receiving approximately 70% of its blood supply from the intestinal venous outflow, the liver represents the first line of defense against gut-derived antigens and is equipped with a broad array of immune cells (i.e., macrophages, lymphocytes, natural killer cells, and dendritic cells) to accomplish this function. In the setting of tissue injury, whereby the liver is otherwise damaged (e.g., viral infection, toxin exposure, ischemic tissue damage, etc.), these same immune cell populations and their interactions with the infiltrating gut bacteria likely contribute to and promote these pathologies. The following paper will highlight recent studies investigating the relationship between the gut microbiota, liver biology, and pathobiology. Defining these connections will likely provide new targets for therapy or prevention of a wide variety of acute and chronic liver pathologies.

Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis.

UNLABELLED: The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced. CONCLUSION: Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Demonstration of cooperative contribution of MET- and EGFR-mediated STAT3 phosphorylation to liver regeneration by exogenous suppressor of cytokine signalings.

BACKGROUND/AIMS: As conditional knockout mice for stat3 are impaired in liver regeneration after partial hepatectomy while those for gp130 have defects in early STAT3 phosphorylation but have normal DNA synthesis, late STAT3 phosphorylation induced independently of gp130 seems to be essential for liver regeneration. Since HGF and EGF can activate STAT3 via gp130-independent MET and EGFR, respectively, we assumed that these factors account for STAT3-dependent liver regeneration. Here, we investigated this hypothesis by introducing suppressor of cytokine signaling (SOCS)-1 and SOCS3, potent negative regulators of STAT3 signaling, selectively in hepatocytes. METHODS: We generated recombinant adenoviruses expressing socs1 and socs3. RESULTS: Hepatocytes infected with socs1-virus lacked STAT3 phosphorylation in response to IL-6 and HGF, while cells infected with socs3-virus lacked the response to all of IL-6, HGF and EGF, indicating that those SOCS proteins differently regulate EGFR signaling. Mice infected with socs3-virus exhibited severe and persistent impairment while those with socs1-virus showed only delayed regeneration, indicating requirement of both MET and EGFR signalings. CONCLUSIONS: These results clearly demonstrated that MET- and EGFR-mediated STAT3 signalings cooperatively contribute to liver regeneration and could provide new insights into tissue homeostasis.

Adeno-associated virus vector-mediated production of hepatocyte growth factor attenuates liver fibrosis in mice.

PURPOSE: Adeno-associated virus (AAV) vectors can achieve long-term gene expression and are now feasible for use in human gene therapy. We constructed hepatocyte growth factor (HGF) expressing AAV (AAV5-HGF) and examined its effect in two mouse hepatic fibrosis models. METHODS: A model of hepatic fibrosis was established by carbon tetrachloride (CCl(4)) administration in Balb/c mice. After the establishment of liver fibrosis, AAV5-HGF was injected once into the portal vein. Mice were killed 3, 6, 9, and 12 weeks after injection. Another model was established by bile duct ligation (BDL). Seven weeks after AAV5-HGF injection, mice underwent BDL, and were then killed 2 weeks after BDL. RESULTS: Mice that received AAV5-HGF achieved stable HGF expression both in the serum and liver for at least 12 weeks. In both models, significant improvement of the liver fibrosis was found in all mice receiving AAV5-HGF based on Azan-Mallory staining. Suppression of hepatic stellate cells (HSC) was confirmed by immunohistochemistry. Fibrogenic markers were significantly suppressed and collagenase activity increased in the livers of mice receiving AAV5-HGF. CONCLUSIONS: A single injection of AAV vector containing HGF gene achieved long-term expression of HGF and resulted in resolution of mouse liver fibrosis. HGF gene therapy mediated by AAV is feasible for the treatment of liver fibrosis.

Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease.

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.

Role of innate immune response in liver regeneration.

Liver regeneration following partial hepatectomy (PH) requires several steps including innate immune responses, particularly interleukin-6 (IL-6) and tumor necrosis factor-(TNF-)alpha production by Kupffer cells, although the activation processes are still unknown. Toll-like receptors (TLR) act as innate immune signal sensors and play central roles in host defense. Myeloid differentiation factor (MyD) 88 is a common adaptor molecule required for signaling mediated by TLR. When the receptors are activated, cells bearing TLR produce various pro-nflammatory cytokines in a MyD88-dependent manner. The authors investigated whether TLR/MyD88 signaling is critical for induction of innate immune responses after PH. In Myd88(-/-) mice after PH, induction of expression of immediate early genes involved in hepatocyte replication and phosphorylation of signal transducer and activators of transcription 3 (STAT3) in the liver, and production of TNF-alpha/IL-6 by and activation of NF-kappaB in the Kupffer cells were grossly subnormal and were associated with impaired liver regeneration, while TLR2, 4 and 9, which recognize Gram-negative and -positive bacterial products, are not essential for NF-kappaB activation and IL-6 production after PH. In conclusion, the TLR/MyD88 pathway is essential for liver restoration after PH, particularly its early phase.

Hepatocyte growth factor promotes remodeling of murine liver fibrosis, accelerating recruitment of bone marrow-derived cells into the liver.

AIM: Hepatocyte growth factor (HGF) ameliorates liver fibrosis/cirrhosis in animal models, while the participation of bone marrow-derived cells (BMC) in the repair process of injured organs has recently been reported. In this study we investigated the roles of HGF and BMC in a remodeling process of liver fibrosis. METHODS: C57BL/6 J mice were treated with carbon tetrachloride (CCl(4)) for 10 weeks. At week six, the mice underwent whole body irradiation and transplantation with bone marrow cells from syngenic LacZ-transgenic mice. After the transplantation, gene transfer of HGF into skeletal muscles was performed once a week for four weeks. In the control group, sterile saline was injected. RESULTS: HGF gene transfer ameliorated the CCl(4)-induced liver fibrosis, accelerating recruitment of LacZ-expressing cells into the liver. This phenomenon was accompanied byincreased gelatinase activity in the liver. A large number of the LacZ-positive cells expressed markers of vascular endothelial cells, while some of them had a marker of macrophages. Expression of stromal cell-derived factor (SDF)-1 in the liver was upregulated around the central veins, especially in the HGF gene-transferred animals, recruiting chemokine (C-X-C motif) receptor (CXCR) 4-positive cells in this area. CONCLUSION: Transplanted BMC participate in the HGF-induced remodeling process of liver fibrosis. The roles of HGF in this process include the recruitment of BMC, possibly through increased expression of SDF-1 in part, as well as anti-apoptotic, mitogenic and antifibrotic activities on liver cells.

Oncostatin M gene therapy attenuates liver damage induced by dimethylnitrosamine in rats.

To assess the usefulness of oncostatin M (osm) gene therapy in liver regeneration, we examined whether the introduction of OSM cDNA enhances the regeneration of livers damaged by dimethylnitrosamine (DMN) in rats. Repeated injection of OSM cDNA enclosed in hemagglutinating virus of Japan envelope into the spleen resulted in the exclusive expression of OSM protein in Kupffer cells of the liver, which was accompanied by increases in body weight, liver weight, and serum albumin levels and the reduction of serum liver injury parameters (bilirubin, aspartate aminotransferase, and alanine aminotransferase) and a serum fibrosis parameter (hyaluronic acid). Histological examination showed that osm gene therapy reduced centrilobular necrosis and inflammatory cell infiltration and augmented hepatocyte proliferation. The apoptosis of hepatocytes and fibrosis were suppressed by osm gene therapy. Time-course studies on osm gene therapy before or after DMN treatment showed that this therapy was effective not only in enhancing regeneration of hepatocytes damaged by DMN but in preventing hepatic cytotoxicity caused by subsequent treatment with DMN. These results indicate that OSM is a key mediator for proliferation and anti-apoptosis of hepatocytes and suggest that osm gene therapy is useful, as preventive and curative means, for the treatment of patients with liver damage.

Selective inactivation of NF-kappaB in the liver using NF-kappaB decoy suppresses CCl4-induced liver injury and fibrosis.

Sustained hepatic inflammation induced by various causes can lead to liver fibrosis. Transcription factor NF-kappaB is important in regulating inflammatory responses, especially in macrophages. We presently investigated whether an NF-kappaB decoy, a synthetic oligodeoxynucleotide (ODN) imitating the NF-kappaB binding site, inhibited the inflammatory response after CCl(4) intoxication to prevent CCl(4)-induced hepatic injury and fibrosis. The NF-kappaB decoy was introduced into livers by injecting the spleens of mice, using a hemagglutinating virus of Japan (HVJ)-liposome method. ODN was transferred mainly to macrophages in normal or fibrotic livers. Increases in serum transaminases and production of inflammatory cytokines after a single challenge with CCl(4) were inhibited by the NF-kappaB decoy, which suppressed nuclear translocation of NF-kappaB in liver macrophages. Liver fibrosis induced by CCl(4) administration for 8 wk was suppressed by the NF-kappaB decoy, accompanied by diminished mRNA expression for transforming growth factor (TGF)-beta, procollagen type 1 alpha(1), and alpha-smooth muscle actin (SMA). In vitro, isolated liver macrophages showed increased DNA binding activity of NF-kappaB and inflammatory cytokine production after hydrogen peroxide treatment; both increases were inhibited significantly by the NF-kappaB decoy. In contrast, NF-kappaB decoy transferred to isolated hepatic stellate cells (HSC) had no effect on their morphological activation or alpha-SMA expression, although the decoy accelerated tumor necrosis factor (TNF)-alpha-induced apoptosis in activated HSC. The effect of NF-kappaB decoy suppressing fibrosis probably results mainly from anti-inflammatory effects on liver macrophages, with a possible minor contribution from its direct proapoptotic effect on activated HSC.

Blockage of HGF/c-Met system by gene therapy (adenovirus-mediated NK4 gene) suppresses hepatocellular carcinoma in mice.

BACKGROUND/AIMS: Hepatocyte growth factor promotes cancer development through cell motility-promoting and angiogenic effects. NK4, a fragment of hepatocyte growth factor, acts as its receptor antagonist. We assessed effects of NK4 gene therapy against human hepatocellular carcinoma cells (HUH7) transplanted into mice. METHODS: NK4 gene transduction was mediated by adenovirus (AdCMV.NK4). LacZ expression adenovirus (AdCMV.LacZ) was used as a control. NK4 effects on HUH7 cells first were studied in vitro. Subcutaneous HUH7 tumors established in athymic nude mice were injected with AdCMV.NK4 (n=6) or AdCMV.Lacz (n=6). Finally, after HUH7 cells were injected into the portal vein in mice with severe combined immunodeficiency to establish hepatic tumors, mice systemically were injected with AdCMV.NK4 (n=6) or AdCMV.LacZ (n=6). RESULTS: NK4 inhibited hepatocyte growth factor-induced phosphorylation of c-Met in HUH7 cells. Invasion and migration of HUH7 cells were inhibited by NK4 transfection, which also suppressed growth of transplanted subcutaneous and liver tumors (p<0.001, p<0.01 respectively), and improved mouse survival (p<0.05). Angiogenesis assessed by small vessel density was significantly decreased in the NK4-treated group. CONCLUSIONS: NK4 inhibited tumor cell motility and angiogenesis, greatly suppressing growth of HUH7 tumors transplanted into mouse liver. NK4 gene therapy thus showed apparent promise for treatment of hepatocellular carcinoma.

Contribution of Toll-like receptor/myeloid differentiation factor 88 signaling to murine liver regeneration.

Toll-like receptors (TLRs) act as innate immune signal sensors and play central roles in host defense. Myeloid differentiation factor (MyD) 88 is a common adaptor molecule required for signaling mediated by TLRs. When the receptors are activated, cells bearing TLRs produce various proinflammatory cytokines in a MyD88-dependent manner. Liver regeneration following partial hepatectomy (PH) requires innate immune responses, particularly interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells, although the recognition and activation processes are still unknown. We investigated whether TLR/MyD88 signaling is critical for induction of innate immune responses after PH. In Myd88(-/-) mice after PH, induction of expression of immediate early genes involved in hepatocyte replication and phosphorylation of STAT3 in the liver, and production of TNF-alpha/IL-6 by and activation of NF-kappaB in the Kupffer cells were grossly subnormal and were associated with impaired liver regeneration. However, TLR2, 4 and 9, which recognize gram-negative and -positive bacterial products, are not essential for NF-kappaB activation and IL-6 production after PH, which excludes a possible contribution of TLR2/TLR4 or TLR9 to MyD88-mediated pathways. In conclusion, the TLR/MyD88 pathway is essential for incidental liver restoration, particularly its early phase.

Nuclear factor kappa B decoy oligodeoxynucleotides prevent endotoxin-induced fatal liver failure in a murine model.

Endotoxin syndrome is a systemic inflammatory response mediated by inflammatory cytokines. Nuclear factor kappa B (NF-kappa B) is the dominant regulator of the production of these cytokines by inflammatory cells. The aim of this study was to assess the efficacy of in vivo transfer of synthetic double-stranded oligodeoxynucleotides (ODN) with high affinity against NF-kappa B (NF-kappa B/decoy/ODN) as a therapeutic strategy for treating endotoxin-induced fatal liver injury. Liver injury was induced by administration of lipopolysaccharide (LPS) to Propionibacterium acnes-primed BALB/C mice. NF-kappa B/decoy/ODN was transferred into the portal vein using a fusigenic liposome with hemagglutinating virus of Japan. NF-kappa B/decoy/ODN was preferentially transferred to Kupffer cells, and activation of NF-kappa B after the LPS challenge was suppressed, leading to decreased inflammatory cytokine production. As a result, the massive necrosis and hepatocyte apoptosis observed in the control mice was dramatically attenuated and the survival rate improved. In conclusion, NF-kappa B/decoy/ODN transfer in vivo effectively suppressed endotoxin-induced fatal liver injury in mice.