Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice.

AIM: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. METHODS: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. RESULTS: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. CONCLUSION: This study showed that HB-EGF plays a protective role during acute liver injury.

Greater omentum gastrointestinal stromal tumor with PDGFRA-mutation and hemoperitoneum.

Although gastrointestinal stromal tumor (GIST) occurs generally in the digestive tract, omental GIST is very rare. We report the first case of an adult greater omental GIST with a new platelet-derived growth factor receptor α gene (PDGFRA)-mutation with hemoperitoneum. A 43-year-old man was admitted to our hospital complaining of acute abdominal pain. Abdominal contrast-enhanced computed tomography revealed a huge mass in the right abdominal cavity, and a large accumulation of fluid in the pelvic cavity, suggesting hemoperitoneum. We diagnosed the rupture as an intra-abdominal tumor, and an emergency tumorectomy was performed with resection of the greater omentum. This tumor was located in the distal right side of the greater omentum, and showed no continuity with the gastric wall. The tumor occurred primarily in the greater omentum. The resected tumor was about 19 cm × 12 cm × 14 cm in diameter, and weighed 1529 g. Histologically, the tumor was composed of epithelioid-shaped cells with high cellularity, and was positive for CD117 and CD34, and negative for S-100, α-smooth muscle actin. The mitosis was 6/50 under high power field. This case showed exon 18 mutation of PDGFRA with 846 (Asp to Glu) substitution, 848 (Asn to Lys) substitution. This is the first report of this PDGFRA mutation in omental GIST, and this might play an important role in the tumorigenesis of this case. Based on these findings, the tumor was diagnosed as high risk GIST primarily occurring in the greater omentum. The patient was treated with imatinib at a dose of 400 mg/d as adjuvant chemotherapy, and has been followed up for 24 mo with no evidence of recurrence.

Adiponectin prevents progression of steatohepatitis in mice by regulating oxidative stress and Kupffer cell phenotype polarization.

AIM: We reported previously that hypoadiponectinemia enhances hepatic oxidative stress and accelerates progression of nonalcoholic steatohepatitis (NASH) in mice. However, the precise mechanism and preventive effects of adiponectin on NASH remain unclear. The aim of this study was to examine the effects of adiponectin on steatohepatitis using adiponectin-knockout (KO) mice and adenovirus-mediated adiponectin expression system. METHODS: We used male KO mice and C57BL6/J (WT) mice fed methionine choline-deficient (MCD)-diet as a steatohepatitis model. Liver histology, hepatic oxidative stress markers, and hepatic gene expression levels were investigated. In addition, Hepa 1-6 cells, a mouse liver cell line, were cultured with or without recombinant adiponectin, and gene expressions were investigated by real-time RT-PCR. RESULTS: After 2-week feeding of MCD diet, hepatic steatosis was enhanced and plasma alanine aminotransferase elevated in KO mice than in WT mice. In KO mice liver, thiobarbituric acid reactive substances increased, glutathione levels decreased, and mRNA expression levels of antioxidant enzymes (catalase, superoxide dismutase-1) downregulated. Adenovirus-mediated adiponectin expression prevented these changes in KO mice. Moreover, Kupffer cell infiltration was enhanced and mRNA levels of anti-inflammatory M2 macrophage markers (interleukin-10, arginase-1) were decreased in KO mice liver. In the in vitro study, adiponectin significantly increased catalase gene expression in Hepa 1-6 cells. CONCLUSIONS: Lack of adiponectin enhanced, and adiponectin administration prevented steatohepatitis progression in mice. These changes were due to the anti-oxidative effects of adiponectin, and its effects on Kupffer cells recruitment and phenotype polarization. Augmentation of adiponectin effects could be a useful preventive approach for NASH progression.

Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice.

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) alpha and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.

Transplantation of basic fibroblast growth factor-pretreated adipose tissue-derived stromal cells enhances regression of liver fibrosis in mice.

Adipose tissue-derived stromal cells (ADSC) potentially differentiate into various cell types similar to bone marrow-derived mesenchymal stromal cells (BMSC). Unlike BMSC, ADSC can be harvested easily and repeatedly. However, the advantages of ADSC for cell transplantation in liver disease remain unclear. To investigate this, we developed a novel culture system for ADSC, as well as effective methods for transplantation of ADSC into mice liver. ADSC were isolated from subcutaneous adipose tissues of male C57BL6/J mice and cultured on plastic dishes with or without basic fibroblast growth factor (bFGF). In the in vivo study, ADSC isolated from green fluorescent protein-transgenic mice were transplanted into carbon tetrachloride-injured C57BL6/J mice liver. bFGF-treated ADSC expressed several liver-specific marker genes and demonstrated liver-related functions such as albumin secretion, glycogen synthesis, urea production, and low-density lipoprotein uptake. Importantly, pretreatment of ADSC with bFGF for 1 wk enhanced the repopulation rate of ADSC in mice liver, attenuated liver fibrosis, and restored normal serum alanine aminotransferase and albumin levels. The results indicate that basic FGF facilitates transdifferentiation of ADSC into hepatic lineage cells in vitro and that transplantation of bFGF-pretreated ADSC reduced hepatic fibrosis in mice. ADSC are a potentially valuable source of cells for transplantation therapy.

Effects of growth factors on the growth and differentiation of mouse fetal liver epithelial cells in primary cultures.

BACKGROUND AND AIMS: Growth factors (GF) are thought to affect the growth and differentiation of hepatocytes during liver development. However, in the midfetal liver, little is known concerning the role of GF. METHODS: The DNA synthesis of fetal liver epithelial cells (FLEC) in monolayer culture and the liver-specific gene expressions of FLEC in 3-D culture were examined in medium supplemented with various GF. RESULTS: DNA synthesis of FLEC was higher than that of adult hepatocytes without GF, and was increased by hepatocyte growth factor (HGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). However, FLEC responded less to GF in terms of DNA synthesis than adult hepatocytes. The liver-specific gene expressions were increased in the presence of HGF, HB-EGF, bFGF and EGF. In embryonic day (E) 13.5 FLEC, this increase was more apparent in the presence of HB-EGF, whereas in E14.5 FLEC, it was more apparent in the presence of HGF. CONCLUSIONS: Hepatocyte growth factor, HB-EGF, bFGF, EGF and TGF-alpha increased DNA synthesis of FLEC. HGF, HB-EGF, bFGF and EGF led to an increase in liver-specific gene expressions; and their effects on differentiation differ as a function of gestation age.

Basic fibroblast growth factor promotes the trans-differentiation of mouse bone marrow cells into hepatic lineage cells via multiple liver-enriched transcription factors.

BACKGROUND/AIMS: Evidence that bone marrow cells have trans-differentiating potential to hepatocytes has been described in recent reports. However, the molecular mechanism underlying this phenomenon is unclear. To address this issue, we investigated the parameters involved in the trans-differentiation of bone marrow cells into a hepatic lineage. METHODS: Mouse BM cells were cultured in a collagen gel without or with growth factors including basic fibroblast growth factor. The expression of hepatocyte-specific markers, cholangiocyte-specific marker and liver-enriched transcription factors was identified by RT-PCR and immunohistochemistry. RESULTS: Basic fibroblast growth factor was found to be the most effective for inducing albumin in cultured BM cells. Furthermore, on stimulation of basic fibroblast growth factor, BM cells were found to express other hepatocyte-specific markers and a cholangiocyte-specific marker. This conversion was found to be associated with the induction of transcription factors including hepatocyte nuclear factors and GATA family proteins. CONCLUSIONS: We established an in vitro culture system in which mouse bone marrow cells could trans-differentiate to hepatic lineage cells in response to growth factors, without cell fusion. In particular, basic fibroblast growth factor has the ability to induce the trans-differentiation into hepatic lineage cells from BM cells.

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin.

BACKGROUND & AIMS: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS: When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.