Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Hypo-osmolarity induces apoptosis resistance via TRPV2-mediated AKT-Bcl-2 pathway.

In cirrhosis, several molecular alterations such as resistance to apoptosis could accelerate carcinogenesis. Recently, mechanotransduction has been attracting attention as one of the causes of these disturbances. In patients with cirrhosis, the serum sodium levels progressively decrease in the later stage of cirrhosis, and hyponatremia leads to serum hypo-osmolality. Since serum sodium levels in patients with cirrhosis with liver cancer are inversely related to cancer's number, size, stage, and cumulative survival, we hypothesized that hypo-osmolality-induced mechanotransduction under cirrhotic conditions might contribute to oncogenesis and/or progression of hepatocellular carcinoma (HCC). In this study, we adjusted osmosis of culture medium by changing the sodium chloride concentration and investigated the influence of hypotonic conditions on the apoptosis resistance of an HCC cell line, HepG2, using a serum-deprivation-induced apoptosis model. By culturing the cells in a serum-free medium, the levels of an antiapoptotic protein Bcl-2 were downregulated. In contrast, the hypotonic conditions caused apoptosis resistance by upregulation of Bcl-2. Next, we examined which pathway was involved in the apoptosis resistance. Hypotonic conditions enhanced AKT signaling, and constitutive activation of AKT in HepG2 cells led to upregulation of Bcl-2. Moreover, we revealed that the enhancement of AKT signaling was caused by intracellular calcium influx via a mechanosensor, TRPV2. Our findings suggested that hyponatremia-induced serum hypotonic in patients with cirrhosis promoted the progression of hepatocellular carcinoma.NEW & NOTEWORTHY Our study first revealed that hypo-osmolarity-induced mechanotransduction enhanced calcium-mediated AKT signaling via TRPV2 activation, resulting in contributing to apoptosis resistance. The finding indicates a possible view that liver cirrhosis-induced hyponatremia promotes hepatocellular carcinogenesis.

Cancer cells produce liver metastasis via gap formation in sinusoidal endothelial cells through proinflammatory paracrine mechanisms.

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species.

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

TGF-ß1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-ß1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH). METHODS: Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (•OH) and oxidative DNA damage were measured using an •OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. RESULTS: In culture, TGF-ß1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H2O2-triggered •OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct •OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions +2-+13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-ß1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA+pSMAD2+- and αSMA+NOX4+-positive hepatic stellate cells from patients with NASH and advanced fibrosis. CONCLUSIONS: Downregulation of CYGB by the TGF-ß1/pSMAD2/SP3-M1 pathway brings about •OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH. LAY SUMMARY: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-ß1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated ß-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling.

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN-targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5'-TGA(C/G)TCA), located -218 to -222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct-ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.

Interstitial fluid flow-induced growth potential and hyaluronan synthesis of fibroblasts in a fibroblast-populated stretched collagen gel culture.

BACKGROUND: Tensioned collagen gels with dermal fibroblasts (DFs) as a dermis model are usually utilized in a static culture (SC) that lacks medium flowing. To make the model closer to its in vivo state, we created a device to perfuse the model with media flowing at a physiological velocity and examined the effects of medium flow (MF) on the cultures. METHODS: We constructed a medium perfusion device for human DF-embedded stretched collagen gels (human dermis model), exposed the model to media that flows upwardly at ~1mL/day, and examined water retention of the gels, cells' growth ability, metabolic activity, expression profiles of nine extracellular matrix (ECM)-related genes. The obtained data were compared with those from the model in SC. RESULTS: MF increases the gels' water retention and cells' growth potential but had little effect on their metabolic activities. MF robustly enhanced hyaluronan synthase 2 (HAS2) and matrix metalloprotease 1 (MMP1) gene expressions but not of the other genes (MMP2, HYAL1, HYAL2, HYAL3, COL1A1, COL3A1, and CD44). MF significantly increased the amounts of cellular hyaluronan and adenosine triphosphate. CONCLUSIONS: The MF at a physiological speed significantly influences the nature of ECMs and their resident fibroblasts and remodels ECMs by regulating hyaluronan metabolism. GENERAL SIGNIFICANCE: Fibroblasts in tensioned collagen gels altered their phenotypes in a MF rate-dependent manner. Collagen gel culture with tension and MF could be utilized as an appropriate in vitro model of interstitial connective tissues to evaluate the pathophysiological significance of mechanosignals generated by fluid flow and cellular/extracellular tension.

Possible Involvement of Nitric Oxide in Enhanced Liver Injury and Fibrogenesis during Cholestasis in Cytoglobin-deficient Mice.

This study clarified the role of Cygb, the fourth globin in mammals originally discovered in rat hepatic stellate cells (HSCs), in cholestatic liver disease. Bile duct ligation (BDL) augmented inflammatory reactions as revealed by increased infiltrating neutrophils, CD68+-macrophages, and chemokine expression in Cygb-/- mice. In these mice, impairment of bile canalicular indicated by the loss of CD10 expression, down-regulation of bile salt transporters, increased total bile acid, and massive apoptotic and necrotic hepatocytes occurred with the release of cytochrome c, activation of caspase 3, resulting in reduced animal survival compared to wild-type mice. In Cygb-/- mouse liver, all of NO metabolites and oxidative stress were increased. Treatment with NO inhibitor restrained all above phenotypes and restored CD10 expression in BDL Cygb-/- mice, while administration of NO donor aggravated liver damage in BDL-wild type mice to the same extent of BDL-Cygb-/- mice. N-acetylcysteine administration had a negligible effect in all groups. In mice of BDL for 1-3 weeks, expression of all fibrosis-related markers was significantly increased in Cygb-/- mice compared with wild-type mice. Thus, Cygb deficiency in HSCs enhances hepatocyte damage and inflammation in early phase and fibrosis development in late phase in mice subjected to BDL, presumably via altered NO metabolism.

Nitration is exclusive to defense-related PR-1, PR-3 and PR-5 proteins in tobacco leaves.

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr(36) of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

Absence of cytoglobin promotes multiple organ abnormalities in aged mice.

Cytoglobin (Cygb) was identified in hepatic stellate cells (HSCs) and pericytes of all organs; however, the effects of Cygb on cellular functions remain unclear. Here, we report spontaneous and age-dependent malformations in multiple organs of Cygb(-/-) mice. Twenty-six percent of young Cygb(-/-) mice (<1 year old) showed heart hypertrophy, cystic disease in the kidney or ovary, loss of balance, liver fibrosis and lymphoma. Furthermore, 71.3% (82/115) of aged Cygb(-/-) mice (1-2 years old) exhibited abnormalities, such as heart hypertrophy and cancer development in multiple organs; by contrast, 5.8% (4/68) of aged wild-type (WT) mice had abnormalities (p < 0.0001). Interestingly, serum and urine analysis demonstrated that the concentration of nitric oxide metabolites increased significantly in Cygb(-/-) mice, resulting in an imbalance in the oxidative stress and antioxidant defence system that was reversed by N(G)-monomethyl-L-arginine treatment. A senescent phenotype and evidence of DNA damage were found in primary HSCs and the liver of aged Cygb(-/-) mice. Moreover, compared with HSC(+/+), HSC(-/-) showed high expression of Il-6 and chemokine mRNA when cocultured with mouse Hepa 1-6 cells. Thus, the absence of Cygb in pericytes provokes organ abnormalities, possibly via derangement of the nitric oxide and antioxidant defence system and through accelerated cellular senescence.

Involvement of hepatic stellate cell cytoglobin in acute hepatocyte damage through the regulation of CYP2E1-mediated xenobiotic metabolism.

Oxygen (O2) is required for cytochrome P450 (CYP)-dependent drug metabolism. Cytoglobin (CYGB) is a unique globin expressed exclusively in hepatic stellate cells (HSCs). However, its role in O2-dependent metabolism in neighboring hepatocytes remains unknown. This study provides evidence that CYGB in HSCs is involved in acetaminophen (N-acetyl-p-aminophenol; APAP)-induced hepatotoxicity. Serum alanine aminotransferase levels were higher in wild-type mice than in Cygb-null mice. Wild-type mice exhibited more severe hepatocyte necrosis around the central vein area compared with Cygb-null mice, thus indicating that CYGB deficiency protects against APAP-induced liver damage. Although no difference in the hepatic expression of CYP2E1, a key enzyme involved in APAP toxicity, was observed between wild-type and Cygb-null mice, the serum levels of the APAP metabolites cysteinyl-APAP and N-acetyl-cysteinyl-APAP were decreased in Cygb-null mice, suggesting reduced APAP metabolism in the livers of Cygb-null mice. In primary cultures, APAP-induced hepatocyte damage was increased by co-culturing with wild-type HSCs but not with Cygb-null HSCs. In addition, cell damage was markedly alleviated under low O2 condition (5% O2), suggesting the requirement of O2 for APAP toxicity. Carbon tetrachloride-induced liver injury (CYP2E1-dependent), but not lipopolysaccharide/D-galactosamine-induced injury (CYP2E1-independent), was similarly alleviated in Cygb-null mice. Considering the function of CYGB as O2 carrier, these results strongly support the hypothesis that HSCs are involved in the CYP2E1-mediated xenobiotic activation by augmenting O2 supply to hepatocytes. In conclusion, CYGB in HSCs contributes to the CYP-mediated metabolism of xenobiotics in hepatocytes by supplying O2 for enzymatic oxidation.

Cytoglobin deficiency promotes liver cancer development from hepatosteatosis through activation of the oxidative stress pathway.

This study was conducted to clarify the role of cytoglobin (Cygb), a globin expressed in hepatic stellate cells (HSCs), in the development of liver fibrosis and cancer in nonalcoholic steatohepatitis (NASH). Cygb expression was assessed in patients with NASH and hepatocellular carcinoma. Mouse NASH model was generated in Cygb-deficient (Cygb(-/-)) or wild-type (WT) mice by giving a choline-deficient amino acid-defined diet and, in some of them, macrophage deletion and N-acetyl cysteine treatment were used. Primary-cultured mouse HSCs isolated from WT (HSCs(Cygb-wild)) or Cygb(-/-) (HSCs(Cygb-null)) mice were characterized. As results, the expression of CYGB was reduced in patients with NASH and hepatocellular carcinoma. Choline-deficient amino acid treatment for 8 weeks induced prominent inflammation and fibrosis in Cygb(-/-) mice, which was inhibited by macrophage deletion. Surprisingly, at 32 weeks, despite no tumor formation in the WT mice, all Cygb(-/-) mice developed liver cancer, which was ameliorated by N-acetyl cysteine treatment. Altered expression of 31 genes involved in the metabolism of reactive oxygen species was notable in Cygb(-/-) mice. Both HSCs(Cygb-null) and Cygb siRNA-transfected-HSCs(Cygb-wild) exhibited the preactivation condition. Our findings provide important insights into the role that Cygb, expressed in HSCs during liver fibrosis, plays in cancer development with NASH.

Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver.

Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.

Repopulation of the immunosuppressed retrorsine-treated infant rat liver with human hepatocytes.

BACKGROUND: We previously generated humanized chimeric mice by transplanting h-hepatocytes into the livers of the diseased-liver transgenic mouse model with immunodeficient background. These mice with livers mostly replaced by human (h) hepatocytes have been proved to be useful for research on drug metabolism and toxicity and on intrahepatic pathogens such as hepatitis. However, their small body size prohibited collecting sufficient biological samples and made surgical manipulation difficult, which motivated us to produce humanized larger animal(s) bearing h-hepatocytes. METHODS: Fischer 344 (F344) rats at 2 weeks of age were administrated with hepatotoxin retrorsine (RS) and then transplanted with syngeneic F344 rat (r)- or h-hepatocytes via the portal vein. The hosts were injected daily with FK506 immunosuppressant. The livers were harvested periodically for determining donor-cell replacement ratios and compared with those of the humanized chimeric mice, and liver-specific mRNA and protein expressions by immunohistochemistry and reverse-transcription PCR. RESULTS: RS treatment of infant rats inhibited hepatocyte proliferation, resulting in decreased liver weight and megalocytic changes in hepatocytes. R-hepatocytes transplanted into RS-treated rats engrafted into and repopulated the liver at ratios of 16.4 ± 6.7% and 48.3 ± 29.3% at 3 and 6 weeks after transplantation, respectively. H-hepatocytes also engrafted into the rat liver and showed a repopulation ratio of 2.5 ± 1.5% at 3 weeks post-transplantation, which was comparable to the ratio in the humanized chimeric mouse model at least until 3 weeks. Propagated h-hepatocytes in the rat liver expressed hepatocyte-specific mRNA and proteins at least 3 weeks after transplantation. CONCLUSIONS: Xenogeneic hepatocytes were able to engraft rat liver and grow well therein for at least 3 weeks post-transplantation in rats when immunosuppression was combined appropriately with liver injury at comparable levels to the well-characterized humanized chimeric mouse model.

Cytoglobin may be involved in the healing process of gastric mucosal injuries in the late phase without angiogenesis.

BACKGROUND AND AIMS: Cytoglobin (Cygb) is the newest globin family and is upregulated during hypoxia to maintain the oxygen status. Herein, we investigated Cygb expression in both acute and chronic gastric mucosal injuries. METHODS: Acute gastric mucosal injuries in rats were produced by oral administration of indomethacin, followed by sacrifice at 1, 3, 6, 24, and 48 h. Gastric ulcer was produced by acetic acid, followed by sacrifice on days 3, 7, 11, 18, and 25. Each protein expression of Cygb and hypoxia-inducible factor (HIF)-1α was evaluated by western blotting. We measured vascular endothelial growth factor (VEGF) mRNA by RT-PCR and examined localization of Cygb by immunofluorescence. RESULTS: In indomethacin-induced injury, Cygb protein was significantly increased at 24 h. In ulcerated tissues, HIF-1α protein was significantly increased on days 7 and 11 (1.83 ± 0.11 and 2.12 ± 0.19 folds, respectively, p < 0.05 and 0.01), which corresponded to the early healing phase. In contrast, Cygb protein was significantly increased on days 11 and 18 (1.87 ± 0.13 and 1.60 ± 0.06 folds, respectively, p < 0.05), which demonstrated late phase. Though these proteins peaked on day 11, VEGF mRNA was gradually increased from day 11 to 18. Cygb was expressed in fibroblasts and myofibroblasts in both acute and chronic models. Cygb and HIF-1α were abundantly colocalized at the ulcer margin before angiogenesis development. However, faint localization was observed with angiogenesis. CONCLUSIONS: Cygb may be involved in the healing process of gastric mucosal injuries in the late phase without angiogenesis.

Transplantation of engineered chimeric liver with autologous hepatocytes and xenobiotic scaffold.

OBJECTIVE: Generation of human livers in pigs might improve the serious shortage of grafts for human liver transplantation, and enable liver transplantation without the need for deceased or living donors. We developed a chimeric liver (CL) by repopulation of rat hepatocytes in a mouse and successfully transplanted it into a rat recipient with vessel reconstruction. This study was designed to investigate the feasibility of CL for supporting the recipient after auxiliary liver grafting. METHODS: Hepatocytes from luciferase transgenic or luciferase/LacZ double-transgenic rats were transplanted into 20- to 30-day-old urokinase-type plasminogen activator/severe-combined immunodeficiency (uPA/SCID) mice (n = 40) to create CLs with rat-origin hepatocytes. After replacement of mouse hepatocytes with those from rats, the CLs were transplanted into wild-type Lewis (n = 30) and analbuminemia (n = 10) rats, followed by immunosuppression using tacrolimus (TAC) with/without cyclophosphamide (CPA) or no immunosuppression. Organ viability was traced by in vivo bioimaging and Doppler ultrasonography in the recipient rats for 4 to 6 months. Rat albumin production was also evaluated in the analbuminemia rats for 4 months. In addition, histological analyses including Ki67 proliferation staining were performed in some recipients. RESULTS: Both immunosuppressive protocols significantly improved graft survival and histological rejection of CLs as compared to the nonimmunosuppressed group. Although rat albumin production was maintained in the recipients for 4 months after transplantation, ultrasonography revealed patent circulation in the grafts for 6 months. Ki67 staining analysis also revealed the regenerative potential of CLs after a hepatectomy of the host native liver, whereas immune reactions still remained in the mouse-origin structures. CONCLUSIONS: This is the first report showing that engineered CLs have potential as alternative grafts to replace the use of grafts from human donors.

A novel animal model for in vivo study of liver cancer metastasis.

AIM: To establish an animal model with human hepatocyte-repopulated liver for the study of liver cancer metastasis. METHODS: Cell transplantation into mouse livers was conducted using alpha-fetoprotein (AFP)-producing human gastric cancer cells (h-GCCs) and h-hepatocytes as donor cells in a transgenic mouse line expressing urokinase-type plasminogen activator (uPA) driven by the albumin enhancer/promoter crossed with a severe combined immunodeficient (SCID) mouse line (uPA/SCID mice). Host mice were divided into two groups (A and B). Group A mice were transplanted with h-GCCs alone, and group B mice were transplanted with h-GCCs and h-hepatocytes together. The replacement index (RI), which is the ratio of transplanted h-GCCs and h-hepatocytes that occupy the examined area of a histological section, was estimated by measuring h-AFP and h-albumin concentrations in sera, respectively, as well as by immunohistochemical analyses of h-AFP and human cytokeratin 18 in histological sections. RESULTS: The h-GCCs successfully engrafted, repopulated, and colonized the livers of mice in group A (RI = 22.0% ± 2.6%). These mice had moderately differentiated adenocarcinomatous lesions with disrupted glandular structures, which is a characteristics feature of gastric cancers. The serum h-AFP level reached 211.0 ± 142.2 g/mL (range, 7.1-324.2 g/mL). In group B mice, the h-GCCs and h-hepatocytes independently engrafted, repopulated the host liver, and developed colonies (RI = 12.0% ± 6.8% and 66.0% ± 12.3%, respectively). h-GCC colonies also showed typical adenocarcinomatous glandular structures around the h-hepatocyte-colonies. These mice survived for the full 56 day-study and did not exhibit any metastasis of h-GCCs in the extrahepatic regions during the observational period. The mice with an h-hepatocyte-repopulated liver possessed metastasized h-GCCs and therefore could be a useful humanized liver animal model for studying liver cancer metastasis in vivo. CONCLUSION: A novel animal model of human liver cancer metastasis was established using the uPA/SCID mouse line. This model could be useful for in vivo testing of anti-cancer drugs and for studying the mechanisms of human liver cancer metastasis.

Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse.

UNLABELLED: The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural killer (NK) cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development of massive liver cell apoptosis. CONCLUSION: Our findings provide the first direct evidence that DC-activated NK cells induce massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B.