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Progenitor Phenotype by Melatonin During Alcoholic Liver Injury
930
Deficiency of Intestinal Mucin-2 Protects From Alcoholic Liver Disease in Mice Phillipp Hartmann, Katharina Brandl, Claus Hellerbrand, Hidekazu Tsukamoto, Samuel B. Ho, Bruce Beutler, Bernd Schnabl
Regulation of Hepatic Stellate Cell Activation by MicroRNA Let-7 Family During Alcoholic Liver Injury Christopher Johnson, Heather L. Francis, Shannon Glaser, Yuyan Han, Julie Venter, Chang-Gong Liu, Mellanie White, Jennifer McCarra, Hidekazu Tsukamoto, Gianfranco Alpini, Fanyin Meng
Background: The intestinal mucus layer protects the epithelium against noxious agents and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease is dependent on the translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. The aim of our study was to investigate the impact of the intestinal mucus layer and in particular Muc2 on alcoholic liver disease. Methods and Results: We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet (n=4-5) or alcohol for 1 week in Muc2-/- (n=9) and wildtype (WT) mice (n=10). Muc2 was abundantly expressed in the small and large intestine of WT mice, but undetectable in the liver. The intestinal mucus layer was considerably thinner in Muc2 deficient mice as shown by PAS staining. Alcohol feeding did not result in a compensatory upregulation of other intestinal mucins in Muc2-/- mice. After alcohol feeding mice deficient in Muc2 were protected from steatohepatitis as evidenced by significantly lower ALT levels and hepatic triglyceride concentrations. In addition, hepatic oxidative stress was significantly reduced in Muc2-/- mice following intragastric alcohol feeding as shown by immunohistochemistry for 4-hydroxynonenal (4-HNE) and by TBARS assay. There was no significant difference in plasma alcohol levels or hepatic alcohol metabolizing enzymes alcohol dehydrogenase 1 (ADH1) and cytochrome p450 enzyme 2E1 (Cyp2E1) in alcohol fed Muc2-/- as compared to WT mice. Most notably, Muc2-/- mice had significantly lower systemic plasma LPS levels after alcohol feeding. In contrast to WT mice, Muc2-/mice did not exhibit intestinal bacterial overgrowth, but a higher amount of intestinal probiotic Lactobacillus after alcohol administration as shown by qPCR for 16S rRNA and Lactobacillus spp. The antimicrobial proteins Reg3b and Reg3g were found to be expressed at significantly higher levels in the proximal small intestine in isocaloric diet and alcohol fed Muc2-/- mice relative to WT mice as assessed by qPCR and Western blotting. As Reg3b and Reg3g are bactericidal c-type lectins, an increase in their expression might contribute to the observed reduction of the bacterial burden and suppression of bacterial overgrowth in the intestine. Conclusion: Intestinal mucin-2 deficiency protects from alcoholic steatohepatitis. We suggest a pathway that involves higher expression of enteric antimicrobial molecules which suppresses alcohol-associated intestinal bacterial overgrowth. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation and cause less alcoholic liver disease.
Background: microRNAs are endogenous molecules, which constitute a new class of negative regulators of gene expression. The activation of hepatic stellate cells (HSCs) is recognized as the key event of alcoholic liver injury and hepatic fibrosis, and new evidence implicated that ethanol-sensitive microRNAs (miRNAs) are indeed regulatory master-switches during liver injury. The current study aimed to characterize the functional role of specific miRNAs regulating hepatic stellate activity during intragastric overfeeding of mice In Vivo and activated human hepatic stellate cells In Vitro. Methods: miRNA expression in LPS treated human hepatic stellate cells, as well as in 4 weeks chronic alcohol feeding mouse liver specimen and control HSCs and liver tissue, was assessed using a hybridization based microarray. Expression of selected mature miRNAs was evaluated by real-time PCR, respectively. Human hepatic stellate cell activation and transdifferentiation was monitored by Western blot and real-time PCR analysis through specific markers such as α-SMA, laminin, fibronectin and MMPs. Results: The total liver histopathology score increased in ethanol group relative to control mice, whereas TUNEL assay revealed increased hepatocellular apoptosis with hepatic stellate activation after 4 weeks ethanol feeding. We identified that ethanol feeding significantly down-regulated 0.9% of known miRNAs, including let-7a (0.26 ±0.05-fold, p=0.003), let-7b (0.39 ±0.04-fold, p=0.021), and let-7g (0.49±0.07-fold, p=0.018), an important miRNA family in liver pathology, were further verified by Taqman real-time PCR assay. The expression of let-7a and let-7b was significantly reduced in during human hepatic stellate cell activation when cultured in uncoated plastic culture dishes for 5 weeks. Treatment of HSCs with LPS (20 μg/ml) for 72 hours induced a significant decrease of let-7a and let-7b in both activated and control state. Transfection of let-7a and let-7b precursors markedly attenuated the expression of laminin and fibronectin mRNAs and additionally blunted the increased expression of α-SMA, MMP-2 and MMP-9, which are key genes involved in the activation of HSCs. Additionally, overexpression of let-7a and let-7b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Furthermore, the expression of activated hepatic stellate markers and verified let-7 target genes, including α-SMA, laminin, fibronectin, MMP-2, MMP-9 and LIN28, were significantly altered in chronic ethanol feeding mouse liver specimens compared to controls. Conclusion: Our results show that miRNAs are critical regulators of human hepatic stellate activation and transdifferentiation during alcoholic liver injury. The findings provide new insight into the function of specific miRNAs and the mechanisms of alcohol induced liver injury and fibrosis.
929
931
Type XVIII Collagen Plays a Critical Role in Liver Regeneration and is Regulated by Transforming Growth Factor Beta 1 Michael Duncan, Priyanka Thakur, Abdul Rehman
Regulation of Hepatic Stem/Progenitor Phenotype by Melatonin During Alcoholic Liver Injury Yuyan Han, Shannon Glaser, Jennifer McCarra, Julie Venter, Mellanie White, Giuseppina Dusio, Heather L. Francis, Christopher Johnson, Hidekazu Tsukamoto, Fanyin Meng, Gianfranco Alpini
A central feature of liver function is its ability to metabolize foreign substances and sense pathogens in the circulating blood. In order to maintain tissue homeostasis during this process, the liver has the capacity to mount a rapid response to toxin mediated injury or pathogen infection and to regenerate itself. Type XVIII collagen is a prominent liver ECM component and a member of the multiplexin family of collagens that is highly expressed in liver, and levels have been shown to increase further during disease states including fibrosis, cirrhosis, and cancers of the liver. In a previous study, we have demonstrated that when challenged with the hepatoxin, carbon tetrachloride (CCl4), mice deficient in type XVIII collagen suffer severe acute liver dysfunction. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and critical for liver regeneration after toxin induced injury. In the current study we examined the kinetics and regulatory mechanisms that govern type XVIII collagen synthesis during the regeneration process. A survey of common liver ECM molecules including laminin, nidogen, and type IV collagen demonstrates variable expression in and around the normal central vein region, a region most susceptible to toxicant-mediated injury. By immunofluorescence analysis we found type XVIII collagen to be highly expressed in this region. A single injection of CCl4 results in centrilobular necrosis and degradation of type XVIII collagen after 48 hours. At this same time point we observed an increase in mRNA expression of the extracellular matrix associated short chain variant of type XVIII collagen in the injured liver, while the levels on the serum associated long chain variant of type XVIII collagen are decreased. The type XVIII collagen protein levels subsequently increase and return to normal during the recovery phase post-CCl4 injection. Considering the expression pattern and remodeling of type XVIII collagen in the normal and CCl4 injured livers, we asked whether the pleiotropic cytokine transforming growth factor beta 1 could regulate type XVIII collagen expression in hepatocytes. In Vitro, the AML12 hepatocyte cell line treated with recombinant TGF beta 1 recapitulates the In Vivo expression pattern after liver injury. We observed a significant increase in short chain variant expression and a significant decrease in expression for the long chain variant. While the role of TGF beta mediated signaling in the regenerating liver are complex, these results point to a critical role for this signaling pathway in restoring the proper balance of extracellular matrix associated type XVIII collagen in the hepatic microenvironment following injury.
Background: Human livers have maturational lineages of cells within liver acini, beginning periportally in stem cell niches and hepatic progenitors such as small cholangiocytes in small bile ducts. Melatonin, an endogenously produced neurohormone secreted by the pineal gland, has a variety of protective effects during organ injury including promoting the proliferation and differentiation of stem cells. However, its effect and mechanism on the hepatic progenitors during alcoholic liver injury remains to be explored. The objective of this study was to evaluate the role of melatonin regulated hepatic progenitor phenotype during alcoholic liver injury. Methods: The mRNA expression of melatonin upstream enzymes, serotonin N-acetyltransferase (AANAT) and N-acetylserotonin O-methyltransferase (ASMT), was assessed in ethanol and LPS treated human liver stem cells (HLSCs) and cholangiocytes, as well as in 4 weeks chronic alcohol feeding mouse liver specimen and control liver tissue by real-time PCR assay. The secretion of melatonin was verified by ELISA assay. Cell proliferation was measured by MTS assay, whereas apoptosis was monitored through flow cytometry analysis. The hepatic expressions of the clock circadian genes such as PER1, BMAL1 and CRY1, as well as matrix metalloproteinases (MMPs) were also determined by real-time PCR assay. Results: The total liver histopathology score increased in ethanol group relative to control mice, along with the significant reduction of melatonin and AANAT in isolated liver tissues after 4 weeks ethanol feeding. AANAT is mainly expressed in bile ducts of normal mice liver. The basal expression of AANAT and ASMT are significantly higher in HLSCs as well as undifferentiated small murine cholangiocytes (SMCCs) when compared H69 human cholangiocytes and differentiated large murine cholangiocytes (LMCCs). Treatment with ethanol (50 mM) and LPS (20 μg/ml) for 24 hours induced the significant reduction of AANAT and ASMT in liver stem cells, but not in mature H69 human cholangiocytes. Application of melatonin (10-11 M for 24 hours) to HLSCs also prevented alcohol-induced cell death and subsequently recovered the mesenchymal enzyme MMP-1, MMP-2 and MMP-11 that are required for normal liver morphogenesis. Furthermore, the expressions of melatonin regulated clock genes, including CLOCK, PER1, BMAL1 and CRY1, were significantly altered in melatonin treated HLSCs In Vitro, and in mice livers after four weeks of ethanol feeding In Vivo relative to the controls. Conclusion: The discovery that melatonin plays a significant role in the regulation of hepatic progenitor phenotype provides the basis for an exciting field in which the melatonin related upstream enzymes (AANAT and ASMT) as well as the downstream clock circadian genes may be manipulated with potential therapeutic benefits for alcoholic liver injury.
S-935
AASLD Abstracts
AASLD Abstracts
928
Deficiency of Intestinal Mucin-2 Protects From Alcoholic Liver Disease in Mice Phillipp Hartmann, Katharina Brandl, Claus Hellerbrand, Hidekazu Tsukamoto, Samuel B. Ho, Bruce Beutler, Bernd Schnabl
Regulation of Hepatic Stellate Cell Activation by MicroRNA Let-7 Family During Alcoholic Liver Injury Christopher Johnson, Heather L. Francis, Shannon Glaser, Yuyan Han, Julie Venter, Chang-Gong Liu, Mellanie White, Jennifer McCarra, Hidekazu Tsukamoto, Gianfranco Alpini, Fanyin Meng
Background: The intestinal mucus layer protects the epithelium against noxious agents and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease is dependent on the translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. The aim of our study was to investigate the impact of the intestinal mucus layer and in particular Muc2 on alcoholic liver disease. Methods and Results: We used the Tsukamoto-French mouse model which involves continuous intragastric feeding of isocaloric diet (n=4-5) or alcohol for 1 week in Muc2-/- (n=9) and wildtype (WT) mice (n=10). Muc2 was abundantly expressed in the small and large intestine of WT mice, but undetectable in the liver. The intestinal mucus layer was considerably thinner in Muc2 deficient mice as shown by PAS staining. Alcohol feeding did not result in a compensatory upregulation of other intestinal mucins in Muc2-/- mice. After alcohol feeding mice deficient in Muc2 were protected from steatohepatitis as evidenced by significantly lower ALT levels and hepatic triglyceride concentrations. In addition, hepatic oxidative stress was significantly reduced in Muc2-/- mice following intragastric alcohol feeding as shown by immunohistochemistry for 4-hydroxynonenal (4-HNE) and by TBARS assay. There was no significant difference in plasma alcohol levels or hepatic alcohol metabolizing enzymes alcohol dehydrogenase 1 (ADH1) and cytochrome p450 enzyme 2E1 (Cyp2E1) in alcohol fed Muc2-/- as compared to WT mice. Most notably, Muc2-/- mice had significantly lower systemic plasma LPS levels after alcohol feeding. In contrast to WT mice, Muc2-/mice did not exhibit intestinal bacterial overgrowth, but a higher amount of intestinal probiotic Lactobacillus after alcohol administration as shown by qPCR for 16S rRNA and Lactobacillus spp. The antimicrobial proteins Reg3b and Reg3g were found to be expressed at significantly higher levels in the proximal small intestine in isocaloric diet and alcohol fed Muc2-/- mice relative to WT mice as assessed by qPCR and Western blotting. As Reg3b and Reg3g are bactericidal c-type lectins, an increase in their expression might contribute to the observed reduction of the bacterial burden and suppression of bacterial overgrowth in the intestine. Conclusion: Intestinal mucin-2 deficiency protects from alcoholic steatohepatitis. We suggest a pathway that involves higher expression of enteric antimicrobial molecules which suppresses alcohol-associated intestinal bacterial overgrowth. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation and cause less alcoholic liver disease.
Background: microRNAs are endogenous molecules, which constitute a new class of negative regulators of gene expression. The activation of hepatic stellate cells (HSCs) is recognized as the key event of alcoholic liver injury and hepatic fibrosis, and new evidence implicated that ethanol-sensitive microRNAs (miRNAs) are indeed regulatory master-switches during liver injury. The current study aimed to characterize the functional role of specific miRNAs regulating hepatic stellate activity during intragastric overfeeding of mice In Vivo and activated human hepatic stellate cells In Vitro. Methods: miRNA expression in LPS treated human hepatic stellate cells, as well as in 4 weeks chronic alcohol feeding mouse liver specimen and control HSCs and liver tissue, was assessed using a hybridization based microarray. Expression of selected mature miRNAs was evaluated by real-time PCR, respectively. Human hepatic stellate cell activation and transdifferentiation was monitored by Western blot and real-time PCR analysis through specific markers such as α-SMA, laminin, fibronectin and MMPs. Results: The total liver histopathology score increased in ethanol group relative to control mice, whereas TUNEL assay revealed increased hepatocellular apoptosis with hepatic stellate activation after 4 weeks ethanol feeding. We identified that ethanol feeding significantly down-regulated 0.9% of known miRNAs, including let-7a (0.26 ±0.05-fold, p=0.003), let-7b (0.39 ±0.04-fold, p=0.021), and let-7g (0.49±0.07-fold, p=0.018), an important miRNA family in liver pathology, were further verified by Taqman real-time PCR assay. The expression of let-7a and let-7b was significantly reduced in during human hepatic stellate cell activation when cultured in uncoated plastic culture dishes for 5 weeks. Treatment of HSCs with LPS (20 μg/ml) for 72 hours induced a significant decrease of let-7a and let-7b in both activated and control state. Transfection of let-7a and let-7b precursors markedly attenuated the expression of laminin and fibronectin mRNAs and additionally blunted the increased expression of α-SMA, MMP-2 and MMP-9, which are key genes involved in the activation of HSCs. Additionally, overexpression of let-7a and let-7b led HSCs to remain in a quiescent state, as evidenced by their quiescent star-like cell morphology. Furthermore, the expression of activated hepatic stellate markers and verified let-7 target genes, including α-SMA, laminin, fibronectin, MMP-2, MMP-9 and LIN28, were significantly altered in chronic ethanol feeding mouse liver specimens compared to controls. Conclusion: Our results show that miRNAs are critical regulators of human hepatic stellate activation and transdifferentiation during alcoholic liver injury. The findings provide new insight into the function of specific miRNAs and the mechanisms of alcohol induced liver injury and fibrosis.
929
931
Type XVIII Collagen Plays a Critical Role in Liver Regeneration and is Regulated by Transforming Growth Factor Beta 1 Michael Duncan, Priyanka Thakur, Abdul Rehman
Regulation of Hepatic Stem/Progenitor Phenotype by Melatonin During Alcoholic Liver Injury Yuyan Han, Shannon Glaser, Jennifer McCarra, Julie Venter, Mellanie White, Giuseppina Dusio, Heather L. Francis, Christopher Johnson, Hidekazu Tsukamoto, Fanyin Meng, Gianfranco Alpini
A central feature of liver function is its ability to metabolize foreign substances and sense pathogens in the circulating blood. In order to maintain tissue homeostasis during this process, the liver has the capacity to mount a rapid response to toxin mediated injury or pathogen infection and to regenerate itself. Type XVIII collagen is a prominent liver ECM component and a member of the multiplexin family of collagens that is highly expressed in liver, and levels have been shown to increase further during disease states including fibrosis, cirrhosis, and cancers of the liver. In a previous study, we have demonstrated that when challenged with the hepatoxin, carbon tetrachloride (CCl4), mice deficient in type XVIII collagen suffer severe acute liver dysfunction. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and critical for liver regeneration after toxin induced injury. In the current study we examined the kinetics and regulatory mechanisms that govern type XVIII collagen synthesis during the regeneration process. A survey of common liver ECM molecules including laminin, nidogen, and type IV collagen demonstrates variable expression in and around the normal central vein region, a region most susceptible to toxicant-mediated injury. By immunofluorescence analysis we found type XVIII collagen to be highly expressed in this region. A single injection of CCl4 results in centrilobular necrosis and degradation of type XVIII collagen after 48 hours. At this same time point we observed an increase in mRNA expression of the extracellular matrix associated short chain variant of type XVIII collagen in the injured liver, while the levels on the serum associated long chain variant of type XVIII collagen are decreased. The type XVIII collagen protein levels subsequently increase and return to normal during the recovery phase post-CCl4 injection. Considering the expression pattern and remodeling of type XVIII collagen in the normal and CCl4 injured livers, we asked whether the pleiotropic cytokine transforming growth factor beta 1 could regulate type XVIII collagen expression in hepatocytes. In Vitro, the AML12 hepatocyte cell line treated with recombinant TGF beta 1 recapitulates the In Vivo expression pattern after liver injury. We observed a significant increase in short chain variant expression and a significant decrease in expression for the long chain variant. While the role of TGF beta mediated signaling in the regenerating liver are complex, these results point to a critical role for this signaling pathway in restoring the proper balance of extracellular matrix associated type XVIII collagen in the hepatic microenvironment following injury.
Background: Human livers have maturational lineages of cells within liver acini, beginning periportally in stem cell niches and hepatic progenitors such as small cholangiocytes in small bile ducts. Melatonin, an endogenously produced neurohormone secreted by the pineal gland, has a variety of protective effects during organ injury including promoting the proliferation and differentiation of stem cells. However, its effect and mechanism on the hepatic progenitors during alcoholic liver injury remains to be explored. The objective of this study was to evaluate the role of melatonin regulated hepatic progenitor phenotype during alcoholic liver injury. Methods: The mRNA expression of melatonin upstream enzymes, serotonin N-acetyltransferase (AANAT) and N-acetylserotonin O-methyltransferase (ASMT), was assessed in ethanol and LPS treated human liver stem cells (HLSCs) and cholangiocytes, as well as in 4 weeks chronic alcohol feeding mouse liver specimen and control liver tissue by real-time PCR assay. The secretion of melatonin was verified by ELISA assay. Cell proliferation was measured by MTS assay, whereas apoptosis was monitored through flow cytometry analysis. The hepatic expressions of the clock circadian genes such as PER1, BMAL1 and CRY1, as well as matrix metalloproteinases (MMPs) were also determined by real-time PCR assay. Results: The total liver histopathology score increased in ethanol group relative to control mice, along with the significant reduction of melatonin and AANAT in isolated liver tissues after 4 weeks ethanol feeding. AANAT is mainly expressed in bile ducts of normal mice liver. The basal expression of AANAT and ASMT are significantly higher in HLSCs as well as undifferentiated small murine cholangiocytes (SMCCs) when compared H69 human cholangiocytes and differentiated large murine cholangiocytes (LMCCs). Treatment with ethanol (50 mM) and LPS (20 μg/ml) for 24 hours induced the significant reduction of AANAT and ASMT in liver stem cells, but not in mature H69 human cholangiocytes. Application of melatonin (10-11 M for 24 hours) to HLSCs also prevented alcohol-induced cell death and subsequently recovered the mesenchymal enzyme MMP-1, MMP-2 and MMP-11 that are required for normal liver morphogenesis. Furthermore, the expressions of melatonin regulated clock genes, including CLOCK, PER1, BMAL1 and CRY1, were significantly altered in melatonin treated HLSCs In Vitro, and in mice livers after four weeks of ethanol feeding In Vivo relative to the controls. Conclusion: The discovery that melatonin plays a significant role in the regulation of hepatic progenitor phenotype provides the basis for an exciting field in which the melatonin related upstream enzymes (AANAT and ASMT) as well as the downstream clock circadian genes may be manipulated with potential therapeutic benefits for alcoholic liver injury.
S-935
AASLD Abstracts
AASLD Abstracts
928