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Metalloproteinases activation involvement in the progression of liver injury from NAFLD to NASH
Abstracts / Digestive and Liver Disease 47S (2015) e226–e235
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METALLOPROTEINASES ACTIVATION INVOLVEMENT IN THE PROGRESSION OF LIVER INJURY FROM NAFLD TO NASH
A DISTINCT MIRNA EXPRESSION PROFILE IN HEPATOCELLULAR CARCINOMA ARISING IN CARRIERS OF THE I148M PNPLA3 GENE VARIANT
C. Berardo 1,∗ , G. Palladini 1 , L.G. Di Pasqua 1 , V. Rizzo 2 , S. Perlini 1 , P. Richelmi 1 , M. Vairetti 1 , A. Ferrigno 1
B. Donati 1,∗ , V. Vaira 2,3 , A. Donnangelo 3 , M. Maggioni 3 , C. Augello 3 , P. Dongiovanni 4 , M. Milano 1,4 , M. Roncalli 5 , S. Fargion 1 , S. Bosari 3 , L. Valenti 1,4
1 Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy 2 Department of Molecular Medicine, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy
Background: Matrix metalloproteinases (MMPs) are a large family of zinc-containing endopeptidases, which are responsible for the degradation of extracellular matrix proteins. Recent studies reported an induction of MMPs in the progression of liver damage such as I/R injury, acute allograft rejection and chronic viral hepatitis. Aim: This study investigated whether, in a rat model of non-alcoholic steatohepatitis (NASH) using a methionine/cholinedeficient (MCD) diet, changes in serum and tissue MMPs occur during the progression of liver injury. Materials and methods: Male Wistar rats underwent to NASH induced by 8 weeks of feeding with MCD diet. Serum and hepatic biopsies, at 2, 4 and 8 weeks, were used for monitoring MMP-2 and MMP-9 activation. Serum hepatic enzymes (AST, ALT), Ca2+ , Fe2+ and uric acid were evaluated. Tissue total lipids, lipid peroxides (TBARS), glutathione and reactive oxygen species (ROS) were also quantified. Results: A significantly lower serum levels of uric acid and Ca2+ and increase in Fe2+ were found in MCD rats compared with control group. A time-dependent increase in tissue MMP-2 occurs in MCD rats and the same trend, although not significant, was found for MMP-9. Serum MMP-2 and MMP-9 showed no significant changes when compared MCD group versus control animals. The hepatic decrease in glutathione content was concomitant with an increase in TBARS and ROS levels. As expected, a time dependent increase in serum AST and ALT in MCD animals was detected. Conclusions: MCD rats exhibit a marked oxidative stress concomitant with an hepatic MMP-2 activation. The reported spontaneously development to severe NASH observed in MCD rats might be associated to these events already detectable in the early period of treatment. Funding: Supported by Fondazione Cariplo, grant no 2011-0439. http://dx.doi.org/10.1016/j.dld.2015.07.035
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Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milano, Italy 2 Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, 20122 Milano, Italy 3 Department of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, 20122 Milano, Italy 4 Internal Medicine and Metabolic Diseases, Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, 20122 Milano, Italy 5 Pathology Unit, Humanitas Clinical and Research hospital, Milano, Italy Background: The I148M PNPLA3 variant is the major genetic determinant of hepatic fat and liver enzymes and a validated risk factor for progression to hepatocellular carcinoma (HCC). Furthermore, carrying the I148M variant worsens HCC prognosis. PNPLA3 regulates lipid droplets metabolism, but the mechanism linking the I148M variant to carcinogenesis is unknown. The expression profile of miRNAs is associated with cancer development, progression and response to therapy. Aims: We investigated whether the PNPLA3 I148M variant is associated with a specific miRNA expression profile in HCC. Methods: We performed a TaqMan MicroRNA arrays (cards A and B, v3.0) experiment to compare miRNA profiles (664 miRNAs) of HCC and surrounding cirrhotic tissue of six 148M/M and six 148I/I patients matched for sex, age and etiology of liver disease. I148M genotype was assessed by TaqMan assays in cirrhotic and tumor tissue. Results: Multiclass comparison analysis revealed 49 miRNAs differentially expressed between HCC tissues of 148M/M and I/I patients (p < 0.01). Supervised dynamic analysis based on miRNA signatures of intra-extralesional tissues of patients with different I148M status, revealed distinct miRNA profiles for cirrhotic compared to cancer tissues independently of genetic background, but showed that PNPLA3 I148M influences the expression of a subset of miRNAs in HCCs. In particular, 82 miRNAs were differentially expressed in cancer lesions according to I148M genotype (p < 0.05), suggesting that this genetic variant is associated with a specific carcinogenic pathway leading to tumors with distinct biological features. Among the differentially expressed miRNAs there was enrichment in molecules regulating cell cycle and carcinogenesis, angiogenesis and lipid metabolism. Conclusions: We detected a panel of miRNAs specifically deregulated in HCC of patients homozygous for the PNPLA3 I148M variant. While results await an independent replication in a confirmatory cohort, they may contribute elucidating the epigenetic mechanism underlying hepatic carcinogenesis and highlight novel diagnostic and therapeutic targets. http://dx.doi.org/10.1016/j.dld.2015.07.036
P11
P12
METALLOPROTEINASES ACTIVATION INVOLVEMENT IN THE PROGRESSION OF LIVER INJURY FROM NAFLD TO NASH
A DISTINCT MIRNA EXPRESSION PROFILE IN HEPATOCELLULAR CARCINOMA ARISING IN CARRIERS OF THE I148M PNPLA3 GENE VARIANT
C. Berardo 1,∗ , G. Palladini 1 , L.G. Di Pasqua 1 , V. Rizzo 2 , S. Perlini 1 , P. Richelmi 1 , M. Vairetti 1 , A. Ferrigno 1
B. Donati 1,∗ , V. Vaira 2,3 , A. Donnangelo 3 , M. Maggioni 3 , C. Augello 3 , P. Dongiovanni 4 , M. Milano 1,4 , M. Roncalli 5 , S. Fargion 1 , S. Bosari 3 , L. Valenti 1,4
1 Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy 2 Department of Molecular Medicine, Fondazione IRCCS Policlinico S. Matteo and University of Pavia, Pavia, Italy
Background: Matrix metalloproteinases (MMPs) are a large family of zinc-containing endopeptidases, which are responsible for the degradation of extracellular matrix proteins. Recent studies reported an induction of MMPs in the progression of liver damage such as I/R injury, acute allograft rejection and chronic viral hepatitis. Aim: This study investigated whether, in a rat model of non-alcoholic steatohepatitis (NASH) using a methionine/cholinedeficient (MCD) diet, changes in serum and tissue MMPs occur during the progression of liver injury. Materials and methods: Male Wistar rats underwent to NASH induced by 8 weeks of feeding with MCD diet. Serum and hepatic biopsies, at 2, 4 and 8 weeks, were used for monitoring MMP-2 and MMP-9 activation. Serum hepatic enzymes (AST, ALT), Ca2+ , Fe2+ and uric acid were evaluated. Tissue total lipids, lipid peroxides (TBARS), glutathione and reactive oxygen species (ROS) were also quantified. Results: A significantly lower serum levels of uric acid and Ca2+ and increase in Fe2+ were found in MCD rats compared with control group. A time-dependent increase in tissue MMP-2 occurs in MCD rats and the same trend, although not significant, was found for MMP-9. Serum MMP-2 and MMP-9 showed no significant changes when compared MCD group versus control animals. The hepatic decrease in glutathione content was concomitant with an increase in TBARS and ROS levels. As expected, a time dependent increase in serum AST and ALT in MCD animals was detected. Conclusions: MCD rats exhibit a marked oxidative stress concomitant with an hepatic MMP-2 activation. The reported spontaneously development to severe NASH observed in MCD rats might be associated to these events already detectable in the early period of treatment. Funding: Supported by Fondazione Cariplo, grant no 2011-0439. http://dx.doi.org/10.1016/j.dld.2015.07.035
e231
1
Department of Pathophysiology and Transplantation, Università degli Studi di Milano, 20122 Milano, Italy 2 Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, 20122 Milano, Italy 3 Department of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, 20122 Milano, Italy 4 Internal Medicine and Metabolic Diseases, Fondazione IRCCS Ca’ Granda Ospedale Policlinico Milano, 20122 Milano, Italy 5 Pathology Unit, Humanitas Clinical and Research hospital, Milano, Italy Background: The I148M PNPLA3 variant is the major genetic determinant of hepatic fat and liver enzymes and a validated risk factor for progression to hepatocellular carcinoma (HCC). Furthermore, carrying the I148M variant worsens HCC prognosis. PNPLA3 regulates lipid droplets metabolism, but the mechanism linking the I148M variant to carcinogenesis is unknown. The expression profile of miRNAs is associated with cancer development, progression and response to therapy. Aims: We investigated whether the PNPLA3 I148M variant is associated with a specific miRNA expression profile in HCC. Methods: We performed a TaqMan MicroRNA arrays (cards A and B, v3.0) experiment to compare miRNA profiles (664 miRNAs) of HCC and surrounding cirrhotic tissue of six 148M/M and six 148I/I patients matched for sex, age and etiology of liver disease. I148M genotype was assessed by TaqMan assays in cirrhotic and tumor tissue. Results: Multiclass comparison analysis revealed 49 miRNAs differentially expressed between HCC tissues of 148M/M and I/I patients (p < 0.01). Supervised dynamic analysis based on miRNA signatures of intra-extralesional tissues of patients with different I148M status, revealed distinct miRNA profiles for cirrhotic compared to cancer tissues independently of genetic background, but showed that PNPLA3 I148M influences the expression of a subset of miRNAs in HCCs. In particular, 82 miRNAs were differentially expressed in cancer lesions according to I148M genotype (p < 0.05), suggesting that this genetic variant is associated with a specific carcinogenic pathway leading to tumors with distinct biological features. Among the differentially expressed miRNAs there was enrichment in molecules regulating cell cycle and carcinogenesis, angiogenesis and lipid metabolism. Conclusions: We detected a panel of miRNAs specifically deregulated in HCC of patients homozygous for the PNPLA3 I148M variant. While results await an independent replication in a confirmatory cohort, they may contribute elucidating the epigenetic mechanism underlying hepatic carcinogenesis and highlight novel diagnostic and therapeutic targets. http://dx.doi.org/10.1016/j.dld.2015.07.036