| Abstract |
Chronic liver injury occurs in response to a variety of causes, including viral hepatitis B and C. During liver fibrosis the pathological accumulation of extracellular matrix occurs as a consequence of the modifications that follow the synthesis and degradation of matrix proteins. The exact mechanism, by which specific matrix-degrading enzymes act during ECM remodelling, is not clear. Evidence of the involvement of MMPs, the most important family of ECM-degradative enzymes, in various liver diseases exists in a number of studies. The majority of the findings regarding matrix metalloproteinase (MMP) expression in hepatitis B and C infected patients indicate the fluctuations of MMP's RNA, DNA or active enzyme levels in relation to the viral presence and chronic infections. However, better understanding is crucial for the management of these diseases since there are still many points to be answered. No study has attempted to study matrix metalloproteinase expression in non-alcoholic steatohepatitis (NASH). To achieve more information about fibroproliferation in hepatitis B, C and NASH patients, the expression of MMPs in these diseases was studied checking the hypothesis that different MMPs might be involved in different liver diseases during the process of fibrogenesis. For that purpose, we assessed the expression levels of MMPs by reverse transcription polymerase chain (RT-PCR) reaction and examined the presence of MMP-2, -9, -10, -11 total RNAs in the tissues from patients with viral and non viral hepatitis.73 patients were examined in this study: non-diseased controls (10), patients with chronic hepatitis B (14), chronic hepatitis C (33) and non-alcoholic steatohepatitis (16). All patients studied consumed no alcohol. Ten liver samples were taken at operation for cholecystectomy and used as healthy controls. Total RNA was isolated after immersion of the liver biopsies in liquid nitrogen and it was reversely transcribed by cDNA synthesis. Consecutive amplification of cDNA was performed by PCR and products were electrophorised through an 8% polyacrylamide gel and silver stained. PCR products were semi-quantitatively analyzed by densitometric analysis using b-actin or b2-microglobulin as the house keeping gene. The RNA levels of each gene were expressed as a ratio of the intensity of the bands in diseased tissues versus the corresponding levels of the internal control. Non-parametric tests (Kruskal-Wallis and Mann-Whitney) were performed to examine differences on metalloproteinase expression amongst diseases and controls. When we grouped together the expression values for metalloproteinases from HCV and HBV viral hepatitis patients and compared it with non-viral patients a statistically significant increase in levels of MMP-9 and MMP-10 in NASH (P<0.05) was obtained. In our studies in viral diseases a statistically significant increase in MMP-2 expression (P<0.05) was observed when compared to control values. We observed statistically significant increases in MMP-9 expression values in NASH when compared to controls (P<0.05). We examined expression patterns of metalloproteinases in different histological stages of chronic hepatitis due to HBV or HCV viruses. In HBV cases, MMP-9 expression showed a statistically significant decrease (P<0.05) in the final stages of the viral infection. When we investigated HCV related liver disease, a stronger presence of MMP-2 and MMP-9 in final disease stages was detected. MMP-10 and MMP-11 followed a decreasing pattern during disease development. Statistical significance was obtained only for the MMP-2 expression (P<0.05) at the final stages of HCV. Few studies have assessed the expression of metalloproteinase network in chronic hepatitis. No study has attempted to study in detail their expression in non-alcoholic steatohepatitis. In the present results in non-alcoholic steatohepatitis we showed a significant increase in the expression of MMP-9 and MMP-10, when compared to viral hepatitis B and C. Previous reports on MMP-9 expression in the human liver have demonstrated an increase of MMP-9 gelatinolytic activity in hepatic tissues from patients with viral and alcoholic cirrhosis and an early increase in chronic viral hepatitis C during all fibrotic stages including cirrhosis. We identified similar increase in the expression of MMP-9 when comparisons were made with normal livers. In our viral hepatitis patients we identified MMP-10 expression with an increasing pattern in advanced fibrosis of HBV cases and a decreasing pattern in advanced HCV fibrosis when compared to the early fibrosis. The reason for this discrepancy is not clear. When HCV and HBV related chronic hepatitis were grouped together and compared to the controls, a significant increase in the MMP-2 expression was observed. Results from experimental animal and human studies agree with our findings. In the present study, when the patients were analysed according to the viral aetiology and disease staging using the Ishaks system in the study of late stage HCV we obtained results which showed a different profile, with values of MMP-9 relatively increased. This increase did not reach statistical significance. Our findings in HCV agree with the findings of Lichtinghagen et al. [2003] who also observed an increase in MMP-9 mRNA expression in patients with advanced HCV fibrotic stages. Gelatinases are the enzymes mostly responsible for collagen type IV degradation. We suggest that their increase might be a compensatory mechanism occurring as a result of the over accumulation of type IV collagen previously reported in fibrotic livers. Our results indicate that apart from fibrotic stages, the viral aetiology should also be taken into account when MMP expression is examined. In addition to these findings our results on MMPs expression in NASH indicate that a different pattern emerges in this non-viral potentially fibrotic liver disease. This may be of importance when looking for diagnostic targets and therapy in the case of hepatic fibrosis. Further studies are warranted in order to elucidate the expression of the other MMPs and their role in hepatic diseases.
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