Liver immunolocalization and plasma levels of MMP-9 in non-alcoholic steatohepatitis (NASH) and hepatitis C infection

Summary

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease. Many issues related to the pathogenesis of this disease remain unresolved. Because of NASH association with the activation of liver fibrogenesis, we examined the plasma levels and liver immunolocalization of matrix metalloprotease-9 (MMP-9), a molecule involved in the remodelling processes of fibrogenesis. In addition, patients with chronic hepatitis C (HCV) were analyzed. Plasma concentrations of MMP-9 were determined by ELISA from peripheral blood and immunohistochemistry of the same protein was carried out in formalin-fixed and paraffin wax-embedded liver specimens. The mean value of circulating concentrations of MMP-9 in healthy controls was 39.7 ng/ml (SD: ±4.6). In NASH and HCV-infected patients, MMP-9 concentrations were higher: 69.0 ng/ml (SD: ±14.5) and 61.7 ng/ml (SD: ±11.0), respectively. In NASH livers, MMP-9 was mainly immunolocalized on neutrophils, whereas in HVC-infected livers it was mainly localized over biliary canaliculi, bile ducts and hepatocyte cytoplasm. The different MMP-9 immunolocalization patterns in the examined diseases suggest the presence of a different pathophysiological involvement of this protease in the fibrogenesis underlying these diseases.

Introduction

Among the non-alcoholic fatty liver diseases (NAFLD), non-alcoholic steatohepatitis (NASH) represents a severe progressive form of disease. It is recognized as one of the most common causes of chronic liver disease and its prevalence is on the increase worldwide. Although the pathogenesis of NASH is multifactorial, many issues remain unresolved (Shifflet and Wu, 2009). Histologically, the liver of NASH patients is mainly characterized by micro- and macrovesicular steatosis, hepatocyte degeneration and inflammation with leukocyte infiltration. Moreover, it is known that NASH is associated with the activation of liver fibrogenesis (Gramlich et al., 2004; Preiss and Sattar, 2008). Chronic viral infections, such as hepatitis B and C, represent another common cause of persistent liver disease leading to inflammation and fibrosis (Friedman, 2003; Neuman et al., 2008).

Such viral and non-viral diseases are characterized by hepatic remodelling due to an increased synthesis and decreased collagen degradation (Boker et al., 2000). Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, have been shown to be involved in this remodelling process (Birkedal-Hansen, 1995; Olaso and Friedman, 1998; Clark et al., 2008). MMPs are generally expressed at low levels in normal tissues, but under certain physiological and pathological conditions, they are upregulated (Ries and Petrides, 1995). MMP-9 (gelatinase B; 92-kDa type IV collagenase) is able to degrade gelatins, type III, IV and V collagen and to cleave other matrix and non-matrix molecules with the subsequent release of bioactive molecules (Bergers et al., 2000; Van den Steen et al., 2002; Björklund and Koivunen, 2005).

It is well established that variations of expression of MMPs are correlated to the fibrotic stage of liver diseases. Thus, it is important to understand the mechanisms involved in the participation of MMPs in the course of diseases that induce fibrogenesis. Because of the high risk of progression to cirrhosis and hepatocellular carcinoma by NASH disease (McCullough, 2006), these efforts could eventually lead to development of better diagnoses and new therapeutic strategies.

In this paper we analyzed MMP-9 plasma levels and localization in liver affected with non-alcoholic steatohepatitis, as well as livers with chronic hepatitis C. Both diseases are characterized by progressive development of liver fibrosis, but differences in MMP-9 expression and localization patterns are thought to be related to different physiopathological mechanisms underlying the two diseases.