Abstract
Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Conclusion: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.
Highlights
Liver fibrosis and its end-stage sequela of cirrhosis are major causes of morbidity and mortality worldwide and result from different etiologies of chronic liver injury
There was no difference in the matrix metalloproteinases (MMPs)-9 protein concentrations in the livers of the Hepatocyte growth factor (HGF)-treated or combined HGF/mMMP-9treated livers compared with the untreated fibrotic livers (Figure 1.D)
MMMP-9 is more effective than HGF gene therapy in reducing CCl4-induced liver fibrosis in rats as supported by the documented reduction in all profibrogenic markers
Summary
Liver fibrosis and its end-stage sequela of cirrhosis are major causes of morbidity and mortality worldwide and result from different etiologies of chronic liver injury. The high morbidity and mortality associated with fibrosis/cirrhosis underscores the need for novel preventive and therapeutic approaches [1]. Fibrosis accumulation is a dynamic process resulting from a wound-healing response involving pathways of fibrogenesis and inflammation [2]. Fibrosis reflects the imbalance between matrix production and degradation [3]. Hepatocyte necrosis and apoptosis instigate inflammatory signaling by chemokines and cytokines resulting in recruitment of immune cell populations, and activation of fibrogenic cells, culminating in the deposition of extracellular matrix (ECM) [4]. A major determinant of progressive fibrosis is failure to degrade the increased interstitial matrix [3]
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