Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.
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.


CONCLUSIONS
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.
Department of Surgery, Faculty of Medicine, Minia University, El-Minia, Egypt.
Hussein
H
Atta
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Mahmoud
M
El-Rehany
Department of Pathology, Theodor Bilharz Research Institute, Giza, Egypt.
Olfat
O
Hammam
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Hend
H
Abdel-Ghany
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Maggie
M
Ramzy
Department of Gastroenterology, Justus-Liebig University Giessen, Germany.
Martin
M
Roderfeld
Department of Gastroenterology, Justus-Liebig University Giessen, Germany.
Elke
E
Roeb
Department of Obstetrics and Gynecology, Georgia Regents University, Augusta, Georgia, United States of America.
Ayman
A
Al-Hendy
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Salama Abdel
SA
Raheim
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Hatem
H
Allam
Department of Biochemistry, Faculty of Medicine, Minia University, El-Minia, Egypt.
Heba
H
Marey
D000199:Actins / Q000235:genetics; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002251:Carbon Tetrachloride; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D000071221:Enhancer of Zeste Homolog 2 Protein; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006801:Humans; D008106:Liver Cirrhosis, Experimental / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D009130:Muscle, Smooth; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C066855:HGF protein, human; D050505:Mutant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D017228:Hepatocyte Growth Factor; D002251:Carbon Tetrachloride; D008780:Methyltransferases; C000604006:Ash1l protein, rat; C578253:EZH2 protein, rat; D000071221:Enhancer of Zeste Homolog 2 Protein; D011495:Histone-Lysine N-Methyltransferase; D063151:Polycomb Repressive Complex 2; D020780:Matrix Metalloproteinase 9