Mutant MMP-9 and HGF Gene Transfer Enhance Resolution of CCl₄-Induced Liver Fibrosis in Rats: Role of ASH1 and EZH2 Methyltransferases Repression

<div><p>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.</p></div

Amplified DNA from different tissues of animals transplanted by (+/+) or (−/−) donors.

<p>Amplified DNA from different tissues of animals transplanted by (+/+) or (−/−) donors.</p

Effect of gene therapy on molecular markers of liver fibrosis.

<p>(A, B) Col1α1 mRNA expression was significantly elevated (*, p<0.001) in saline-treated livers compared with healthy livers (n = 10), and was significantly reduced with HGF (n = 14, #, p<0.05), mMMP-9 (n = 10, *, p<0.001) and HGF/mMMP-9 (n = 8, p<0.001) treatments compared with saline-treated fibrotic livers (n = 13). (C, D) α-SMA mRNA expression was significantly reduced with HGF (n = 14, p<0.001), mMMP-9 (n = 10, p<0.001) and HGF/mMMP-9 (n = 9, p<0.001) treatments compared with saline-treated fibrotic livers and was significantly elevated (p<0.001) in saline-treated fibrotic livers compared with healthy livers (n = 10). (E, F) TGF-β1 mRNA expression was significantly reduced with mMMP-9 (n = 10, p<0.01) and HGF/mMMP-9 (n = 9, p<0.01) treatment but not with HGF (n = 13, p = 0.87) compared with saline-treated fibrotic livers (n = 13). (G) TIMP-1 protein concentration was elevated (p<0.01) in saline-treated fibrotic livers (n = 9) and was further elevated in HGF-treated livers (n = 13) compared with healthy liver (n = 9). TIMP-1 concentration in fibrotic livers was significantly (p<0.001) decreased with mMMP-9 (n = 10) but not with HGF (p = 0.09) or with HGF/mMMP-9 (n = 9, p = 0.69) treatment. (H) Hydroxyproline concentration in saline-treated fibrotic livers (n = 10) was significantly elevated (p<0.001) compared with healthy livers (n = 10) and was significantly (p<0.001) decreased only with mMMP-9 treatment (n = 10) but not with HGF (n = 11, p = 0.86) or with HGF/mMMP-9 (n = 7, p = 0.98) treatments. (I) ALT serum concentration in saline-treated fibrotic rats (n = 14) was significantly elevated (p<0.001) compared with healthy rats (n = 10) and was significantly decreased with mMMP-9 (n = 10, p<0.001) and combined HGF/mMMP-9 (n = 9, p<0.01) but not with HGF (n = 14, p = 0.15) treatment.</p

Immunohistochemical staining for α-SMA and PCNA in the liver.

<p>Bar graphs represent α-SMA and PCNA labeling indices (percent of positively stained brown cytoplasm or brown nuclei, respectively in 10 successive fields). Both α-SMA and PCNA indices were significantly elevated (*, p<0.001) in saline-treated fibrotic (n = 14 both) compared with healthy livers (n = 10 both) and both were significantly reduced (*, p<0.001) in mMMP-9-treated (n = 10 both) and in HGF/mMMP-9-treated (n = 9 both, *, p<0.001 and #, p<0.01, respectively) versus saline-treated fibrotic livers. Both α-SMA and PCNA labeling indices were not significantly different in HGF-treated fibrotic (n = 14, p = 0.99 and p = 0.06, respectively) from that in saline-treated fibrotic livers. (Original magnification ×400).</p

Changes of total body weight(A), ovaries(B), uterus(C), vagina and cervix(D), and serum level of FSH(E) and estrogen(F) in treated (Tr) Vs control (Ct) animals at different time points of experiment.

<p>For B, C and D organs weight considered as % of total body weight. Both treated and control group had increase in total body weight but BMT group showed significantly more increase than control group (A, P<0.03). As indicated, reproductive organs which are highly modulated by estrogen, showed remarkable increase in weight at all time points of the experiment except for the first week (for B, C, and D, a P value of less than 0.04 obtained). Bone marrow transplanted animals compare to untreated controls showed 40–50% decrease in serum FSH level (E, P<0.03) and 4–5.5 folds increase in serum estrogen (F, P<0.004) at all time points of experiment.</p

Histopathological findings in rat liver sections stained with H&E and Sirius-red.

<p>Fibrosis score was significantly elevated (*, p<0.001) in saline-treated fibrotic (n = 14) compared with healthy livers (n = 10). Fibrosis scores of HGF-treated livers (n = 14) were not statistical different from those of saline-treated livers (p = 0.71). However, fibrosis score was significantly reduced (**, p<0.01) in mMMP-9-treated (n = 9) and (#, p<0.05) in HGF/mMMP-9-treated (n = 9) compared to saline-treated fibrotic livers. (Original magnification ×200). Sirius red stained collagen area percent was significantly elevated (p<0.001) in saline-treated fibrotic livers (n = 14) compared with healthy livers (n = 10). Collagen area percent was significantly reduced in mMMP-9-treated (n = 9, p<0.001)) and in HGF/mMMP-9-treated (n = 9, p<0.01) but not in HGF-treated (n = 13, p = 0.19) compared to saline-treated fibrotic livers. (Original magnification, ×100).</p

Development of the ovary in BMT animal (A) compared to untreated control animal (B).

<p>Both the total number of follicles and the number of antral follicles are significantly higher in BMT compare to control group. Histological evaluation showed on average 28±4 follicles/ovary in treated group with 8±2 follicles at the antral stage compared to only 6±2 with no follicles at antral stage in untreated control mice. Photos have been taken at the same magnification.</p