Measurement of CO3-610, a Potential Liver Biomarker Derived from Matrix Metalloproteinase-9 Degradation of Collagen Type III, in a Rat Model of Reversible Carbon-Tetrachloride-Induced Fibrosis

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Phosphodiesterase inhibition mediates matrix metalloproteinase activity and the level of collagen degradation fragments in a liver fibrosis <it>ex vivo</it> rat model

<p>Abstract</p> <p>Background</p> <p>Accumulation of extracellular matrix (ECM) and increased matrix metalloproteinase (MMP) activity are hallmarks of liver fibrosis. The aim of the present study was to develop a model of liver fibrosis combining <it>ex vivo</it> tissue culture of livers from CCl₄ treated animals with an ELISA detecting a fragment of type III collagen generated <it>in vitro</it> by MMP-9 (C3M), known to be associated with liver fibrosis and to investigate cAMP modulation of MMP activity and liver tissue turnover in this model.</p> <p>Findings</p> <p><it>In vivo:</it> Rats were treated for 8 weeks with CCl₄/Intralipid. Liver slices were cultured for 48 hours. Levels of C3M were determined in the supernatants of slices cultured without treatment, treated with GM6001 (positive control) or treated with IBMX (phosphodiesterase inhibitor). Enzymatic activity of MMP-2 and MMP-9 were studied by gelatin zymography. <it>Ex vivo:</it> The levels of serum C3M increased 77% in the CCl₄-treated rats at week 8 (p < 0.01); Levels of C3M increased significantly by 100% in fibrotic liver slices compared to controls after 48 hrs (p < 0.01). By adding GM6001 or IBMX to the media, C3M was restored to control levels. Gelatin zymography demonstrated CCl₄-treated animals had highly increased MMP-9, but not MMP-2 activity, compared to slices derived from control animals.</p> <p>Conclusions</p> <p>We have combined an <it>ex vivo</it> model of liver fibrosis with measurement of a biochemical marker of collagen degradation in the condition medium. This technology may be used to evaluate the molecular process leading to structural fibrotic changes, as collagen species are the predominant structural part of fibrosis. These data suggest that modulation of cAMP may play a role in regulation of collagen degradation associated with liver fibrosis.</p

Activation of thyroid hormone receptor-β improved disease activity and metabolism independent of body weight in a mouse model of non-alcoholic steatohepatitis and fibrosis

Background and Purpose: Activation of hepatic thyroid hormone receptor β (THR-β) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-β agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-β agonist, as a prototype to investigate the effects of THR-β activation in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis. Experimental Approach: C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg−1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-β activation. Key Results: Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis. Conclusion and Implications: Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity

Serum Markers of Liver Fibrosis: Combining the BIPED Classification and the Neo-Epitope Approach in the Development of New Biomarkers

Background: Fibrosis is a central histological feature of chronic liver diseases and is characterized by the accumulation and reorganization of the extracellular matrix. The gold standard for assessment of fibrosis is histological evaluation of a percutaneous liver biopsy. Albeit a considerable effort have been invested in finding alternative non-invasive approaches, these have not been sufficiently succesfull to replace biopsy assessment

Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy

AIM: To characterize development of diet-induced nonalcoholic steatohepatitis (NASH) by performing liver biopsy in wild-type and genetically obese mice. METHODS: Male wild-type C57BL/6J (C57) mice (DIO-NASH) and male Lep(ob)/Lep(ob) (ob/ob) mice (ob/ob-NASH) were maintained on a diet high in trans-fat (40%), fructose (22%) and cholesterol (2%) for 26 and 12 wk, respectively. A normal chow diet served as control in C57 mice (lean chow) and ob/ob mice (ob/ob chow). After the diet-induction period, mice were liver biopsied and a blinded histological assessment of steatosis and fibrosis was conducted. Mice were then stratified into groups counterbalanced for steatosis score and fibrosis stage and continued on diet and to receive daily PO dosing of vehicle for 8 wk. Global gene expression in liver tissue was assessed by RNA sequencing and bioinformatics. Metabolic parameters, plasma liver enzymes and lipids (total cholesterol, triglycerides) as well as hepatic lipids and collagen content were measured by biochemical analysis. Non-alcoholic fatty liver disease activity score (NAS) (steatosis/inflammation/ballooning degeneration) and fibrosis were scored. Steatosis and fibrosis were also quantified using percent fractional area. RESULTS: Diet-induction for 26 and 12 wk in DIO-NASH and ob/ob-NASH mice, respectively, elicited progressive metabolic perturbations characterized by increased adiposity, total cholesterol and elevated plasma liver enzymes. The diet also induced clear histological features of NASH including hepatosteatosis and fibrosis. Overall, the metabolic NASH phenotype was more pronounced in ob/ob-NASH vs DIO-NASH mice. During the eight week repeated vehicle dosing period, the metabolic phenotype was sustained in DIO-NASH and ob/ob-NASH mice in conjunction with hepatomegaly and increased hepatic lipids and collagen accumulation. Histopathological scoring demonstrated significantly increased NAS of DIO-NASH mice (0 vs 4.7 ± 0.4, P < 0.001 compared to lean chow) and ob/ob-NASH mice (2.4 ± 0.3 vs 6.3 ± 0.2, P < 0.001 compared to ob/ob chow), respectively. Furthermore, fibrosis stage was significantly elevated for DIO-NASH mice (0 vs 1.2 ± 0.2, P < 0.05 compared to lean chow) and ob/ob NASH (0.1 ± 0.1 vs 3.0 ± 0.2, P < 0.001 compared to ob/ob chow). Notably, fibrosis stage was significantly (P < 0.001) increased in ob/ob-NASH mice, when compared to DIO-NASH mice. CONCLUSION: These data introduce the obese diet-induced DIO-NASH and ob/ob-NASH mouse models with biopsy-confirmed individual disease staging as a preclinical platform for evaluation of novel NASH therapeutics