Empagliflozin Improves Metabolic and Hepatic Outcomes in a Non-Diabetic Obese Biopsy-Proven Mouse Model of Advanced NASH.

Empagliflozin, an established treatment for type 2 diabetes (T2DM), has shown beneficial effects on liver steatosis and fibrosis in animals and in humans with T2DM, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). However, little is known about the effects of empagliflozin on liver function in advanced NASH with liver fibrosis and without diabetes. This study aimed to assess the effects of empagliflozin on hepatic and metabolic outcomes in a diet-induced obese (DIO) and insulin-resistant but non-diabetic biopsy-confirmed mouse model of advanced NASH. Male C57BL/6JRj mice with a biopsy-confirmed steatosis and fibrosis on AMLN diet (high fat, fructose and cholesterol) for 36-weeks were randomized to receive for 12 weeks: (a) Empagliflozin (10 mg/kg/d p.o.), or (b) vehicle. Metabolic outcomes, liver pathology, markers of Kupffer and stellate cell activation and lipidomics were assessed at the treatment completion. Empagliflozin did not affect the body weight, body composition or insulin sensitivity (assessed by intraperitoneal insulin tolerance test), but significantly improved glucose homeostasis as assessed by oral glucose tolerance test in DIO-NASH mice. Empagliflozin improved modestly the NAFLD activity score compared with the vehicle, mainly by improving inflammation and without affecting steatosis, the fibrosis stage and markers of Kupffer and stellate cell activation. Empagliflozin reduced the hepatic concentrations of pro-inflammatory lactosylceramides and increased the concentrations of anti-inflammatory polyunsaturated triglycerides. Empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic DIO-NASH mice and thus may be effective against NASH even in non-diabetic conditions.

Activation of thyroid hormone receptor-ß 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 ß (THR-ß) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-ß agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-ß agonist, as a prototype to investigate the effects of THR-ß 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-ß 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.

Interferon lambda 4 genotype and pathway in alcoholic hepatitis.

OBJECTIVES: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) gene influence liver inflammation and fibrosis in chronic liver disease. We investigated whether this is also the case during acute liver disease, alcoholic hepatitis. We, therefore, related variants within the IFNL4 gene to the clinical course of acute alcoholic hepatitis, and characterized the activation state of the IFN lambda system in these patients. METHODS: In this pilot study, 58 patients with alcoholic hepatitis were genotyped for the rs368234815IFNL4 single nucleotide polymorphism (deltaG, deltaG/TT: IFN lambda 4 positive, TT/TT: IFN lambda 4 negative). The genotypes were related to mortality, infection and inflammation and expression of the IFNL receptor 1 and IFN inducible genes were measured in liver and peripheral leukocytes. RESULTS: Amongst the alcoholic hepatitis patients who died, the IFN negative patients live longer after diagnosis, and also the IFN negative patients tended to have an overall short-term survival benefit compared to IFN lambda positive patients (p = .058). The IFN lambda 4 negative patients at diagnosis had fewer circulating monocytes and lower plasma soluble CD163. The patients with alcoholic hepatitis had reduced expression of the IFNL receptor 1in both liver and blood compared with healthy controls. In blood, the expression of IFN stimulated genes was lower than in healthy controls and most so in the patients, who died. CONCLUSIONS: The IFN lambda 4 pathway seems involved in the acute disease processes of alcoholic hepatitis and patients without IFN lambda expression seem to have a short-term survival benefit.

Low Interleukin-22 Binding Protein Is Associated With High Mortality in Alcoholic Hepatitis and Modulates Interleukin-22 Receptor Expression.

INTRODUCTION: In alcoholic hepatitis (AH), high interleukin (IL)-22 production is associated with disease improvement, purportedly through enhanced infection resistance and liver regeneration. IL-22 binding protein (BP) binds and antagonizes IL-22 bioactivity, but data on IL-22BP in liver disease suggest a complex interplay. Despite the scarcity of human data, IL-22 is in clinical trial as treatment of AH. We, therefore, in patients with AH, described the IL-22 system focusing on IL-22BP and associations with disease course, and mechanistically pursued the human associations in vitro. METHODS: We prospectively studied 41 consecutive patients with AH at diagnosis, days 7 and 90, and followed them for up to 1 year. We measured IL-22 pathway proteins in liver biopsies and blood and investigated IL-22BP effects on IL-22 in hepatocyte cultures. RESULTS: IL-22BP was produced in the gut and was identifiable in the patients with AH' livers. Plasma IL-22BP was only 50% of controls and the IL-22/IL-22BP ratio thus elevated. Consistently, IL-22-inducible genes were upregulated in AH livers at diagnosis. Low plasma IL-22BP was closely associated with high 1-year mortality. In vitro, IL-22 stimulation reduced IL-22 receptor (R) expression, but coincubation with IL-22BP sustained IL-22R expression. In the AH livers, IL-22R mRNA expression was similar to healthy livers, although IL-22R liver protein was higher at diagnosis. DISCUSSION: Plasma IL-22BP was associated with an adverse disease course, possibly because its low level reduces IL-22R expression so that IL-22 bioactivity was reduced. This suggests the IL-BP interplay to be central in AH pathogenesis, and in future treatment trials (see Visual abstract, Supplementary Digital Content 5, http://links.lww.com/CTG/A338).

The Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator CHS-131 Improves Liver Histopathology and Metabolism in a Mouse Model of Obesity and Nonalcoholic Steatohepatitis.

CHS-131 is a selective peroxisome proliferator-activated receptor gamma modulator with antidiabetic effects and less fluid retention and weight gain compared to thiazolidinediones in phase II clinical trials. We investigated the effects of CHS-131 on metabolic parameters and liver histopathology in a diet-induced obese (DIO) and biopsy-confirmed mouse model of nonalcoholic steatohepatitis (NASH). Male C57BL/6JRj mice were fed the amylin liver NASH diet (40% fat with trans-fat, 20% fructose, and 2% cholesterol). After 36 weeks, only animals with biopsy-confirmed steatosis and fibrosis were included and stratified into treatment groups (n = 12-13) to receive for the next 12 weeks (1) low-dose CHS-131 (10 mg/kg), (2) high-dose CHS-131 (30 mg/kg), or (3) vehicle. Metabolic parameters, liver pathology, metabolomics/lipidomics, markers of liver function and liver, and subcutaneous and visceral adipose tissue gene expression profiles were assessed. CHS-131 did not affect body weight, fat mass, lean mass, water mass, or food intake in DIO-NASH mice with fibrosis. CHS-131 improved fasting insulin levels and insulin sensitivity as assessed by the intraperitoneal insulin tolerance test. CHS-131 improved total plasma cholesterol, triglycerides, alanine aminotransferase, and aspartate aminotransferase and increased plasma adiponectin levels. CHS-131 (high dose) improved liver histology and markers of hepatic fibrosis. DIO-NASH mice treated with CHS-131 demonstrated a hepatic shift to diacylglycerols and triacylglycerols with a lower number of carbons, increased expression of genes stimulating fatty acid oxidation and browning, and decreased expression of genes promoting fatty acid synthesis, triglyceride synthesis, and inflammation in adipose tissue. Conclusion: CHS-131 improves liver histology in a DIO and biopsy-confirmed mouse model of NASH by altering the hepatic lipidome, reducing insulin resistance, and improving lipid metabolism and inflammation in adipose tissue.

Validity of biopsy-based drug effects in a diet-induced obese mouse model of biopsy-confirmed NASH.

BACKGROUND: Compounds in clinical development for nonalcoholic steatohepatitis (NASH) improve liver histopathology in diet-induced obese mouse models of biopsy-confirmed NASH. Since the biopsy section used for histopathological evaluation represents only < 1% of the whole mouse liver, we evaluated how well biopsy-based quantitative image analyses correlate to stereology-based whole-liver quantitative changes upon drug treatment. METHODS: Male leptin-deficient Lepob/Lepob mice were fed the Amylin liver NASH (AMLN) diet for 16 weeks before stratification into treatment groups using a biopsy-based evaluation of type I collagen αI (col1a1) levels. Mice were treated for 8 weeks with either vehicle (PO, QD), liraglutide (0.4 mg/kg, SC, QD), elafibranor (30 mg/kg, PO, QD) or INT-767 (10 mg/kg, PO, QD). Terminal quantitative histological assessment of liver lipid (hematoxylin-eosin staining), inflammation (galectin-3 immunohistochemistry (IHC); gal-3), and fibrosis (col1a1 IHC) was performed on terminal liver biopsies and compared with stereologically sampled serial sections spanning the medial, left and right lateral lobe of the liver. RESULTS: The distribution of liver lipid and fibrosis was markedly consistent across lobes, whereas inflammation showed some variability. While INT-767 and liraglutide significantly reduced total liver weight by 20 and 48%, respectively, elafibranor tended to exacerbate hepatomegaly in Lepob/Lepob-NASH mice. All three compounds markedly reduced biopsy-based relative liver lipid content. Elafibranor and INT-767 significantly reduced biopsy-based relative gal-3 levels (P < 0.001), whereas INT-767 and liraglutide tended to reduce relative col1a1 levels. When changes in liver weight was accounted for, both INT-767 and liraglutide significantly reduced biopsy-based total col1a1 content. Although minor differences in absolute and relative liver lipid, inflammation and fibrosis levels were observed across lobes, the interpretation of drug-induced effects were consistent with biopsy-based conclusions. Notably, the incorporation of changes in total liver mass revealed that liraglutide's efficacy reached statistical significances for all analyzed parameters. CONCLUSIONS: In conclusion, in-depth analyses of liver homogeneity demonstrated that drug-induced improvement in liver biopsy-assessed histopathology is representative for overall liver effects assessed using stereology. Importantly, these findings reveal how changes in whole-liver mass should be considered to provide a deeper understanding of apparent drug treatment efficacy in preclinical NASH studies.

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.

Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases.

BACKGROUND: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR. RESULTS: Biglycan was cleaved in vitro by MMP-9 and -12 and the 344'YWEVQPATFR'353 peptide (BGM) was chosen as a potential neo-epitope. A technically sound competitive ELISA for the measurement of BGM was generated and the assay was validated in a bovine cartilage explant culture (BEX), in a collagen induced model of rheumatoid arthritis (CIA) and in two different rat models of liver fibrosis: the carbon tetrachloride (CCL4)-induced fibrosis model, and the bile duct ligation (BDL) model. Significant elevation in serum BGM was found in CIA rats compared to controls, in rats treated with CCL4 for 16 weeks and 20 weeks compared to the control groups as well as in all groups of rats subject to BDL compared with sham operated groups. Furthermore, there was a significant correlation of serum BGM levels with the extent of liver fibrosis determined by the Sirius red staining of liver sections in the CCL4 model. CONCLUSION: We demonstrated that the specific tissue remodeling product of MMPs-degraded biglycan, namely the neo-epitope BGM, is correlated with pathological ECMR. This assay represents both a novel marker of ECM turnover and a potential new tool to elucidate biglycan role during the pathological processes associated with ECMR.

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

BACKGROUND: 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 ex vivo tissue culture of livers from CCl(4) treated animals with an ELISA detecting a fragment of type III collagen generated in vitro 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. FINDINGS: In vivo: Rats were treated for 8 weeks with CCl(4)/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. Ex vivo: The levels of serum C3M increased 77% in the CCl(4)-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 CCl(4)-treated animals had highly increased MMP-9, but not MMP-2 activity, compared to slices derived from control animals. CONCLUSIONS: We have combined an ex vivo 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.

Circulating levels of citrullinated and MMP-degraded vimentin (VICM) in liver fibrosis related pathology.

AIM: To investigate whether increased levels of vimentin citrullinated peptides identified by MS in articular cartilage can be measured in pathologies other than rheumatoid arthritis and be utilised for diagnostic purposes. METHODS: A monoclonal antibody against the sequence RLRSSVPGV-citrulline (VICM) was developed and evaluated in a carbon tetrachloride (CCl(4)) (n=52 + 28 controls) rat model of liver fibrosis and two clinical cohorts of adult patients with hepatitis C (HCV) (n=92) and non-alcoholic fatty liver disease (NAFLD) (n=62), and compared to healthy controls. RESULTS: In CCl(4)-treated rats, mean systemic VICM levels increased 31% at week 12 (176 ng/mL, P<0.001), 41.7% at weeks 16 (190 ng/mL, P<0.001), 49.2% at weeks 20 (200 ng/ml, P<0.001), compared to controls (134 ng/mL). VICM levels correlated with total hepatic collagen determined by Sirius red staining of rat livers (r=0.75, P<0.05). In the HCV cohort, when stratified according to the METAVIR F score, VICM levels were 63% higher in F0 (632 ng/mL ±75, p<0.05), 54% in F1 (597 ng/mL ±41.3, p<0.05) and 62% in F2 (628 ng/mL ±59, p<0.05) all compared to controls. In the NAFLD cohort, VICM levels were 20.6% higher in F0 (339 ±12 ng/mL, P<0.05), 23.8% in F1 (348 ±12 ng/mL, P<0.05) and 28.8% in F2 (362 ±25 P<0.05). CONCLUSION: We demonstrated increased serological levels of citrullinated and MMP degraded vimentin in an animal model of liver fibrosis and in early fibrosis associated with HCV and NAFLD patients. These data suggest that citrullinated and MMP degraded proteins are also present in liver fibrosis.

MMP mediated degradation of type VI collagen is highly associated with liver fibrosis--identification and validation of a novel biochemical marker assay.

BACKGROUND AND AIMS: During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis. METHODS: Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats. RESULTS: Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl(4)-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R(2) = 0.58, p<0.0001) in CCl(4)- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003). CONCLUSIONS: This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.

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.

BACKGROUND AND AIM: The current study utilized a carbon tetrachloride (CCl(4))-induced liver fibrosis model to measure levels of the MMP9-mediated collagen type III degradation fragment CO3-610 (site of cleavage: KNGETGPQGP), during disease progression and regression, and to investigate a potential prognostic role of the biomarker. MATERIALS AND METHODS: 72 female Sprague-Dawley rats aged 6 months old were injected with CCl(4) twice a week over different periods of time to induce varying degrees of liver fibrosis. After 4, 6 and 8 weeks of treatment, administration of CCl(4) was stopped. The 6- and 8-week treatment groups were left to regress for a further 6 or 12 weeks at which point they were sacrificed and livers removed and sectioned. Liver fibrosis was quantified using Visiopharm software to analyse Sirius red-stained sections. Serum levels of CO3-610 were measured in all animals using an ELISA assay as described by Barascuk et al.1 RESULTS: Quantitative histology revealed total collagen deposition in the liver increased as fibrosis progressed. In animals treated with CCl(4) for 4 weeks, collagen comprised on average 4.94% of the total tissue in liver sections, while after 6 weeks the mean was 8.25%, and after 8 weeks, 9.11%. During the regression phase, the total collagen deposition gradually decreased to a mean of 6.9% and 5.09% for animals regressing 6 and 12 weeks respectively after 6 weeks treatment, and 6.27% for animals regressed 12 weeks after 8 weeks treatment. CO3-610 values increased progressively in rats treated for 4 weeks (by a mean of 55.0 ng/ml), 6 weeks (mean 61.1 ng/ml) and 8 weeks (mean 70.2 ng/ml). During the regression phase, CO3-610 values rapidly decreased by a mean of 28.9 ng/ml at 6 weeks and 21.6 ng/ml at 12 weeks in animals previously treated for 6 weeks, and by a mean of 19.52 ng/ml in animals treated for 8 weeks and regressed for 12 weeks. CO3-610 levels were statistically significantly correlated with total collagen during disease progression (r = 0.5701, P < 0.0001). No statistically significant correlation was observed during regression (r = 0.2081, P = 0.1138). CONCLUSION: Levels of the MMP-9 generated fragment of collagen type III, CO3-610, correlated with the degree of liver fibrosis in rats during the progression phase, but were not correlated with total collagen levels during regression. CO3-610 seems to be produced only under the CCL(4) stimulus, and signifies CO3-610 as a potential marker of progression rather than regression. The corresponding steep elevations in levels of CO3-610 total collagen and collagen type III during liver fibrosis progression underline a potential prognostic capacity of the biomarker.

Measurement of matrix metalloproteinase 9-mediated collagen type III degradation fragment as a marker of skin fibrosis.

BACKGROUND: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies. METHODS: Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels. RESULTS: CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65). CONCLUSION: Increased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice.

Procollagen type I N-terminal propeptide (PINP) is a marker for fibrogenesis in bile duct ligation-induced fibrosis in rats.

BACKGROUND: Fibrosis can be described as the excess deposition of extracellular matrix (ECM) components, such as collagens and proteoglycans. Fibrosis of the liver, which eventually leads to cirrhosis, is a major global health problem. Being able to measure fibrosis progression may enable timely preventative intervention. The aim of the current study was to investigate the utility of serum procollagen type I N-terminal propeptide (PINP) as a marker of hepatic fibrosis, as distinct from bone formation, during three different periods of fibrosis development following hepatic injury induced by bile duct ligation (BDL) in rats. METHODS: BDL was performed on 30 female Sprague-Dawley rats aged 6 months, and sham operations on 30 controls. Animals were killed after 14, 28, or 35 days. The extent of liver fibrosis was evaluated by quantitative histology after Sirus Red staining. Levels of serum PINP and osteocalcin (a marker solely for osteoblastic bone formation) were determined using ELISA at baseline and post termination. RESULTS: Collagen formation increased by 30% compared to 3% in sham-operated animals (P < 0.0001). PINP levels increased significantly in all BDL groups compared with baseline (14 days: baseline 13.9 ng/ml, termination 17.7 ng/ml, P = 0.047; 28 days: baseline 17.9 ng/ml, termination 26.2 ng/ml, P = 0.005; 35 days: baseline 18.0 ng/ml, termination 27.4 ng/ml P = 0.015, an increase of 52%). PINP levels did not change from baseline in the sham-operated rats, indicating that the increased PINP levels were due to hepatic injury. The bone-specific marker, osteocalcin, did not increase in either BDL or sham-operated rats. PINP measured in serum correlated to the extent of liver fibrosis as evaluated by quantitative histology (R2 = 0.42, P < 0.001). CONCLUSION: PINP was associated with the development of liver fibrosis, but not bone formation, in mature rats subjected to BDL. Thus, PINP may be useful in studying the pathogenesis of liver fibrosis. However, caution should be applied when interpreting PINP levels in other disease states.

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 successful to replace biopsy assessment. AIM: To identify the extracellular matrix proteins of interest, that as protein degradation fragments produced during extracellular matrix metabolism neo-epitopes, may be targeted for novel biochemical marker development in fibrosis. We used the recently proposed BIPED system (Burden of disease, Investigative, Prognostic, Efficacy and Diagnostic) to characterise present serological markers. METHODS: Pubmed was search for keywords; Liver fibrosis, neo-epitopes, biomarkers, clinical trail, extra cellular matrix, protease, degradation, fragment. RESULTS AND CONCLUSION: Implementation of BIPED categorization in the development and validation of fibrosis biomarkers to simplify and standardize the use of existing and future biomarkers seems advantageous. In addition, a systematic use of the neo-epitope approach, i.e. the quantification of peptide epitopes generated from enzymatic cleavage of proteins during extracellular remodeling, may prove productive in the quest to find new markers of liver fibrosis.