Biomarkers of Type IV Collagen Turnover Reflect Disease Activity in Patients with Early-Stage Non-Alcoholic Fatty Liver (NAFL).

BACKGROUND: Identification of progressive liver disease necessitates the finding of novel non-invasive methods to identify and monitor patients in need of early intervention. Investigating patients with early-liver injury may help identify unique biomarkers. Early-liver injury is characterized by remodeling of the hepatocyte basement membrane (BM) of the extracellular matrix. Thus, we quantified biomarkers targeting two distinct neo-epitopes of the major BM collagen, type IV collagen (PRO-C4 and C4M), in patients spanning the non-alcoholic fatty liver disease (NAFLD) spectrum. METHODS: We evaluated PRO-C4 and C4M in a cross-sectional study with 97 patients with NAFLD confirmed on histology. Serological levels of PRO-C4 and C4M were quantified using validated competitive enzyme-linked immunosorbent assays (ELISA). Using the fatty liver inhibition of progression (FLIP) algorithm, we stratified patients into two groups: non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). Biomarker levels were investigated in the two groups in patients stratified by the NAFLD activity score (NAS). In both groups, biomarker measurements were analyzed in relation to histological scorings of steatosis, inflammation, ballooning, and fibrosis. RESULTS: Patients had a body mass index (BMI) of 30.9 ± 5.6 kg/m2, age of 53 ± 13 years and a NAS range of 1-8. Upon stratification by FLIP, the NASH patients had higher platelets, ALT, and AST levels than the NAFL group. Both PRO-C4 (p = 0.0125) and C4M (p = 0.003) increased with increasing NAS solely within the NAFL group; however, a large variability was present in the NASH group. Furthermore, both markers were significantly associated with lobular inflammation (p = 0.020 and p = 0.048) and steatosis (p = 0.004 and p = 0.015) in patients with NAFL. CONCLUSIONS: This study found that type IV collagen turnover increased with the increase in NAS in patients with NAFL; however, this was not the case in patients with NASH. These findings support the assessments of the BM turnover using biomarkers in patients with early-disease development. These biomarkers may be used to track specific processes involved in the early pathobiology of NAFL.

Biomarkers reflecting pericellular fibrosis improve together with liver histology after bariatric surgery in early non-alcoholic fatty liver disease.

AIM: Non-invasive tests for non-alcoholic fatty liver disease (NAFLD) are needed for assessing disease stage, prognosis and treatment efficacy. Extracellular matrix biomarkers, such as PRO-C3, are useful as biomarkers of advanced liver fibrosis. However, non-invasive biomarkers of early-stage NAFLD, characterized by pericellular fibrosis, are lacking. Here, we measured serological biomarkers of type IV and VIII collagens reflecting the remodeling of the pericellular basement membrane to explore the effect of bariatric surgery on pericellular fibrosis in patients with early NAFLD. METHODS: Seventy patients with severe obesity underwent bariatric surgery. The cohort consisted of 61 % females who had a mean age of 44. Patients had a median NAFLD activity score of 3 and mild-to-moderate fibrosis F0 (3 %), F1 (86 %), and F2 (11 %). Blood samples were taken at baseline, three, six and 12 months after surgery. At 12 months, 40 patients had a follow-up liver biopsy. The biomarkers PRO-C3, PRO-C4, C4M, and PRO-C8 were measured using indirect competitive ELISAs. RESULTS: Twelve months after surgery patients had significantly lower levels of ALT, GGT, HbA1c, fasting glucose, and CRP. The pericellular fibrosis biomarkers, C4M, PRO-C4, and PRO-C8 decreased by 24 %, 18 % and 44 %, respectively (p < 0.0001), while the interstitial matrix fibrosis marker PRO-C3 remained unchanged. Furthermore, baseline C4M was associated with histologically assessed hepatocyte ballooning and lobular inflammation in patients with (p = 0.032) and without (p = 0.032) steatosis, respectively. CONCLUSION: Biomarkers of pericellular fibrosis decrease in early-stage NAFLD after patients undergo bariatric surgery and potentially reflect an improvement in liver histology.

Serologically assessed heat shock protein 47 is related to fibrosis stage in early compensated alcohol-related liver disease.

BACKGROUND AND AIMS: Heat shock protein (HSP)47 is a collagen-specific chaperone, essential for the correct formation of fibrillar procollagens. Collagen accumulation in the extracellular matrix (ECM) is a hallmark of fibrogenesis. The expression of HSP47 is proportional to the rate of collagen formation. Thus, HSP47 is a potential drug target for fibrotic diseases. We hypothesized that a C-terminal fragment of HSP47 (HSP47-C) could be quantified serologically and related to liver fibrosis stage. For this, a novel competitive enzyme-linked immunosorbent assay (ELISA) was developed. METHOD: An ELISA employing a monoclonal antibody targeting HSP47-C was developed and technically validated. The assay was evaluated in serum from a cross-sectional biopsy-controlled study of 281 patients with alcohol-related liver disease (ALD) and 50 gender, age and BMI matched healthy controls (HC). All liver biopsies from ALD patients were scored by one pathologist according to fibrosis stage (F0-4). RESULTS: The HSP47-C assay was technically robust and specific for the target sequence. HSP47-C was 39% higher in ALD patients (median 17.7 ng/mL, IQR 12.4-24.0 ng/mL) compared to HC (median 12.7 ng/mL, IQR 9.4-15.7 ng/mL, p < 0.0001). In addition, HSP47-C was elevated in patients with severe fibrosis (F3-4, median 22.8 ng/mL, IQR 17.5-33.3 ng/mL) compared to none-to-moderate fibrosis (F0-2, median 16.5 ng/mL, IQR 11.8-22.5 ng/mL) with an AUROC of 0.72 (p < 0.0001). HSP47-C also correlated with other liver disease parameters, albumin, bilirubin and aspartate transaminase. CONCLUSION: We developed a competitive ELISA for serological detection of HSP47-C. The study supports HSP47 as a potential marker of liver fibrosis in ALD.

Obesity Modifies the Performance of Fibrosis Biomarkers in Nonalcoholic Fatty Liver Disease.

CONTEXT: Guidelines recommend blood-based fibrosis biomarkers to identify advanced nonalcoholic fatty liver disease (NAFLD), which is particularly prevalent in patients with obesity. OBJECTIVE: To study whether the degree of obesity affects the performance of liver fibrosis biomarkers in NAFLD. DESIGN: Cross-sectional cohort study comparing simple fibrosis scores [Fibrosis-4 Index (FIB-4); NAFLD Fibrosis Score (NFS); aspartate aminotransferase to platelet ratio index; BARD (body mass index, aspartate-to-alanine aminotransferase ratio, diabetes); Hepamet Fibrosis Score (HFS)] and newer scores incorporating neo-epitope biomarkers PRO-C3 (ADAPT, FIBC3) or cytokeratin 18 (MACK-3). SETTING: Tertiary referral center. PATIENTS: We recruited overweight/obese patients from endocrinology (n = 307) and hepatology (n = 71) clinics undergoing a liver biopsy [median body mass index (BMI) 40.3 (interquartile range 36.0-44.7) kg/m2]. Additionally, we studied 859 less obese patients with biopsy-proven NAFLD to derive BMI-adjusted cutoffs for NFS. MAIN OUTCOME MEASURES: Biomarker area under the receiver operating characteristic (AUROC), sensitivity, specificity, and predictive values to identify histological stage ≥F3 fibrosis or nonalcoholic steatohepatitis with ≥F2 fibrosis [fibrotic nonalcoholic steatohepatitis (NASH)]. RESULTS: The scores with an AUROC ≥0.85 to identify ≥F3 fibrosis were ADAPT, FIB-4, FIBC3, and HFS. For fibrotic NASH, the best predictors were MACK-3 and ADAPT. The specificities of NFS, BARD, and FIBC3 deteriorated as a function of BMI. We derived and validated new cutoffs for NFS to rule in/out ≥F3 fibrosis in groups with BMIs <30.0, 30.0 to 39.9, and ≥40.0 kg/m2. This optimized its performance at all levels of BMI. Sequentially combining FIB-4 with ADAPT or FIBC3 increased specificity to diagnose ≥F3 fibrosis. CONCLUSIONS: In obese patients, the best-performing fibrosis biomarkers are ADAPT and the inexpensive FIB-4, which are unaffected by BMI. The widely used NFS loses specificity in obese individuals, which may be corrected with BMI-adjusted cutoffs.

Biomarkers of extracellular matrix formation are associated with acute-on-chronic liver failure.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by organ failure(s), high short-term mortality, and, pathophysiologically, deranged inflammatory responses. The extracellular matrix (ECM) is critically involved in regulating the inflammatory response. This study aimed to determine alterations in biomarkers of ECM turnover in ACLF and their association with inflammation, organ failures, and mortality. METHODS: We studied 283 patients with cirrhosis admitted for acute decompensation (AD) with or without ACLF, 64 patients with stable cirrhosis, and 30 healthy controls. A validation cohort (25 ACLF, 9 healthy controls) was included. Plasma PRO-C3, PRO-C4, PRO-C5, PRO-C6, and PRO-C8 (i.e. collagen type III-VI and VIII formation) and C4M and C6M (i.e. collagen type IV and VI degradation) were measured. Immunohistochemistry of PRO-C6 was performed on liver biopsies (AD [n = 7], ACLF [n = 5]). A competing-risk regression analysis was performed to explore the prognostic value of biomarkers of ECM turnover with 28- and 90-day mortality. RESULTS: PRO-C3 and PRO-C6 were increased in ACLF compared to AD (p = 0.089 and p <0.001, respectively), whereas collagen degradation markers C4M and C6M were similar. Both PRO-C3 and PRO-C6 were strongly associated with liver function and inflammatory markers. Only PRO-C6 was associated with extrahepatic organ failures and 28- and 90-day mortality (hazard ratio [HR; on log-scale] 6.168, 95% CI 2.366-16.080, p <0.001, and 3.495, 95% CI 1.509-8.093, p = 0.003, respectively). These findings were consistent in the validation cohort. High PRO-C6 expression was observed in liver biopsies of patients with ACLF. CONCLUSIONS: This study shows, for the first time, evidence of severe net interstitial collagen deposition in ACLF and makes the novel observation of the association between PRO-C6 and (extrahepatic) organ failures and mortality. Further studies are needed to define the pathogenic significance of these observations. LAY SUMMARY: This study describes a disrupted turnover of collagen type III and VI in Acute-on-chronic liver failure (ACLF). Plasma biomarkers of these collagens (PRO-C3 and PRO-C6) are associated with the severity of liver dysfunction and inflammation. PRO-C6, also known as the hormone endotrophin, has also been found to be associated with multi-organ failure and prognosis in acute decompensation and ACLF.

Potential role of adenylyl cyclase 8 signaling complexes in regulating insulin secretion from pancreatic beta cells.

Glucose-stimulated insulin secretion from pancreatic ß cells is mediated by Ca2+ influx and amplified by stimulation of GLP-1-receptors through cAMP-based signaling pathways. Interestingly, it has been found that glucose-induced Ca2+ signals can induce concurrent adenylyl cyclase isoform 8 (AC8)-mediated cAMP signals and, conversely, that GLP-1-receptor-mediated cAMP signals are able to induce Ca2+ signals. In this review, we explore the signaling complexes revolving around AC8 in modulating insulin release, from the initial discovery of the importance of this AC isoform to recent investigations of its interacting molecular partners. We suggest that investigating the structural assembly of the proteins associated with AC8 in ß cells might reveal how this particular protein complex could be targeted to modify insulin secretion. Specifically, we suggest that disrupting the protein-protein interaction between A-kinase anchoring protein 79 (AKAP79) and AC8 could lead to disinhibition of AC8 activity and increased insulin secretion. Potentially, AC8 protein interactions could become a future target in type 2 diabetic patients with dysfunction of insulin secretion.