M.J. Nielsen

Extracellular Matrix Remodeling: The Common Denominator in Connective Tissue DiseasesPossibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture, but a Key Player in Tissue Failure

Increased attention is paid to the structural components of tissues. These components are mostly collagens and various proteoglycans. Emerging evidence suggests that altered components and noncoded modifications of the matrix may be both initiators and drivers of disease, exemplified by excessive tissue remodeling leading to tissue stiffness, as well as by changes in the signaling potential of both intact matrix and fragments thereof. Although tissue structure until recently was viewed as a simple architecture anchoring cells and proteins, this complex grid may contain essential information enabling the maintenance of the structure and normal functioning of tissue. The aims of this review are to (1) discuss the structural components of the matrix and the relevance of their mutations to the pathology of diseases such as fibrosis and cancer, (2) introduce the possibility that post-translational modifications (PTMs), such as protease cleavage, citrullination, cross-linking, nitrosylation, glycosylation, and isomerization, generated during pathology, may be unique, disease-specific biochemical markers, (3) list and review the range of simple enzyme-linked immunosorbent assays (ELISAs) that have been developed for assessing the extracellular matrix (ECM) and detecting abnormal ECM remodeling, and (4) discuss whether some PTMs are the cause or consequence of disease. New evidence clearly suggests that the ECM at some point in the pathogenesis becomes a driver of disease. These pathological modified ECM proteins may allow insights into complicated pathologies in which the end stage is excessive tissue remodeling, and provide unique and more pathology-specific biochemical markers.

Novel insights into the function and dynamics of extracellular matrix in liver fibrosis

Emerging evidence suggests that altered components and posttranslational modifications of proteins in the extracellular matrix (ECM) may both initiate and drive disease progression. The ECM is a complex grid consisting of multiple proteins, most of which play a vital role in containing the essential information needed for maintenance of a sophisticated structure anchoring the cells and sustaining normal function of tissues. Therefore, the matrix itself may be considered as a paracrine/endocrine entity, with more complex functions than previously appreciated. The aims of this review are to 1) explore key structural and functional components of the ECM as exemplified by monogenetic disorders leading to severe pathologies, 2) discuss selected pathological posttranslational modifications of ECM proteins resulting in altered functional (signaling) properties from the original structural proteins, and 3) discuss how these findings support the novel concept that an increasing number of components of the ECM harbor signaling functions that can modulate fibrotic liver disease. The ECM entails functions in addition to anchoring cells and modulating their migratory behavior. Key ECM components and their posttranslational modifications often harbor multiple domains with different signaling potential, in particular when modified during inflammation or wound healing. This signaling by the ECM should be considered a paracrine/endocrine function, as it affects cell phenotype, function, fate, and finally tissue homeostasis. These properties should be exploited to establish novel biochemical markers and antifibrotic treatment strategies for liver fibrosis as well as other fibrotic diseases.

Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degraded extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation.

Serological investigation of the collagen degradation profile of patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis.

In both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), abnormally high collagen remodeling occurs within the lung tissue. Matrix metalloproteinase (MMP)-degraded type I, III, IV, V and VI collagen and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-degraded type III collagen were assessed in serum of patients diagnosed with mild COPD (n= 10) or IPF (n= 30), and healthy controls (n= 15). The collagen degradation markers C1M, C3M, C5M and C6M were significantly elevated in serum of both mild COPD and IPF patients, versus controls. C3A and C4M were only elevated in patients with mild COPD, compared with controls. The most reliable indicators of mild COPD versus controls were: C1M (area under the receiver-operating characteristics (AUROC = 0.94, P < 0.0001), C3M (AUROC = 0.95, P < 0.0001), and C5M (AUROC = 0.95, P < 0.0001). The most reliable markers for the diagnosis of IPF were achieved by C1M (AUROC = 0.90, P < 0.0001) and C3M (AUROC = 0.93, P < 0.0001). Collagen degradation was highly up-regulated in patients with IPF and mild COPD, indicating that degradation fragments of collagens are potential markers of pulmonary diseases. Interestingly, C4M and C3A were only elevated in patients with mild COPD, indicating that these markers could be used to distinguish between the two pathologies.

Enzyme‐linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S): A marker related to the extracellular matrix remodeling during liver fibrogenesis

Aim:  The present study describes the ability of a newly developed N‐terminal pro‐peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme‐linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100% homologous in the human, rat, and mouse species.

Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression in patients with chronic hepatitis C

Fibrogenesis results in release of certain extracellular matrix protein fragments into the circulation. We evaluated the diagnostic and prognostic performance of two novel serological markers, the precisely cleaved N‐terminal propeptide of type III collagen (Pro‐C3) and a peptide of helical collagen type III degradation (C3M), in chronic hepatitis C (CHC) patients.

Serum markers of the extracellular matrix remodeling reflect antifibrotic therapy in bile-duct ligated rats.

Background: Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibers at a specific site and thereby generate soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes might reflect the stage of liver fibrosis and may allow monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins. Methods: Bile duct ligation (BDL) was performed on wild type rats, which received atorvastatin (15 mg/kg*d) for 1 week starting at 1, 2, 3, 4 and 5 weeks after BDL (T1–T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohistochemistry and hepatic hydroxyproline content. TGFβ levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of degradation MMP driven type I, III, IV and VI collagen degradation (C1M, C3M, C4M, and C6M) and type III and IV collagen formation (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes. Results: Serum markers of ECM neo-epitopes reflected significantly the deposition of ECM in the liver and were able to distinguish between early (T1–T3) and severe fibrosis (T4–T5). Statin treatment resulted in reduction of neo-epitope markers, especially when therapy was started in the stage of severe fibrosis (T4–T5). Furthermore, these markers correlated with hepatic expression of profibrotic cytokines TGFβ1 and TGFβ2. Formation markers of type III and IV collagen (PRO-C3 and P4NP7S) and degradation markers C4M and C6M correlated significantly with hepatic MMP-2 activity in rats with severe fibrosis. Conclusion: Determination of ECM remodeling turnover markers in serum allowed a distinction between mild and severe fibrosis. With respect to statin therapy, the markers may serve as read-out for efficacy of anti-fibrotic treatment.

Fibrogenesis assessed by serological type III collagen formation identifies patients with progressive liver fibrosis and responders to a potential antifibrotic therapy

There are no approved treatments for liver fibrosis. To aid development of antifibrotic therapies, noninvasive biomarkers that can identify patients with progressive fibrosis and that permit monitoring of the response to antifibrotic therapy are much needed. Samples from a phase II antifibrotic trial of the glitazone farglitazar in patients with advanced hepatitis C, with matched follow-up liver biopsies, and from a phase III study of balaglitazone in patients with late-stage Type 2 diabetes (BALLET study) were analyzed for serological Pro-C3 levels in conjunction with other disease parameters. In the farglitazar study, a predefined cutoff value for Pro-C3 as a selection criterion led to the identification of subjects who 1) progressed by histological scores and 2) responded to therapy, as documented by attenuated fibrosis in liver biopsies. In the BALLET trial, subjects with the highest tertile of Pro-C3 levels responded to balaglitazone with reductions in levels of alanine aminotransferase and Pro-C3, as well as improved insulin sensitivity and lipid profile. Elevated Pro-C3 levels are indicative of active fibrogenesis and structural progression of fibrosis, and it can potentially identify patients most likely to benefit from antimetabolic and antifibrotic treatments. Serum Pro-C3 may facilitate patient selection and could help to speed up antifibrotic drug development and validation.

Markers of Collagen Remodeling Detect Clinically Significant Fibrosis in Chronic Hepatitis C Patients.

Background and Aim Detection of advanced fibrosis (Metavir F≥3) is important to identify patients with a high urgency of antiviral treatments vs. those whose treatment could be deferred (F≤2). The aim was to assess the diagnostic value of novel serological extracellular matrix protein fragments as potential biomarkers for clinically significant and advanced fibrosis. Methods Specific protein fragments of matrix metalloprotease degraded type I, III, IV and VI collagen (C1M, C3M, C4M, C6M) and type III and IV collagen formation (Pro-C3 and P4NP7S) were assessed in plasma from 403 chronic hepatitis C patients by specific ELISAs. Patients were stratified according to Metavir Fibrosis stage; F0 (n = 46), F1 (n = 161), F2 (n = 95), F3 (n = 44) and F4 (n = 33) based on liver biopsy. Results Pro-C3 was significantly elevated in patients with significant fibrosis (≥F2) compared to F0-F1 (p<0.05), while the markers C3M, C4M, C6M and P4NP7S were significantly elevated in patients with advanced fibrosis (≥F3) compared to F0-F2 (p<0.05). C1M showed no difference between fibrosis stages. Using Receiver Operating Characteristics analysis, the best marker for detecting ≥F2 and ≥F3 was Pro-C3 with AUC = 0.75 and AUC = 0.86. Combination of Pro-C3 and C4M with age, BMI and gender in a multiple ordered logistic regression model improved the diagnostic value for detecting ≥F2 and ≥F3 with AUC = 0.80 and AUC = 0.88. Conclusion The Pro-C3 protein fragment provided clinically relevant diagnostic accuracy as a single marker of liver fibrosis. A model combining Pro-C3 and C4M along with patient’s age, body mass index and gender increased the diagnostic power for identifying clinically significant fibrosis.

Pro-C5, a marker of true type V collagen formation and fibrillation, correlates with portal hypertension in patients with alcoholic cirrhosis

Abstract Background and aims: The hepatic venous pressure gradient (HVPG) is an important but invasive diagnostic and prognostic marker in cirrhotic patients. The aim of the study was to evaluate a novel biochemical plasma marker of true type V collagen formation (Pro-C5) for describing HVPG. Methods. Ninety-four patients mainly with alcoholic cirrhosis and fourteen liver-healthy controls were included in a retrospective study. Relevant clinical and routine laboratory data and hemodynamics were determined. Plasma Pro-C5 was correlated to HVPG and liver function parameters. Furthermore, Pro-C5 was combined in a linear regression model. Results. Plasma Pro-C5 correlated to HVPG, indocyanine green clearance, sustained vascular resistance and mean arterial pressure (r = -0.68–0.33, p < 0.0001). A multiple regression analysis including Pro-C5, alanine aminotransferase, bilirubin and model for end-stage liver disease (MELD) improved the correlation to HVPG (r = 0.74, p < 0.0001). Plasma Pro-C5 was positively or negatively correlated to a number of routine liver function markers and MELD score (r = 0.27–0.68; p < 0.05–0.0001). Furthermore, plasma Pro-C5 could clearly separate patients with a HVPG <10 mmHg or HVPG ≥10 mmHg (p < 0.001, area under the curve (AUC) = 0.73), HVPG 10-<16 mmHg or HVPG ≥16 mmHg (p < 0.001, AUC = 0.68) and controls from diseased patients (p < 0.0001, AUC = 0.88). Finally, there was a clear relation to increasing Child score A-C and plasma Pro-C5 (ANOVA p < 0.001). Conclusion. Plasma Pro-C5 reflects liver hemodynamics, liver function, disease stage and clinically significant portal hypertension (PH). A multimarker model in combination with clinical scores predicted HVPG and separated clinical relevant HVPG thresholds. Plasma Pro-C5 may be suitable for the noninvasive evaluation of PH in patients with cirrhosis.