Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

Turnover of type I and III collagen predicts progression of idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of fibrillar collagens in the alveolar space resulting in reduced pulmonary function and a high mortality rate. Biomarkers measuring the turnover of type I and III collagen could provide valuable information for prognosis and treatment decisions in IPF. METHODS: Serological biomarkers reflecting the formation of type III collagen (PRO-C3) and degradation of type I (C1M) and III collagen (C3M) were evaluated in a real-world cohort of 178 newly diagnosed IPF patients. Blood samples and clinical data were collected at baseline, six, and 12 months. Baseline and longitudinal biomarker levels were related to disease progression of IPF (defined as ≥ 5% decline in forced vital capacity (FVC) and/or ≥ 10% decline in diffusing capacity for carbon monoxide (DLco) and/or all-cause mortality at 12 months). Furthermore, we analysed differences in percentage change of biomarker levels from baseline between patients receiving antifibrotic treatment or not. RESULTS: Increased baseline levels of type I and III collagen turnover biomarkers were associated with a greater risk of disease progression within 12 months compared to patients with a low baseline type I and III collagen turnover. Patients with progressive disease had higher serum levels of C1M (P = 0.038) and PRO-C3 (P = 0.0022) compared to those with stable disease over one year. There were no differences in biomarker levels between patients receiving pirfenidone, nintedanib, or no antifibrotics. CONCLUSION: Baseline levels of type I and III collagen turnover were associated with disease progression within 12 months in a real-world cohort of IPF patients. Longitudinal biomarker levels of type I and III collagen turnover were related to progressive disease. Moreover, antifibrotic therapy did not affect type I and III collagen turnover biomarkers in these patients. PRO-C3 and C1M may be potential biomarkers for a progressive disease behavior in IPF.

The novel collagen matrikine, endotrophin, is associated with mortality and cardiovascular events in patients with atherosclerosis.

BACKGROUND: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. OBJECTIVE: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. METHODS: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan-Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. RESULTS: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 -1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 -1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 -1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 -1.117], p = 0.017). CONCLUSION: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts.

Can biomarkers of extracellular matrix remodelling and wound healing be used to identify high risk patients infected with SARS-CoV-2?: lessons learned from pulmonary fibrosis.

Pulmonary fibrosis has been identified as a main factor leading to pulmonary dysfunction and poor quality of life in post-recovery Severe Acute Respiratory Syndrome (SARS) survivor's consequent to SARS-Cov-2 infection. Thus there is an urgent medical need for identification of readily available biomarkers that in patients with SARS-Cov-2 infection are able to; (1) identify patients in most need of medical care prior to admittance to an intensive care unit (ICU), and; (2) identify patients post-infection at risk of developing persistent fibrosis of lungs with subsequent impaired quality of life and increased morbidity and mortality. An intense amount of research have focused on wound healing and Extracellular Matrix (ECM) remodelling of the lungs related to lung function decline in pulmonary fibrosis (PF). A range of non-invasive serological biomarkers, reflecting tissue remodelling, and fibrosis have been shown to predict risk of acute exacerbations, lung function decline and mortality in PF and other interstitial lung diseases (Sand et al. in Respir Res 19:82, 2018). We suggest that lessons learned from such PF studies of the pathological processes leading to lung function decline could be used to better identify patients infected with SARS-Co-V2 at most risk of acute deterioration or persistent fibrotic damage of the lung and could consequently be used to guide treatment decisions.

A serological marker of the N-terminal neoepitope generated during LOXL2 maturation is elevated in patients with cancer or idiopathic pulmonary fibrosis.

OBJECTIVES: Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation. DESIGN AND METHODS: An ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16). RESULTS: A technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001-0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49-84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001). CONCLUSIONS: A specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.

A biomarker of collagen type I degradation is associated with cardiovascular events and mortality in patients with atherosclerosis.

OBJECTIVE: Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix (ECM) proteins in the arterial wall. Collagen type I (COL1), a component of the arterial ECM, is cleaved by matrix metalloproteinases (MMPs) and known to be remodelled in atherosclerosis. We explored whether the MMP-mediated COL1 biomarker, C1M, was associated with cardiovascular events, cardiovascular mortality and all-cause mortality in a large prospective cohort of patients with known atherosclerosis. METHODS: Serum from 787 patients who underwent a carotid endarterectomy was included. Circulating levels of C1M were measured in serum. A total of 473 patients were followed for 6 years after surgery. Associations between C1M and incidence of cardiovascular events, cardiovascular mortality and all-cause mortality were assessed by Kaplan-Meier curves and Cox regression analysis. RESULTS: A total of 101 (21.4%) patients suffered from nonfatal cardiovascular events during the follow-up period, and 64 (13.5%) patients died. Of these, 39 (60.9%) died from cardiovascular diseases. Patients with C1M levels above the median were significantly associated with cardiovascular events, cardiovascular mortality and all-cause mortality (P < 0.001, P = 0.004 and P < 0.001, respectively). C1M was included in the final model for prediction of cardiovascular events (HR 2.15, 95% CI 1.40-3.32, P = 0.001), cardiovascular mortality (HR 2.20, 95% CI 1.07-4.51, P = 0.031) and all-cause mortality (HR 2.98 95% CI 1.67-5.33, P = < 0.001). CONCLUSIONS: In patients with atherosclerotic carotid lesions, high levels of C1M predicted cardiovascular events, cardiovascular mortality and all-cause mortality. These findings emphasize the importance of remodelling mechanisms in atherosclerosis that are now becoming more and more explored.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer - implications of a new collagen chaperone function of SPARC.

The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B.

BACKGROUND: Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. AIMS: To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. METHODS: STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. RESULTS: STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. CONCLUSION: Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.

Serological markers of extracellular matrix remodeling predict transplant-free survival in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis is a progressive liver disease with a remarkably variable course. Biomarkers of disease activity or prognostic models predicting outcome at an individual level are currently not established. AIM: To evaluate the prognostic utility of four biomarkers of basement membrane and interstitial extracellular matrix remodeling in patients with primary sclerosing cholangitis. METHODS: Serum samples were available from 138 large-duct primary sclerosing cholangitis patients (of which 102 [74%] with IBD) recruited 2008-2012 and 52 ulcerative colitis patients (controls). The median follow-up time was 2.2 (range 0-4.3) years. Specific biomarkers of type III and V collagen formation (PRO-C3 and PRO-C5, respectively) and type III and IV collagen degradation (C3M and C4M, respectively) were assessed. The Enhanced Liver Fibrosis test, including procollagen type III N-terminal peptide, tissue inhibitor of metalloproteinase-1 and hyaluronic acid was assessed for comparison. RESULTS: All markers were elevated in primary sclerosing cholangitis compared to ulcerative colitis patients (P < 0.001). PRO-C3 showed the largest difference between the two groups with a threefold increase in primary sclerosing cholangitis compared to ulcerative colitis patients. Patients with high baseline serum levels of all markers, except C3M, had shorter survival compared to patients with low baseline serum levels (P < 0.001). Combining PRO-C3 and PRO-C5 the odds ratio for predicting transplant-free survival was 47 compared to the Enhanced Liver Fibrosis test's odds ratio of 11. CONCLUSIONS: Extracellular matrix remodeling is elevated in primary sclerosing cholangitis patients compared to ulcerative colitis patients. Furthermore, the interstitial matrix marker PRO-C3 was identified as a potent prognostic marker and an independent predictor of transplant-free survival in primary sclerosing cholangitis.

Aggrecanase degradation of type III collagen is associated with clinical knee pain.

There is a lack of biochemical markers for non-invasive and objective assessment of symptomatic osteoarthritis (OA). Aggrecanase activity has been shown to be associated with joint deterioration and symptomatic disease through the degradation of extracellular matrix proteins, such as type III collagen. Our study aimed to identify and develop a novel biomarker by measuring an aggrecanase-mediated type III collagen neoepitope, and correlate levels of this biomarker with OA joint pain. Mass spectrometric analysis of purified type III collagen, degraded by the aggrecanase A Disintigrin and Metalloproteinase with Thrombospondin motif (ADAMTS), revealed a fragment generated by ADAMTS-1, -4 and -8. A monoclonal antibody was raised against the neoepitope of this fragment (COL3-ADAMTS) and a competitive ELISA was developed and tested; using serum samples from a cross-sectional cohort of patients with different degrees of knee OA (n = 261). The COL3/ADAMTS ELISA was technically robust and specific for the ADAMTS-1, -4 and -8 generated neoepitope. COL3/ADAMTS was released form cytokine stimulated synovial cultures, indicating a biologic link between the marker and synovium. In OA patients, serum COL3/ADAMTS was independently associated with pain scores (rho = -0.13-0.17, p < 0.05). This association was associated significantly with the presence of radiographic OA. Together, these data indicate that COL3/ADAMTS could be a marker of early osteoarthritis and the underlining pathology.

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders - development and biological validation of a new serum biomarker.

BACKGROUND: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders. METHODS: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls. RESULTS: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively. CONCLUSION: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

The good and the bad collagens of fibrosis - Their role in signaling and organ function.

Usually the dense extracellular structure in fibrotic tissues is described as extracellular matrix (ECM) or simply as collagen. However, fibrosis is not just fibrosis, which is already exemplified by the variant morphological characteristics of fibrosis due to viral versus cholestatic, autoimmune or toxic liver injury, with reticular, chicken wire and bridging fibrosis. Importantly, the overall composition of the ECM, especially the relative amounts of the many types of collagens, which represent the most abundant ECM molecules and which centrally modulate cellular functions and physiological processes, changes dramatically during fibrosis progression. We hypothesize that there are good and bad collagens in fibrosis and that a change of location alone may change the function from good to bad. Whereas basement membrane collagen type IV anchors epithelial and other cells in a polarized manner, the interstitial fibroblast collagens type I and III do not provide directional information. In addition, feedback loops from biologically active degradation products of some collagens are examples of the importance of having the right collagen at the right place and at the right time controlling cell function, proliferation, matrix production and fate. Examples are the interstitial collagen type VI and basement membrane collagen type XVIII. Their carboxyterminal propeptides serve as an adipose tissue hormone, endotrophin, and as a regulator of angiogenesis, endostatin, respectively. We provide an overview of the 28 known collagen types and propose that the molecular composition of the ECM in fibrosis needs careful attention to assess its impact on organ function and its potential to progress or reverse. Consequently, to adequately assess fibrosis and to design optimal antifibrotic therapies, we need to dissect the molecular entity of fibrosis for the molecular composition and spatial distribution of collagens and the associated ECM.

Fibrosis is not just fibrosis - basement membrane modelling and collagen metabolism differs between hepatitis B- and C-induced injury.

BACKGROUND: While morphological patterns differ, the molecular phenotype of liver fibrosis is considered a stereotypical response to chronic liver injury. However, with different cellular triggers and networks regulating fibrosis, the molecular responses of the injured liver may not be identical. AIM: To investigate whether differences in extracellular matrix (ECM) composition of the liver during fibrogenesis in two seemingly similar types of viral hepatitis could be reflected by differences in ECM turnover. METHODS: Utilising a cross-sectional design, we measured specific ECM protein fragments in plasma from 197 chronic hepatitis B (CHB) patients and 403 chronic hepatitis C (CHC) patients matched for inflammation grade and fibrosis stage. Markers of matrix metalloprotease degraded type I, III, IV and VI collagen (C1M, C3M, C4M, C6M) and type III and IV collagen formation (Pro-C3, P4NP7S). RESULTS: P4NP7S, C3M, C4M and C6M were significantly elevated in CHB compared to CHC. In contrast, Pro-C3 was significantly elevated in CHC compared to CHB. Pro-C3, C3M and C4M were increased in parallel with inflammation and fibrosis in both cohorts. C6M and P4NP7S were associated with inflammation and fibrosis only in CHC. Basement membrane collagen fragments P4NP7S and C4M were significantly higher in matched activity and fibrosis cohorts within CHB vs CHC. CONCLUSION: The main parameters to determine extracellular matrix biomarker levels are inflammation, fibrosis, and type of viral insult. Compared to CHC, CHB appears to induce a higher basement membrane turnover. This suggests that there are aetiology-dependent molecular signatures in liver fibrosis that could have pathogenic and diagnostic implications.

Quantification of fibronectin as a method to assess ex vivo extracellular matrix remodeling.

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFß and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies.

Excessive collagen turnover products are released during colorectal cancer progression and elevated in serum from metastatic colorectal cancer patients.

During cancer progression, the homeostasis of the extracellular matrix becomes imbalanced with an excessive collagen remodeling by matrix metalloproteinases. As a consequence, small protein fragments of degraded collagens are released into the circulation. We have investigated the potential of protein fragments of collagen type I, III and IV as novel biomarkers for colorectal cancer. Specific fragments of degraded type I, III and IV collagen (C1M, C3M, C4M) and type III collagen formation (Pro-C3) were assessed in serum from colorectal cancer patients, subjects with adenomas and matched healthy controls using well-characterized and validated ELISAs. Serum levels of the biomarkers were significantly elevated in colorectal cancer patients compared to subjects with adenomas (C1M, Pro-C3, C3M) and controls (C1M, Pro-C3). When patients were stratified according to their tumour stage, all four biomarkers were able to differentiate stage IV metastatic patients from all other stages. Combination of all markers with age and gender in a logistic regression model discriminated between metastatic and non-metastatic patients with an AUROC of 0.80. The data suggest that the levels of these collagen remodeling biomarkers may be a measure of tumour activity and invasiveness and may provide new clinical tools for monitoring of patients with advanced stage colorectal cancer.

Collagen degradation products measured in serum can separate ovarian and breast cancer patients from healthy controls: A preliminary study.

BACKGROUND: During cancer the otherwise tightly controlled homeostasis of the extracellular matrix (ECM) is disturbed. The protein composition changes, the ECM stiffens and increased levels of proteases are secreted. The combination of these processes result in release of specific protein fragments (e.g. collagens) to the circulation, which when measured may reflect disease pathogenesis. OBJECTIVE: To investigate if biomarkers of protease-degraded collagen could differentiate ovarian and breast cancer patients from healthy controls when measured in serum. METHODS: The levels of markers reflecting MMP-degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12) collagen were assessed in serum from ovarian cancer patients (n= 10), breast cancer patients (n= 14) and healthy controls (n= 49) using validated ELISAs. The markers were compared using one way ANOVA and AUC was calculated. RESULTS: All markers were significantly elevated in serum from ovarian cancer patients (p< 0.0001) and breast cancer patients (p< 0.04-0.0001) compared to healthy controls. Furthermore, diagnostically the markers were able to differentiate ovarian (AUROC 90%-93%) and breast cancer patients (AUROC 76%-93%) from healthy controls, with C1M being the strongest differentiator of disease vs. CONCLUSION: Four serum biomarkers reflecting altered MMP-mediated collagen turnover were able to differentiate ovarian and breast cancer patients from healthy controls.

Circulating Elastin Fragments Are Not Affected by Hepatic, Renal and Hemodynamic Changes, But Reflect Survival in Cirrhosis with TIPS.

BACKGROUND AND AIMS: Progressive fibrosis increases hepatic resistance and causes portal hypertension with complications. During progressive fibrosis remodeling and deposition of collagens and elastin occur. Elastin remodeling is crucially involved in fibrosis progression in animal models and human data. This study investigated the association of circulating elastin with the clinical outcome in cirrhotic patients with severe portal hypertension receiving transjugular intrahepatic porto-systemic shunt (TIPS). METHODS: We analyzed portal and hepatic venous samples of 110 cirrhotic patients obtained at TIPS insertion and 2 weeks later. The circulating levels of elastin fragments (ELM) were determined using specific monoclonal ELISA. The relationship of ELM with clinical short-time follow-up and long-term outcome was investigated. RESULTS: Circulating levels of ELM showed a gradient across the liver before TIPS with higher levels in the hepatic vein. Interestingly, the circulating ELM levels remained unchanged after TIPS. The circulating levels of ELM in portal and hepatic veins correlated with platelet counts and inversely with serum sodium. Hepatic venous levels of ELM were higher in CHILD C compared to CHILD A and B and were associated with the presence of ascites. Patients with high levels of ELM in the hepatic veins before TIPS showed poorer survival. In multivariate analysis ELM levels in the hepatic veins and MELD were independent predictors of mortality in these patients. CONCLUSION: This study demonstrated that circulating levels of ELM are not associated with hemodynamic changes, but might reflect fibrosis remodeling and predict survival in patients with severe portal hypertension receiving TIPS independently of MELD.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

OBJECTIVES: Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. DESIGN AND METHODS: Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. RESULTS: The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p<0.0001) and lung cancer (96%, p<0.0001) compared to matched controls. CONCLUSIONS: MMP-7-generated elastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases.

The importance of extracellular matrix for cell function and in vivo likeness.

OBJECTIVE: Fibrotic diseases may be described as a disease of the extracellular matrix, where the balance between matrix formation and degradation has been shifted leading to an accumulation of matrix. Currently a fit for purpose model and readily available approach are adapted when doing cell cultures, which may not reflect physiology and pathophysiology optimally. The aim of this review is to draw special attention to the similarities and differences of current state of the art in vitro and ex vivo models, with special focus on the proteins, cell-cell interactions, and correct matrix composition, which may be a better representative of in vivo conditions in a disease where the extracellular matrix is the central player. METHODS: We reviewed current literature with emphasis on the role of the extracellular matrix in health and disease, different fibrotic disease models, and highlighting the importance of this when looking at translational science. CONCLUSION: To further our fibrotic research one paramount problem is to fundamentally understand the core of the disease, the production and degradation of the extracellular matrix. For a surprisingly long time the ECM has been underestimated until recently, with the discovery that the ECM may control cell phenotype through cell-matrix interactions. This highlights the need of a native ECM when investigating pathways and response to potential therapy. Clearly, both in vitro and in vivo models provide fit to purpose benefits, but in particular for the fibrosis field we may ask, do single cell cultures in monolayers recapitulate the complicated ECM environment controlling cell fate?

Review article: the efficacy of biomarkers in chronic fibroproliferative diseases - early diagnosis and prognosis, with liver fibrosis as an exemplar.

BACKGROUND: Nearly 45% of all deaths are associated with chronic fibroproliferative diseases, of which the primary characteristic is altered remodelling of the extracellular matrix. A major difficulty in developing anti-fibrotic therapies is the lack of accurate and established techniques to estimate dynamics of fibrosis, regression or progression, in response to therapy. AIM: One of the most pressing needs in modern clinical chemistry for fibroproliferative disorders is the development of biomarkers for early diagnosis, prognosis, and early efficacy for the benefit of patients and to facilitate improved drug development. The aim of this article was to review the serological biomarkers that may assist in early diagnosis of patients, separate fast from slow- or nonprogressors, and possibly assist in drug development for fibroproliferative diseases, exemplified by liver fibrosis. The lack of success of biochemical markers and the possible reasons for this is discussed in the context of other fields with biomarker success. METHOD: This is a personal opinion review article. RESULTS: Biochemical markers, originating from the fibrotic structure, may have increased specificity and sensitivity for disease. Assessment of the tissue turnover balance by measurement of tissue formation and tissue degradation separately by novel technologies may provide value. CONCLUSIONS: Novel technologies focused on the protein fingerprint in addition to biomarker classification, may increase the quality of biomarker development and provide the much needed biomarkers to further the fibroproliferative field. This is in direct alignment with the Food and Drug Administration and European Medicinal Agencies initiatives of personal health care.