Jannie Marie Bülow Sand

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.

MMP mediated degradation of type IV collagen alpha 1 and alpha 3 chains reflects basement membrane remodeling in experimental and clinical fibrosis--validation of two novel biomarker assays.

Objectives Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of various growth factors, cytokines and proteases. We hypothesized that matrix metalloproteinase (MMP) mediated degradation of type IV collagen, a main component of the basement membrane, will release peptide fragments (neo-epitopes) into the circulation. Here we present the development of two competitive enzyme-linked immunosorbent assays (ELISAs) for assessing the levels of specific fragments of type IV collagen α1 (C4M12a1) and α3 (C4M12a3) chains in serum as indicators of fibrosis. Methods Fragments of type IV collagen cleaved in vitro by MMP-12 were identified by mass spectrometry, and two were chosen for ELISA development due to their unique sequences. The assays were evaluated using samples from a carbon tetrachloride (CCl4) rat model of liver fibrosis and from patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD). Results Two technically robust ELISAs were produced using neo-epitope specific monoclonal antibodies. Mean serum C4M12a1 levels were significantly elevated in CCl4-treated rats compared with controls in weeks 12, 16, and 20, with a maximum increase of 102% at week 16 (p < 0.0001). Further, C4M12a1 levels correlated with the total collagen content of the liver in CCl4-treated rats (r = 0.43, p = 0.003). Mean serum C4M12a3 levels were significantly elevated in patients with mild, moderate, and severe IPF, and COPD relative to healthy controls, with a maximum increase of 321% in COPD (p < 0.0001). Conclusions Two assays measuring C4M12a1 and C4M12a3 enabled quantification of MMP mediated degradation of type IV collagen in serum. C4M12a1 was elevated in a pre-clinical model of liver fibrosis, and C4M12a3 was elevated in IPF and COPD patients. This suggests the use of these assays to investigate pathological remodeling of the basement membrane in different organs. However, validations in larger clinical settings are needed.

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.

Accelerated extracellular matrix turnover during exacerbations of COPD

BackgroundExacerbations of chronic obstructive pulmonary disease (COPD) contribute significantly to disease progression. However, the effect on tissue structure and turnover is not well described. There is an urgent clinical need for biomarkers of disease activity associated with disease progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD.Methods69 patients with COPD hospitalised for an exacerbation were recruited at admission and returned for a 4 weeks follow-up. Competitive ELISAs measuring circulating protein fragments in serum or plasma assessed the formation and degradation of collagen types III (Pro-C3 and C3M, respectively), IV (P4NP 7S and C4M, respectively), and VI (Pro-C6 and C6M, respectively), and degradation of elastin (ELM7 and EL-NE) and versican (VCANM).ResultsCirculating levels of C3M, C4M, C6M, ELM7, and EL-NE were elevated during an exacerbation of COPD as compared to follow-up (all P <0.0001), while VCANM levels were decreased (P <0.0001). Pro-C6 levels were decreased and P4NP 7S levels were elevated during exacerbation (P <0.0001). Pro-C3 levels were unchanged. At time of exacerbation, degradation/formation ratios were increased for collagen types III and VI and decreased for collagen type IV.ConclusionsExacerbations of COPD resulted in elevated levels of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression.

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

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.

Systemic Biomarkers of Collagen and Elastin Turnover Are Associated With Clinically Relevant Outcomes in COPD

Background Extracellular matrix (ECM) remodeling of the lung tissue releases protein fragments into the blood, where they may be detected as serologic surrogate markers of disease activity in COPD. Our goal was to assess the association of ECM turnover with severity and outcome of COPD. Methods In a prospective, observational, multicenter study including 506 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease grades II to IV), serum samples were analyzed at stable state, exacerbation, and 4 weeks after exacerbation. The analysis comprised a panel of five novel neoepitopes, including fragments of collagen type III (C3M) and collagen type VI (C6M), pro‐forms of collagen type III (Pro‐C3) and type VI (Pro‐C6), and neutrophil elastase‐generated fragments of elastin (EL‐NE) according to enzyme‐linked immunosorbent assay. These neoepitopes were also measured at stable state in a derivation cohort that included 100 patients with COPD. Results Serum levels of C3M, C6M, Pro‐C3, Pro‐C6, and EL‐NE were associated with lung function. Patients with the lowest levels of Pro‐C3 and Pro‐C6 had more severe airflow limitation, hyperinflation, air trapping, and emphysema. C3M and C6M were associated with dyspnea. All ECM biomarkers, except Pro‐C6, were increased at exacerbation compared with stable state but, except EL‐NE, did not differ between stable state and exacerbation follow‐up in the crude and adjusted analyses. In Cox regression adjusted analyses, Pro‐C3 was associated with a shorter time to exacerbation (hazard ratio, 0.72; CI, 0.59‐0.89; P = .002) and Pro‐C6 with survival (hazard ratio, 2.09; CI, 1.18‐3.71; P = .011). Conclusions Serum biomarkers of ECM turnover were significantly associated with disease severity and clinically relevant outcomes in patients with COPD. Trial Registry No.: ISRCTN99586989; URL: www.controlled‐trials.com.

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.

Clinical Drug Development Using Dynamic Biomarkers to Enable Personalized Health Care in COPD.

Despite massive investments in the development of novel treatments for heterogeneous diseases such as COPD, the resources spent have only benefited a fraction of the population treated. Personalized health care to guide selection of a suitable patient population already in the clinical development of new compounds could offer a solution. This review discusses past successes and failures in drug development and biomarker research in COPD, describes research in COPD phenotypes and the required characteristics of a suitable biomarker for identifying patients at higher risk of progression, and examines the role of extracellular matrix proteins found to be upregulated in COPD. Novel biomarkers of connective tissue remodeling that may provide added value for a personalized approach by detecting subgroups of patients with active disease suitable for pharmacologic intervention are discussed.

Characterization of serological neo-epitope biomarkers reflecting collagen remodeling in clinically stable chronic obstructive pulmonary disease.

OBJECTIVES Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation that leads to excessive remodeling of the lung extracellular matrix (ECM), resulting in release of protein fragments (neo-epitopes) to the blood. Serological markers assessing this have previously been associated with exacerbations of COPD. However, characterization of these in individuals with clinically stable COPD is lacking. The aim of this study was to characterize the collagen remodeling in stable COPD by the serological assessment of neo-epitopes. DESIGN AND METHODS Sixty-eight subjects with clinically stable COPD were included into the study at baseline, and 27 came back for a four weeks follow-up visit. Serum and plasma levels of neo-epitopes were assessed for the evaluation of collagen type III (C3M), IV (C4M, C4Ma3, P4NP 7S), and VI (C6M, Pro-C6) remodeling. RESULTS C3M, C4M, C4Ma3, P4NP 7S, and C6M levels were significantly elevated in COPD subjects compared with healthy controls (p<0.0001 to p=0.044). Each neo-epitope biomarker was significantly correlated between serum and plasma (p<0.0001) and most biomarkers were stable in the majority of patients from baseline to week four. Serum C6M levels were weakly correlated with FEV1% predicted (r=-0.274, p=0.025) and serum Pro-C6 levels were elevated in subjects with previous exacerbations (p=0.014). C3M, C4Ma3, C6M, and P4NP 7S were weakly correlated with MRC dyspnea scores (p<0.01). No associations were seen with BMI, smoking, duration of COPD, blood oxygen saturation, shuttle walk test distance, GOLD grades, or CAT scores. CONCLUSIONS Serological biomarkers of collagen remodeling were elevated in subjects with COPD as compared with healthy individuals. Biomarker levels were significantly correlated with measures of dyspnea, indicating a relationship with degree of symptoms, while only C6M showed a weak but significant association with lung function. Biomarker levels were not related to GOLD grades, which was in line with previous studies indicating that ECM remodeling may be related to disease activity rather than severity.