Diana Leeming - Academia.edu (original) (raw)

Papers by Diana Leeming

Research paper thumbnail of Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

BackgroundDuring the pathological destruction of lung tissue, neutrophil elastase (NE) degrades e... more BackgroundDuring the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.MethodsMonoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).ResultsAnalyte recovery of EL-NE for human serum was between 85% and...

Research paper thumbnail of 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 circul... more 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.

Research paper thumbnail of A MMP derived versican neo-epitope is elevated in plasma from patients with atherosclerotic heart disease

International Journal of Clinical and Experimental Medicine, 2013

Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. ... more Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. Versican, an extracellular matrix proteoglycan present in normal and atherosclerotic arteries is a substrate for matrix metalloproteinases (MMPs) present in macrophage rich areas. The aim of the current study was to develop an immunoassay to detect a specific MMP-12 derived versican degradation fragment (VCANM) and assess its potential as a biomarker for extracellular matrix remodelling in atherosclerosis. A mouse monoclonal antibody raised against VCANM was used for the development of a competitive ELISA for detection of the fragment in plasma. VCANM was measured in plasma of patients with different levels of heart diseases. Patients experiencing I) acute coronary syndrome, II) stable ischemic heart disease and III) demonstrating high levels of coronary calcium deposits had significantly higher plasma levels of VCANM compared to a control group of individuals with no detectable coronary calcium deposits. VCANM was also detected by immunohistochemistry in coronary artery sections of patients with different degrees of atherosclerosis. VCANM ability to separate patients with atherosclerotic diseases from healthy individuals suggested VCANM as a potential biomarker for the pathological arterial matrix remodelling associated with atherosclerosis.

Research paper thumbnail of The extracellular matrix in the kidney: a source of novel non-invasive biomarkers of kidney fibrosis?

Fibrogenesis & Tissue Repair, 2014

Interstitial fibrosis is the common endpoint of end-stage chronic kidney disease (CKD) leading to... more Interstitial fibrosis is the common endpoint of end-stage chronic kidney disease (CKD) leading to kidney failure. The clinical course of many renal diseases, and thereby of CKD, is highly variable. One of the major challenges in deciding which treatment approach is best suited for a patient but also in the development of new treatments is the lack of markers able to identify and stratify patients with stable versus progressive disease. At the moment renal biopsy is the only means of diagnosing renal interstitial fibrosis. Novel biomarkers should improve diagnosis of a disease, estimate its prognosis and assess the response to treatment, all in a non-invasive manner. Existing markers of CKD do not fully and specifically address these requirements and in particular do not specifically reflect renal fibrosis. The aim of this review is to give an insight of the involvement of the extracellular matrix (ECM) proteins in kidney diseases and as a source of potential novel biomarkers of renal fibrosis. In particular the use of the protein fingerprint technology, that identifies neo-epitopes of ECM proteins generated by proteolytic cleavage by proteases or other post-translational modifications, might identify such novel biomarkers of renal fibrosis.

Research paper thumbnail of Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

Fibrogenesis & Tissue Repair, 2013

Background: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragm... more 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 0 YWEVQPATFR 0 353 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.

Research paper thumbnail of The importance of extracellular matrix for cell function and in vivo likeness

Experimental and molecular pathology, Jan 13, 2015

Fibrotic diseases may be described as a disease of the extracellular matrix, where the balance be... more 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. 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. To further our fibrotic research one paramount problem...

Research paper thumbnail of Markers of extracellular matrix remodeling can elucidate the matrix turnover profile of the fibrotic liver

Research paper thumbnail of URINARY AND SEROLOGICAL MARKERS OF COLLAGEN DEGRADATION ARE ASSOCIATED WITH DISEASE SEVERITY AND INFLAMMATION IN IGA NEPHROPATHY PATIENTS

Research paper thumbnail of The role of extracellular matrix quality in pulmonary fibrosis

Respiration; international review of thoracic diseases, 2014

This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmo... more This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmonary fibrosis (PF). In PF, the highly ordered structure of collagens and elastin within the ECM of the lung is severely disrupted and lacks its original tissue quality. Discussions about the ECM have focused on the role of protein quantity in relation to the progression of PF, while the importance of lung ECM quality, defined by the levels of ECM protein modifications and by the protein distribution in lung tissue, has not been properly addressed. The quality and function of proteins may be altered by different post-translational modifications (PTMs), such as cross-linking, proteolytic cleavage, citrullination, misfolding and glycosylation. This paper is the first to review key data from the literature related to the lung ECM at the molecular level, relate these to changes observed at a macroscopic level and evaluate which PTMs most likely contribute to PF. This paper also reviews the ro...

Research paper thumbnail of Molecular Serum Markers of Liver Fibrosis

Biomarker Insights, 2012

Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the exc... more Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the excessive accumulation and reorganizationoftheextracellularmatrix(ECM).Thegoldstandardforassessmentoffibrosisisliverbiopsy.Asthisprocedurehas variouslimitations,includingriskofpatientinjuryandsamplingerror,anon-invasiveserummarkerforliverfibrosisisdesirable.The increasing understanding of the pathogenesis of hepatic fibrosis has suggested several markers which could be useful indicators of hepaticfibrogenesisandfibrosis.Thesemarkersincludeserummarkersofliverfunction,ECMsynthesis,fibrolyticprocesses,ECM degradation and fibrogenesis related cytokines. Recently, neo-epitopes, which are post-translational modifications of proteins, have beensuccessfullyusedinboneandcartilagediseaseswhicharecharacterizedbyextensiveECMremodeling.Increasingnumbersof studiesarebeingundertakentoidentifyneo-epitopesgeneratedduringliverfibrosis,andwhichultimatelymightbeusefulfordiagnosingandmonitoringfibrogenesis.Todate,themetalloproteinasesgeneratedfragmentofcollagenI,III,IVandVIhavebeenproventobe elevatedintworatmodelsoffibrosis.Thisreviewsummarizestherecenteffortsthathavebeenmadetoidentifypotentiallyreliablenoninvasiveserummarkers.WeusedtherecentlyproposedBIPED(Burdenofdisease,Investigative,Prognostic,Efficacyand Diagnostic) systemtocharacterizepotentialserummarkersandneo-epitopemarkersthathavebeenidentifiedtodate.

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

ASSAY and Drug Development Technologies, 2013

Increased attention is paid to the structural components of tissues. These components are mostly ... more 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.

Research paper thumbnail of 76P * SEROLOGICAL DETECTION OF MATRIX METALLOPROTEINASE GENERATED FRAGMENTS OF ELASTIN AND TYPE IV COLLAGEN MAY BE BIOMARKERS OF LUNG METASTATIC BREAST CANCER

Research paper thumbnail of Biochemical markers for CVD risk assessment

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

American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015

Emerging evidence suggests that altered components and post-translational modifications of protei... more Emerging evidence suggests that altered components and post-translational 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 plays 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 post-translational modifications of ECM proteins resulting in altered functional (signalling) 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 harbour signalling functions that can modulate fibrotic liver disease. The ECM entails functions in addition to anchoring cells and modulating their migratory behaviour. Key ECM components and their post-translational modifications often harbour multiple domains with different signalling potential, in particular when modified during inflammation or wound healing. This signalling by the ECM should be considered as 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 anti-fibrotic treatment strategies for liver fibrosis as well as other fibrotic diseases.

Research paper thumbnail of Longitudinal change in collagen degradation biomarkers in idiopathic pulmonary fibrosis: an analysis from the prospective, multicentre PROFILE study

The Lancet Respiratory Medicine, 2015

Background Idiopathic pulmonary fi brosis, a progressive and inevitably fatal disorder, has a hig... more Background Idiopathic pulmonary fi brosis, a progressive and inevitably fatal disorder, has a highly variable clinical course. Biomarkers that refl ect disease activity are urgently needed to inform patient management and for use as biomarkers of therapeutic response (theragnostic biomarkers) in clinical trials. We aimed to determine whether dynamic change in markers of extracellular matrix (ECM) turnover predicts progression of idiopathic pulmonary fi brosis as determined by change in forced vital capacity and death.

Research paper thumbnail of PRO-C3-Levels in Patients with HIV/HCV-Co-Infection Reflect Fibrosis Stage and Degree of Portal Hypertension

PLoS ONE, 2014

Background: Liver-related deaths represent the leading cause of mortality among patients with HIV... more Background: Liver-related deaths represent the leading cause of mortality among patients with HIV/HCV-co-infection, and are mainly related to complications of fibrosis and portal hypertension. In this study, we aimed to evaluate the structural changes by the assessment of extracellular matrix (ECM) derived degradation fragments in peripheral blood as biomarkers for fibrosis and portal hypertension in patients with HIV/HCV co-infection.

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

Scandinavian Journal of Gastroenterology, 2015

Abstract Background and aims: The hepatic venous pressure gradient (HVPG) is an important but inv... more 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.

Research paper thumbnail of Association of Versican Turnover with All-Cause Mortality in Patients on Haemodialysis

PLoS ONE, 2014

Cardiovascular diseases are among the most common causes of mortality in renal failure patients u... more Cardiovascular diseases are among the most common causes of mortality in renal failure patients undergoing haemodialysis. A high turnover rate of the proteoglycan versican, represented by the increased presence of its fragmentation products in plasma, has previously been associated with cardiovascular diseases. The objective of the study was to investigate the association of versican turnover assessed in plasma with survival in haemodialysis patients. A specific matrix metalloproteinase-generated neo-epitope fragment of versican (VCANM) was measured in plasma of 364 haemodialysis patients with a 5-years follow-up, using a robust competitive enzyme-linked immunosorbent assays. Association between VCANM plasma concentration and survival was assessed by Kaplan-Meier analysis and adjusted Cox model. Haemodialysis patients with plasma VCANM concentrations in the lowest quartile had increased risk of death (odds ratio, as compared to the highest quartile: 7.1, p<0.001), with a reduced survival of 152 days compared to 1295 days for patients with plasma VCANM in the highest quartile. Multivariate analysis showed that low VCANM (p<0.001) and older age (p<0.001) predicted death in haemodialysis patients. Low concentrations of the versican fragment VCANM in plasma were associated with higher risk of death among haemodialysis patients. A possible protective role for the examined versican fragment is suggested.

Research paper thumbnail of Association of Systemic Collagen Type IV Formation with Survival among Patients Undergoing Hemodialysis

PLoS ONE, 2013

Objective: The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic col... more Objective: The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic collagen type IV formation. The objective of the study was to investigate the association of systemic collagen type IV formation with survival among patients undergoing hemodialysis.

Research paper thumbnail of 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

PLoS ONE, 2013

Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of var... more 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. 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). 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). 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.

Research paper thumbnail of Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling

BackgroundDuring the pathological destruction of lung tissue, neutrophil elastase (NE) degrades e... more BackgroundDuring the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.MethodsMonoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).ResultsAnalyte recovery of EL-NE for human serum was between 85% and...

Research paper thumbnail of 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 circul... more 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.

Research paper thumbnail of A MMP derived versican neo-epitope is elevated in plasma from patients with atherosclerotic heart disease

International Journal of Clinical and Experimental Medicine, 2013

Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. ... more Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. Versican, an extracellular matrix proteoglycan present in normal and atherosclerotic arteries is a substrate for matrix metalloproteinases (MMPs) present in macrophage rich areas. The aim of the current study was to develop an immunoassay to detect a specific MMP-12 derived versican degradation fragment (VCANM) and assess its potential as a biomarker for extracellular matrix remodelling in atherosclerosis. A mouse monoclonal antibody raised against VCANM was used for the development of a competitive ELISA for detection of the fragment in plasma. VCANM was measured in plasma of patients with different levels of heart diseases. Patients experiencing I) acute coronary syndrome, II) stable ischemic heart disease and III) demonstrating high levels of coronary calcium deposits had significantly higher plasma levels of VCANM compared to a control group of individuals with no detectable coronary calcium deposits. VCANM was also detected by immunohistochemistry in coronary artery sections of patients with different degrees of atherosclerosis. VCANM ability to separate patients with atherosclerotic diseases from healthy individuals suggested VCANM as a potential biomarker for the pathological arterial matrix remodelling associated with atherosclerosis.

Research paper thumbnail of The extracellular matrix in the kidney: a source of novel non-invasive biomarkers of kidney fibrosis?

Fibrogenesis & Tissue Repair, 2014

Interstitial fibrosis is the common endpoint of end-stage chronic kidney disease (CKD) leading to... more Interstitial fibrosis is the common endpoint of end-stage chronic kidney disease (CKD) leading to kidney failure. The clinical course of many renal diseases, and thereby of CKD, is highly variable. One of the major challenges in deciding which treatment approach is best suited for a patient but also in the development of new treatments is the lack of markers able to identify and stratify patients with stable versus progressive disease. At the moment renal biopsy is the only means of diagnosing renal interstitial fibrosis. Novel biomarkers should improve diagnosis of a disease, estimate its prognosis and assess the response to treatment, all in a non-invasive manner. Existing markers of CKD do not fully and specifically address these requirements and in particular do not specifically reflect renal fibrosis. The aim of this review is to give an insight of the involvement of the extracellular matrix (ECM) proteins in kidney diseases and as a source of potential novel biomarkers of renal fibrosis. In particular the use of the protein fingerprint technology, that identifies neo-epitopes of ECM proteins generated by proteolytic cleavage by proteases or other post-translational modifications, might identify such novel biomarkers of renal fibrosis.

Research paper thumbnail of Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

Fibrogenesis & Tissue Repair, 2013

Background: The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragm... more 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 0 YWEVQPATFR 0 353 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.

Research paper thumbnail of The importance of extracellular matrix for cell function and in vivo likeness

Experimental and molecular pathology, Jan 13, 2015

Fibrotic diseases may be described as a disease of the extracellular matrix, where the balance be... more 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. 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. To further our fibrotic research one paramount problem...

Research paper thumbnail of Markers of extracellular matrix remodeling can elucidate the matrix turnover profile of the fibrotic liver

Research paper thumbnail of URINARY AND SEROLOGICAL MARKERS OF COLLAGEN DEGRADATION ARE ASSOCIATED WITH DISEASE SEVERITY AND INFLAMMATION IN IGA NEPHROPATHY PATIENTS

Research paper thumbnail of The role of extracellular matrix quality in pulmonary fibrosis

Respiration; international review of thoracic diseases, 2014

This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmo... more This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmonary fibrosis (PF). In PF, the highly ordered structure of collagens and elastin within the ECM of the lung is severely disrupted and lacks its original tissue quality. Discussions about the ECM have focused on the role of protein quantity in relation to the progression of PF, while the importance of lung ECM quality, defined by the levels of ECM protein modifications and by the protein distribution in lung tissue, has not been properly addressed. The quality and function of proteins may be altered by different post-translational modifications (PTMs), such as cross-linking, proteolytic cleavage, citrullination, misfolding and glycosylation. This paper is the first to review key data from the literature related to the lung ECM at the molecular level, relate these to changes observed at a macroscopic level and evaluate which PTMs most likely contribute to PF. This paper also reviews the ro...

Research paper thumbnail of Molecular Serum Markers of Liver Fibrosis

Biomarker Insights, 2012

Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the exc... more Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the excessive accumulation and reorganizationoftheextracellularmatrix(ECM).Thegoldstandardforassessmentoffibrosisisliverbiopsy.Asthisprocedurehas variouslimitations,includingriskofpatientinjuryandsamplingerror,anon-invasiveserummarkerforliverfibrosisisdesirable.The increasing understanding of the pathogenesis of hepatic fibrosis has suggested several markers which could be useful indicators of hepaticfibrogenesisandfibrosis.Thesemarkersincludeserummarkersofliverfunction,ECMsynthesis,fibrolyticprocesses,ECM degradation and fibrogenesis related cytokines. Recently, neo-epitopes, which are post-translational modifications of proteins, have beensuccessfullyusedinboneandcartilagediseaseswhicharecharacterizedbyextensiveECMremodeling.Increasingnumbersof studiesarebeingundertakentoidentifyneo-epitopesgeneratedduringliverfibrosis,andwhichultimatelymightbeusefulfordiagnosingandmonitoringfibrogenesis.Todate,themetalloproteinasesgeneratedfragmentofcollagenI,III,IVandVIhavebeenproventobe elevatedintworatmodelsoffibrosis.Thisreviewsummarizestherecenteffortsthathavebeenmadetoidentifypotentiallyreliablenoninvasiveserummarkers.WeusedtherecentlyproposedBIPED(Burdenofdisease,Investigative,Prognostic,Efficacyand Diagnostic) systemtocharacterizepotentialserummarkersandneo-epitopemarkersthathavebeenidentifiedtodate.

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

ASSAY and Drug Development Technologies, 2013

Increased attention is paid to the structural components of tissues. These components are mostly ... more 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.

Research paper thumbnail of 76P * SEROLOGICAL DETECTION OF MATRIX METALLOPROTEINASE GENERATED FRAGMENTS OF ELASTIN AND TYPE IV COLLAGEN MAY BE BIOMARKERS OF LUNG METASTATIC BREAST CANCER

Research paper thumbnail of Biochemical markers for CVD risk assessment

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

American Journal of Physiology - Gastrointestinal and Liver Physiology, 2015

Emerging evidence suggests that altered components and post-translational modifications of protei... more Emerging evidence suggests that altered components and post-translational 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 plays 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 post-translational modifications of ECM proteins resulting in altered functional (signalling) 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 harbour signalling functions that can modulate fibrotic liver disease. The ECM entails functions in addition to anchoring cells and modulating their migratory behaviour. Key ECM components and their post-translational modifications often harbour multiple domains with different signalling potential, in particular when modified during inflammation or wound healing. This signalling by the ECM should be considered as 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 anti-fibrotic treatment strategies for liver fibrosis as well as other fibrotic diseases.

Research paper thumbnail of Longitudinal change in collagen degradation biomarkers in idiopathic pulmonary fibrosis: an analysis from the prospective, multicentre PROFILE study

The Lancet Respiratory Medicine, 2015

Background Idiopathic pulmonary fi brosis, a progressive and inevitably fatal disorder, has a hig... more Background Idiopathic pulmonary fi brosis, a progressive and inevitably fatal disorder, has a highly variable clinical course. Biomarkers that refl ect disease activity are urgently needed to inform patient management and for use as biomarkers of therapeutic response (theragnostic biomarkers) in clinical trials. We aimed to determine whether dynamic change in markers of extracellular matrix (ECM) turnover predicts progression of idiopathic pulmonary fi brosis as determined by change in forced vital capacity and death.

Research paper thumbnail of PRO-C3-Levels in Patients with HIV/HCV-Co-Infection Reflect Fibrosis Stage and Degree of Portal Hypertension

PLoS ONE, 2014

Background: Liver-related deaths represent the leading cause of mortality among patients with HIV... more Background: Liver-related deaths represent the leading cause of mortality among patients with HIV/HCV-co-infection, and are mainly related to complications of fibrosis and portal hypertension. In this study, we aimed to evaluate the structural changes by the assessment of extracellular matrix (ECM) derived degradation fragments in peripheral blood as biomarkers for fibrosis and portal hypertension in patients with HIV/HCV co-infection.

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

Scandinavian Journal of Gastroenterology, 2015

Abstract Background and aims: The hepatic venous pressure gradient (HVPG) is an important but inv... more 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.

Research paper thumbnail of Association of Versican Turnover with All-Cause Mortality in Patients on Haemodialysis

PLoS ONE, 2014

Cardiovascular diseases are among the most common causes of mortality in renal failure patients u... more Cardiovascular diseases are among the most common causes of mortality in renal failure patients undergoing haemodialysis. A high turnover rate of the proteoglycan versican, represented by the increased presence of its fragmentation products in plasma, has previously been associated with cardiovascular diseases. The objective of the study was to investigate the association of versican turnover assessed in plasma with survival in haemodialysis patients. A specific matrix metalloproteinase-generated neo-epitope fragment of versican (VCANM) was measured in plasma of 364 haemodialysis patients with a 5-years follow-up, using a robust competitive enzyme-linked immunosorbent assays. Association between VCANM plasma concentration and survival was assessed by Kaplan-Meier analysis and adjusted Cox model. Haemodialysis patients with plasma VCANM concentrations in the lowest quartile had increased risk of death (odds ratio, as compared to the highest quartile: 7.1, p<0.001), with a reduced survival of 152 days compared to 1295 days for patients with plasma VCANM in the highest quartile. Multivariate analysis showed that low VCANM (p<0.001) and older age (p<0.001) predicted death in haemodialysis patients. Low concentrations of the versican fragment VCANM in plasma were associated with higher risk of death among haemodialysis patients. A possible protective role for the examined versican fragment is suggested.

Research paper thumbnail of Association of Systemic Collagen Type IV Formation with Survival among Patients Undergoing Hemodialysis

PLoS ONE, 2013

Objective: The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic col... more Objective: The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic collagen type IV formation. The objective of the study was to investigate the association of systemic collagen type IV formation with survival among patients undergoing hemodialysis.

Research paper thumbnail of 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

PLoS ONE, 2013

Fibrosis is characterized by excessive tissue remodeling resulting from altered expression of var... more 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. 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). 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). 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.