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Background Remodeling of the extracellular matrix (ECM) is a central mechanism in the progression of idiopathic pulmonary fibrosis (IPF), and remodeling of type VI collagen has been suggested to be associated with disease progression. Biomarkers that reflect and predict the progression of IPF would provide valuable information for clinicians when treating IPF patients. Methods Two serological biomarkers reflecting formation (PRO-C6) and degradation (C6M) of type VI collagen were evaluated in a real-world cohort of 178 newly diagnoses IPF patients. All patients were treatment naïve at the baseline visit. Blood samples and clinical data were collected from baseline, six months, and 12 months visit. The biomarkers were measured by competitive ELISA using monoclonal antibodies. Results Patients with progressive disease had higher (P = 0.0099) serum levels of PRO-C6 compared to those with stable disease over 12 months with an average difference across all timepoints of 12% (95% CI 3–22), whereas C6M levels tended (P = 0.061) to be higher in patients with progressive disease compared with stable patients over 12 months with an average difference across all timepoints of 12% (95% CI − 0.005–27). Patients who did not receive antifibrotic medicine had a greater increase of C6M (P = 0.043) compared to treated patients from baseline over 12 months with an average difference across all timepoints of 12% (95% CI − 0.07–47). There were no differences in biomarker levels between patients receiving pirfenidone or nintedanib. Conclusions Type VI collagen formation was related to progressive disease in patients with IPF in a real-world cohort and antifibrotic therapy seemed to affect the degradation of type VI collagen. Type VI collagen formation and degradation products might be potential biomarkers for disease progression in IPF.

Chronic obstructive pulmonary disease (COPD) is characterized by a slow heterogeneous progression. Therefore, improved biomarkers that can accurately identify patients with the highest likelihood of progression and therefore the ability to benefit from a given treatment, are needed. Elastin is an essential structural protein of the lungs. In this study, we investigated whether elastin degradation products generated by the enzymes proteinase 3, cathepsin G, neutrophil elastase, MMP7 or MMP9/12 were prognostic biomarkers for COPD-related outcomes. The elastin degradome was assessed in a subpopulation (n = 1307) of the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort with 3 years of clinical follow-up. Elastin degraded by proteinase 3 could distinguish between COPD participants and non-smoking controls (p = 0.0006). A total of 30 participants (3%) died over the 3 years of observation. After adjusting for confounders, plasma levels of elastin degraded by proteinase 3 and cathepsin G were independently associated with mortality outcome with a hazard ratio per 1 SD of 1.49 (95%CI 1.24–1.80, p

Introduction: Remodeling of the airway wall is a hallmark feature of asthma. Under physiological conditions, a finely tuned balance maintains a functional state of the extracellular matrix. This balance is disrupted in asthma. COL4A3 is reduced 18-fold in lung tissue from asthmatics, however, the mechanism leading to the diminished levels of COL4A3 has remained elusive. Methods: The COL4A3 fibrolysis marker C4Ma3 (Nordic Bioscience, Denmark) was measured in the ALLIANCE cohort study participants (Fuchs et al. 2018). Pediatric controls/cases: 34/134; Adult control/cases 31/149. C4Ma3 data was correlated with clinical data, serum cytokine profiles (27-Bioplex, Biorad). Female 6-8 week old Balb/C mice were sensitized (OVA i.p., day 1, 14, 21) and challenged with aerosol (OVA i.n., day 26, 27, 28) to induce acute allergic airway inflammation. Acute exacerbations were provoked by instillation of poly(cytidylic-inosinic, i.n. day 28). Murine precision cut lung slices (PCLS) were used to elucidate the role of sensitisation for COL4A3 degradation. Results: Increased C4Ma3 correlated with asthma exacerbation (pl0.01) and elevated levels of Th2/Th9 cytokines in the ALLIANCE cohort. Serum levels of C4Ma3 were elevated in OVA challenged mice (pl0.0001) and correlated with lung tissue levels of mast cell chymase (pl0.01), but not with neutrophils or matrix-metallo-proteinase 9. C4MA3 was elevated in PCLS of allergic, HDM challenged mice (pl0.05) with no further increase after PolyIC. Conclusion: COL4A3 degradation (circulating C4Ma3), is elevated in severe, exacerbating type 2 asthmatics. C4Ma3 levels correlate with chymase positive mast cells in a mouse model of asthma exacerbation. These data highlight that the degradation of COL4A3 is a central part of the pathology of asthma.

Background: Neutrophils play a crucial role in pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Upon activation, neutrophils can degranulate multiple protein species in an immune response, herein calprotectin (CP) and human neutrophil elastase (HNE). Thus, the decay-rate of circulating neutrophils is rapid. The aim of this study was to examine neutrophil activity in COPD and IPF in serum samples by quantifying the novel biomarker CPa9-HNE (specific neo-epitope fragment of HNE mediated CP degradation), which reflects neutrophil activity. Methods: A specific monoclonal antibody was raised towards a neoepitope of CP generated by HNE and a robust competitive ELISA (CPa9-HNE) was developed. Two patient cohorts were used to assess serum levels of CPa9-HNE: 1) A cohort of clinically stable COPD patients (n=68), where 25 of these came back for a four weeks follow-up visit. These were compared to a panel of healthy controls (n=36) and; 2) A cohort of IPF patients (n=16) as compared to a panel of healthy controls (n=10). Results: CPa9-HNE levels were significantly elevated in COPD patients (median 247.9 ng/mL) as compared with healthy controls (median 18.32 ng/mL), (pl0.0001, AUROC: 0.9996). At four weeks follow-up, the CPa9-HNE levels remained stable. Moreover, the CPa9-HNE was not dependent on age or sex. Serum CPa9-HNE was also significantly elevated in IPF patients (median 161.0 ng/mL) compared to healthy controls (median 26 ng/mL), (pl0.0001, AUROC: 0.9813). Conclusions: Systemically assessed CPa9-HNE was highly elevated in patients with COPD and IPF as compared to healthy controls, thus indicating that neutrophil activity may be evaluated utilizing this novel assay and that neutrophil activity is indeed elevated in IPF and COPD.

BackgroundAsthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3).ObjectiveTo delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response.MethodsThe serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test.ResultsSerum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5).ConclusionC4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

Currently, more knowledge and novel biomarkers are needed to distinguish asthmatic children who will suffer from persistent asthma from those where childhood asthma will resolve. Asthma pathogenesis is characterized by airway remodeling, which involves the degradation of the extracellular matrix (ECM). As a consequence of persistent tissue inflammation, inflammatory cells degrade the ECM by matrix metallo-proteinases (MMPs), which results in the release of the type I collagen fragment (C1M). We investigated if the neoepitope C1M measured in serum of asthmatic children was able to predict persistent asthma in adolescence/young adulthood. Serum from 387 children with asthma was collected in 2003-2005 (mean age 6.3yrs). Follow-up was performed in 2016-2017 (mean age 18.2yrs) where 169 participants had persistent asthma and 218 did not have current asthma. Serum C1M was assessed by a competitive ELISA at baseline and follow-up. Children with persistent asthma at follow-up had increased serum C1M levels at baseline compared to children with no current asthma (P=0.0456). The percentage change in C1M levels from baseline to follow-up was increased in children with no current asthma compared to children with persistent asthma (P=0.005). No difference in serum C1M levels was observed between children with persistent asthma and children with no current asthma at follow up. These data indicate that increased MMP degradation of type I collagen occurs in children with persistent asthma compared to those in which asthma resolves. Serum C1M could potentially be a biomarker for early identification of children with persistent asthma. But changes in C1M over time may reflect that tissue remodeling is affected by age.

Background: Extracellular matrix (ECM) remodeling is a key component of idiopathic pulmonary fibrosis (IPF). Biomarkers reflecting formation of type III (PRO-C3) and type VI (PRO-C6) collagen may provide valuable information on disease progression. We have investigated whether these biomarkers are related to progression of disease and death in IPF patients. Methods: 184 IPF patients from the prospective Danish PFBIO-cohort had biomarkers measured at the time of diagnosis, before initiation of antifibrotic treatment, and were followed for up to 3 years. Progression was defined as FVC decline > 10% and/or DLCO decline > 15% at any time. Results: IPF patients had higher levels of PRO-C3 and PRO-C6 compared to the normal range. A logistic regression showed baseline PRO-C3 levels to be associated with a combined endpoint of progression or death at 6 months (p=0.005) and 12 months (p=0.048). Baseline PRO-C6 levels were associated with progression or death at 6 months (p=0.031). In Cox proportional hazards analysis, baseline PRO-C3 levels were associated with an increased mortality during follow-up (HR: 2.32, p=0.003), while this was not found for baseline PRO-C6 levels (HR: 1.8, p=0.192). Conclusions: PRO-C3 and, to a lesser extent, PRO-C6 measured at the time of diagnosis were able to predict disease progression and death for IPF patients. These markers of ECM formation could be useful to identify the patient in most need of treatment.

Background: Chronic Obstructive Pulmonary Disease (COPD) is characterized by abnormal epithelial repair resulting in a hypercoagulable state, intra-alveolar accumulation of fibrin and basement membrane destruction. The aim of this study was to investigate if serological assessment of these processes were associated with COPD progression. Methods: Plasmin-mediated degradation of cross-linked fibrin (X-FIB), an end-product of fibrinogen, and MMP-mediated degradation of type IV collagen (C4Ma3) were assessed in plasma of 984 COPD subjects from the ECLIPSE cohort. Biomarker data were dichotomized into high vs low by ROC analysis. Cox proportional hazards regression assessed predictive value of the biomarkers alone and in combination for all-cause mortality after 2 years. Results: High levels of plasma X-FIB and C4Ma3 were associated with increased risk of mortality as compared with low levels, with hazard ratios of 4.88 (95% CI 2.28-10.47) and 4.68 (1.40-15.71), respectively. The combination of markers of fibrin cross-linking and collagen IV degradation increased the hazard ratio to 7.35 (3.48-15.55). Additionally, X-FIB was associated with emphysema while C4Ma3 was associated with acute exacerbations. Conclusions: Serological assessments of lung remodeling related to the abnormal epithelial repair in COPD are associated with a poor prognosis. The combination of two different aspects of COPD pathology, elevated wound healing and basement membrane destruction, may improve prediction. Method: GSK (ECLIPSE; SCO104960, NCT00292552); The Danish Agency for Science, Technology, and Innovation; The Danish Research Foundation; NIHR Manchester Biomedical Research Centre.

Background Lung epithelial damage, activation of the wound healing cascade, and remodeling of the extracellular matrix (ECM) play a major role in chronic obstructive pulmonary disease (COPD). The pro-peptide of type VI collagen has been identified as the hormone endotrophin. Endotrophin has been shown to promote fibrosis and inflammation, whereas von Willebrand factor (VWF) is a crucial part of wound healing initiation. Here, we assessed the released and activated form of VWF and endotrophin, the pro-peptide of type VI collagen, serologically to investigate their association with mortality in COPD subjects alone or in combination. Methods One thousand COPD patients with 3 years of clinical follow-up from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) cohort were included. Serum and heparin plasma were collected at 6 months and 1 year, respectively. Competitive ELISA utilizing specific monoclonal antibodies assessed endotrophin/type VI collagen formation (PRO-C6), VWF release (VWF-N), and activated VWF (VWF-A). Biomarker levels were dichotomized into high and low as defined by receiver operating characteristic (ROC) curves based on mortality data. Kaplan-Meier analysis was used to determine hazard ratios for all-cause mortality for biomarkers alone or in combination. Results High levels of PRO-C6, VWF-A, and VWF-N have previously been shown to be individually associated with a higher risk of mortality with hazard ratios of 5.6 (95% CI 2.4–13.1), 3.7 (1.8–7.6), and 4.6 (2.2–9.6), respectively. The hazard ratios increased when combining the biomarkers: PRO-C6*VWFA 8.8 (2.8–27.7) and PRO-C6*VWFN 13.3 (5.6–32.0). Notably, PRO-C6*VWF-N increased more than 2-fold. Conclusion We demonstrated that by combining two pathological relevant aspects of COPD, tissue remodeling, and wound healing, the predictive value of biomarkers for mortality increased notably.

Background Chronic obstructive pulmonary disease (COPD) is characterized by abnormal epithelial repair resulting in a hypercoagulable state with intra-alveolar accumulation of fibrin and alveolar basement membrane destruction. This study aimed to investigate if the combination of two serological biomarkers evaluating these pathological processes could improve the prediction of mortality risk compared to single biomarkers. Methods Matrix metalloproteinase-mediated degradation of the type IV collagen α3 chain (C4Ma3), located in the alveolar basement membrane, and plasmin-mediated degradation of crosslinked fibrin (X-FIB), an end-product of fibrinogen, were assessed serologically in a subset of the ECLIPSE cohort (n = 982). Biomarker data were dichotomized into high versus low at the median. Cox regression and Kaplan-Meier curves were used to analyze the predictive value of having one or two high biomarkers for all-cause mortality over two years. Results COPD participants with high levels of two biomarkers were at significantly higher risk of all-cause mortality with a hazard ratio of 7.66 (95% CI 1.75–33.48; p = 0.007) while participants with one high biomarker were not at significantly higher risk (HR 3.79 [95% CI 0.85–16.94]; p = 0.08). Conclusions A combination of serological biomarkers of alveolar basement membrane destruction and clot resolution was predictive of all-cause mortality in COPD. The combination of two different pathological aspects may strengthen prognostic accuracy and could be used in conjunction with clinical assessment to guide treatment decisions.

Background Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. Methods Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-β1 with or without treatment with an ALK5/TGF-β1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression. Results TGF-β1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-β1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-β1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. Conclusions TGF-β1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.

Elastin is a signature protein of lungs. Increased elastin turnover driven by altered proteolytic activity is an important part of lung tumorigenesis. Elastin-derived fragments have been shown to be pro-tumorigenic, however, little is known regarding the biomarker potential of such elastin fragments. Here, we present an elastin turnover profile by non-invasively quantifying five specific elastin degradation fragments generated by different proteases. Elastin fragments were assessed in serum from patients with stage I–IV non-small cell lung cancer (NSCLC) (n = 40) and healthy controls (n = 30) using competitive ELISAs targeting different protease-generated fragments of elastin: ELM12 (generated by matrix metalloproteinase MMP-9 and -12), ELM7 (MMP-7), EL-NE (neutrophil elastase), EL-CG (cathepsin G) and ELP-3 (proteinase 3). ELM12, ELM7, EL-NE and EL-CG were all significantly elevated in NSCLC patients (n = 40) when compared to healthy controls (n = 30) (ELM12, p = 0.0191; ELM7, p

Asthma is a chronic inflammatory disease, characterized by symptoms including increased mucus production, reversible airway obstruction and lung inflammation: all of which are exaggerated during asthma exacerbations. Extracellular matrix remodeling is associated with the release of ECM protein fragments (neo-epitopes) to the circulation. We sought to investigate the relationship between serological assessment of ECM remodeling markers (neo-epitopes) and the level of symptoms in a mouse model of exacerbated asthma. The hypothesis was, that an increase in ECM remodeling would be observed as a consequence of airway inflammation during exacerbations. Balb/C mice were sensitized to ovalbumin (OVA) (i.p.), acute exacerbations were provoked by i.n. instillation of poly-cytidylic-inosinic acid. Markers of matrix metalloproteinase (MMP) degraded collagen type I (C1M), type III (C3M), type IV (C4M), elastin (ELM7), and laminin (LAMa5) were assessed in serum. Analysis-software: JMP13 (SAS). Serum levels of C1M and LAMa5 individually correlated with bronchoalveolar lavage cells (BAL). Furthermore, the increase of airway resistance (C4M rs = -0.42, p

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.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal epithelial repair process that may result in intra-airway accumulation of fibrin. Given that plasma fibrinogen is the only FDA approved biomarker that predicts mortality and COPD exacerbations, we hypothesized that changes in the processing of fibrinogen may provide additional characterization of disease phenotype and COPD progression. Methods: A subpopulation of subjects with COPD, (n = 983) smoker (n = 205) and non-smoker controls (n = 98) were included from The Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort. Two biomarkers that specifically target the thrombin-mediated conversion of fibrinogen into fibrin (PRO-FIB), and plasmin-mediated degradation of cross-linked fibrin (X–FIB) were measured and compared with fibrinogen measurements. Results: X–FIB had a predictive value for two-year mortality, with an adjusted hazard ratio of 1.48 per SD (n = 980; 95% Cl 1.18–1.84; p < 0.0001), and comparable to the fibrinogen hazard ratio of 1.59 per SD (n = 983; 95% Cl 1.29–1.96; p = 0.0003). X–FIB (p < 0.001), fibrinogen (p < 0.0001) and PRO-FIB (p < 0.05) were significantly elevated in symptomatic COPD (mMRC ≥ 2) as compared to asymptomatic COPD. X–FIB was the only biomarker that was associated with emphysema (p < 0.001), and only plasma fibrinogen (p < 0.05) was associated with exacerbations. Conclusion: There is a need for biomarkers to characterize the heterogeneity of COPD, to continuously improve clinical trial design and to identify disease progressors for efficient health care utilization. Each of three fibrinogen biomarkers studied provide information representing distinct aspects of COPD which may be used to characterize disease endotypes and to assess mortality risk in COPD.

Lasse L Langholm,1,2 Sarah Rank Rønnow,1,3 Jannie MB Sand,1 Diana Julie Leeming,1 Ruth Tal-Singer,4 Bruce E Miller,4 Jørgen Vestbo,5 Morten A Karsdal,1 Tina Manon-Jensen1 1Nordic Bioscience A/S, Herlev, Denmark; 2University of Copenhagen, Faculty of Health and Medical Sciences, Department of Biomedical Sciences, Copenhagen, Denmark; 3University of Southern Denmark, The Faculty of Health Science, Odense, Denmark; 4Respiratory Medical Innovation, Value Evidence & Outcomes, GSK R&D, Collegeville, PA, USA; 5Division of Infection, Immunity and Respiratory Medicine, University of Manchester, and Manchester University NHS Foundation Trust, Manchester, UKCorrespondence: Lasse L LangholmNordic Bioscience A/S, Herlev Hovedgade 205-207, Herlev 2730, DenmarkTel +45 4452 5252Fax +45 4452 5251Email lla@nordicbio.comBackground: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and lung tissue deterioration. Given the high vascularity of the lung, von Willebrand factor (VWF), a central component of wound healing initiation, has previously been assessed in COPD. VWF processing, which is crucial for regulating the primary response of wound healing, has not been assessed directly. Therefore, this study aimed to characterize wound healing initiation in COPD using dynamic VWF-processing biomarkers and to evaluate how these relate to disease severity and mortality.Methods: A cross-sectional analysis of plasma samples from the ECLIPSE study collected at year 1 from moderate to very severe COPD subjects (GOLD 2– 4, n=984)was performed. We applied competitive neo-epitope ELISAs specifically targeting the formation of and ADAMTS13-processed form of VWF, VWF-N and VWF-A, respectively.Results: VWF-A and VWF-N were significantly increased (VWF-N, p=0.01; VWF-A, p=0.0001) in plasma of symptomatic (mMRC score ≥ 2) compared to asymptomatic/mild symptomatic COPD subjects. Increased VWF-N and VWF-A levels were specifically associated with emphysema (VWF-N, p< 0.0001) or prior exacerbations (VWF-A, p=0.01). When dichotomized, high levels of both biomarkers were associated with increased risk of all-cause mortality (VWF-N, HR 3.5; VWF-A, HR 2.64).Conclusion: We demonstrate that changes in VWF processing were related to different pathophysiological aspects of COPD. VWF-N relates to the chronic condition of emphysema, while VWF-A was associated with the more acute events of exacerbations. This study indicates that VWF-A and VWF-N may be relevant markers for characterization of disease phenotype and are associated with mortality in COPD.Study Identifier: NCT00292552; GSK study code SCO104960.Keywords: von Willebrand factor processing, COPD, emphysema, exacerbations, increased mortality risk

Aim: Fibrosis is characterized by an altered turnover of extracellular matrix (ECM) proteins. Scar-in-a-Jar (SiaJ) is an in vitro fibroblast model used as a drug screening tool for antifibrotics in lung fibrosis (Chen et al. 2009). In this study, TGF-β activation of the fibroblasts was initiated prior to treatment in order to create a fibrotic environment. The aim was to investigate if it was possible to reverse fibrogenesis by different doses of nintedanib rather than prevent it, since that may be more physiologically relevant. Methods: Primary human healthy lung fibroblasts were cultured in presence of ficoll and stimulated with TGF-β at day 0, 4 and 8. Two doses of nintedanib (1µM and 100 nM) were added at day 4 and 8 in combination with TGF-β. Formation of type I (PRO-C1) and type III (PRO-C3) collagen were assessed in the supernatant using neo-epitope specific ELISAs. Metabolic activity was assessed by AlamarBlue. Results: TGF-β stimulated fibroblasts had 12 and 29 fold higher concentrations of PRO-C1 and PRO-C3 respectively compared to the unstimulated fibroblasts. 1 µM nintedanib was added at day 4 and 8, which decreased PRO-C1 (P=0.0004) and PRO-C3 (P=0.004) at day 12 compared to the untreated fibroblasts. 100 nM nintedanib added at day 4 and 8 showed a reduction of PRO-C1 (P=0.02) at day 12, but no reduction of PRO-C3 was observed compared to untreated fibroblasts. Metabolic activity was slightly decreased by both doses of nintedanib compared to the untreated fibroblasts at day 12. Conclusion: We found that nintedanib was able to reverse fibroblast activation induced by TGF-β stimulation. This modified setup of the SiaJ model may be more pathological relevant for treating rather than preventing fibrogenesis.

Background Identifying subjects with chronic obstructive pulmonary disease (COPD) at high risk of exacerbation and mortality is key to aid individual management of COPD. The only FDA approved blood-based drug development biomarker for patients at high risk of mortality, is plasma fibrinogen. In this study, we benchmarked two biomarkers of basement membrane remodeling, a characteristic of COPD, against plasma fibrinogen alone and as a combination. The biomarkers of basement membrane remodeling are two neoepitopes from of the alpha 3 chain of type IV collagen (COL4A3). Materials and methods COL4A3 degradation was assessed by the biomarkers C4Ma3 and tumstatin (TUM) in year 1 plasma samples in 984 COPD subjects, 95 non-smoking controls and 95 smoking controls from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort. They were measured by competitive ELISA using monoclonal antibodies recognizing two specific MMP-generated cleavage site within COL4A3. The level of fibrinogen was previously assessed in year 1 plasma. Results In COPD subjects, plasma C4Ma3 levels were significantly correlated with plasma fibrinogen levels (0.389 (P

Background: Pulmonary fibrosis (PF) is characterized by excessive extracellular matrix (ECM) remodeling. Clinically validated ECM neoepitopes markers, related to progressive PF, may be useful for the evaluation of potential anti-fibrotic effects. Aim: The aim was to evaluate ECM remodelling (ECMR) in a human ex vivo precision-cut lung slice (PCLS) model. Methods: Human PF tissue was collected from two donors during lung transplantation. Within 24hours, the lungs were processed into PCLSs. The slices were cultured 2 pr/well and in triplicates for 48hours in serum free medium with 1nM-10mM nintedanib or 100pM-1µM mTOR/PI3K inhibitor omipalisib (GSK2126458). Responsiveness was tested using Lipopolysaccaride (LPS) and cytotoxicity using lactate dehydrogenase (LDH). Markers of collagen type I, III and VI formation (P1NP, PRO-C3, PRO-C6) and type III collagen degradation (C3M) were assessed in the supernatants. The subpleural and central lung regions were used and evaluated by hematoxylin and eosin staining. Results: The tissue from both donors was responsive to LPS and no toxicity was seen with the selected compound doses using LDH. P1NP, PRO-C3, and C3M were significantly reduced by 1nM-1µM omipalisib (p

Background: Extracellular matrix (ECM) deposition and stiffening have emerged as an important player in cancer progression. ECM deposition is stimulated by transforming growth factor-beta (TGF-β) that is being widely evaluated as a new therapeutic target in cancer. ECM stiffness is characterized by type III collagen deposition and cross-linking in the tumor microenvironment. Cross-linked type III collagen products reflecting ECM stiffness and TGF-β signaling may therefore have biomarker potential in cancer. By developing an assay to quantify cross-linked type III collagen pro-peptides and applying the “Scar-in-a-jar” (SiaJ) model, we evaluated the impact of TGF-β on this novel biomarker and assessed the biomarker potential in colorectal cancer (CRC). Methods: A monoclonal antibody was raised against the N-terminal pro-peptide of type III collagen and a sandwich ELISA was developed and technically validated. The cross-linked type III collagen product levels were assessed in supernatants from the SiaJ culture model in which primary human healthy lung fibroblasts were cultured up to 12 days in the presence of ficoll and 1ng/ml TGF-β, with or without addition of 1µM ALK-5/type I TGF-β receptor kinase inhibitor (iTGF-β). The pathological relevance of cross-linked type III collagen products was assessed in serum from patients with CRC (n=34) and healthy controls (n=39). Levels were compared using unpaired, two-tailed Mann-Whitney test. Results: The newly developed sandwich ELISA was shown to be technically robust and specific towards cross-linked type III collagen pro-peptides. Stimulating lung fibroblasts with TGF-β induced the production of cross-linked type III collagen with levels increasing up to 22-fold compared to without TGF-β (p=0.029), and iTGF-β reduced this induction. Levels of cross-linked type III collagen were significantly elevated in CRC patients compared to healthy controls (7.0 ng/ml vs. 5.6 ng/ml, p=0.0009). Furthermore, the diagnostic accuracy to separate CRC patients from healthy controls was 72% (p=0.0003, AUC: 0.72) with an odds ratio of 8 (95%Cl=2.4-23.2, p=0.0006) for having CRC. Conclusions: Cross-linked pro-peptides of type III collagen can be quantified in serum from CRC patients as a potential surrogate marker of tissue stiffness and stromal reactivity driving tumor progression. In addition, TGF-β induces increased production of cross-linked type III collagen suggesting that this novel biomarker could potentially interrogate the effect of anti-TGF-β therapy. Future studies are needed to investigate the prognostic and predictive potential. Citation Format: Christina Jensen, Sarah Rønnow, Jannie M. Sand, Lars N. Jørgensen, Morten A. Karsdal, Nicholas Willumsen. Non-invasive quantification of cross-linked type III collagen products - A measure of extracellular matrix stiffness related to TGF-β signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1899.