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INTRODUCTION: The pathogenesis of sarcoidosis involves tissue remodelling mediated by the accumulation of abnormal extracellular matrix, which is partly the result of an imbalance in collagen synthesis, cross-linking and degradation. During this process, collagen fragments or neoepitopes, are released into the circulation. The significance of these circulating collagen neoepitopes in sarcoidosis remains unknown. METHODS: We employed plasma samples from patients with sarcoidosis enrolled in A Case Control Etiologic Study of Sarcoidosis (ACCESS) and Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS), and healthy control patients recruited from the Yale community. Plasma concentrations of type III and VI collagen degradation (C3M and C6M) and formation (PRO-C3 and PRO-C6) were quantified via neoepitope-specific competitive ELISA, and statistical associations were sought with clinical phenotypes. RESULTS: Relative to healthy controls, the plasma of both sarcoidosis cohorts was enriched for C3M and C6M, irrespective of corticosteroid use and disease duration. While circulating collagen neoepitopes were independent of Scadding stage, there was a significant association between multiorgan disease and PRO-C3, PRO-C6 and C3M in the ACCESS cohort; PRO-C3 and C6M displayed this property in GRADS. These findings were unrelated to plasma levels of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. Moreover, PRO-C3 was associated with dermatological disease in both cohorts. DISCUSSION: In two well-characterised sarcoidosis cohorts, we discovered that the plasma is enriched for neoepitopes of collagen degradation (C3M and C6M). In multiorgan disease, there was an association with circulating neoepitopes of type III formation (PRO-C3), perhaps mediated by dermatological sarcoidosis. Further investigation in this arena has the potential to foster new insights into the pathogenic mechanisms of this complex disease.
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BACKGROUND: Coronavirus disease 2019 (COVID-19) is a viral pneumonia that can result in serious respiratory illness. It is associated with extensive systemic inflammation, changes to the lung extracellular matrix, and long-term lung impairment such as interstitial lung disease (ILD). In this study, the aim was to investigate whether tissue remodelling, wound healing, and neutrophil activity is altered in patients with COVID-19 and how these relate to the development of post-COVID ILD. METHOD: Serum samples were collected from 63 patients three months after discharge as part of the Research Evaluation Alongside Clinical Treatment study in COVID-19 (REACT COVID-19), 10 of whom developed ILD, and 16 healthy controls. Samples were quantified using neo-epitope specific biomarkers reflecting tissue stiffness and formation (PC3X, PRO-C3, and PRO-C6), tissue degradation (C1M, C3M, and C6M), wound healing (PRO-FIB and X-FIB), and neutrophil activity (CPa9-HNE and ELP-3). RESULTS: Mean serum levels of PC3X (p < 0.0001), PRO-C3 (p = 0.002), C3M (p = 0.009), PRO-FIB (p < 0.0001), CPa9-HNE (p < 0.0001), and ELP-3 (p < 0.0001) were significantly elevated in patients with COVID-19 compared to healthy controls. Moreover, PC3X (p = 0.023) and PRO-C3 (p = 0.032) were significantly elevated in post-COVID ILD as compared to COVID-19. CONCLUSION: Serological biomarkers reflecting type III collagen remodelling, clot formation, and neutrophil activity were significantly elevated in COVID-19 and type III collagen formation markers were further elevated in post-COVID ILD. The findings suggest an increased type III collagen remodelling in COVID-19 and warrants further investigations to assess the potential of tissue remodelling biomarkers as a tool to identify COVID-19 patients at high risk of developing ILD.
Subject(s)
Biomarkers , COVID-19 , Lung Diseases, Interstitial , SARS-CoV-2 , Humans , COVID-19/complications , COVID-19/blood , Male , Biomarkers/blood , Female , Lung Diseases, Interstitial/blood , Lung Diseases, Interstitial/physiopathology , Middle Aged , Aged , Wound Healing , Case-Control Studies , Neutrophils , AdultABSTRACT
Fibrosis, driven by fibroblast activities, is an important contributor to morbidity and mortality in most chronic diseases. Endotrophin, a signaling molecule derived from processing of type VI collagen by highly activated fibroblasts, is involved in fibrotic tissue remodeling. Circulating levels of endotrophin have been associated with an increased risk of mortality in multiple chronic diseases. We conducted a systematic literature review collecting evidence from original papers published between 2012 and January 2023 that reported associations between circulating endotrophin (PROC6) and mortality. Cohorts with data available to the study authors were included in an Individual Patient Data (IPD) meta-analysis that evaluated the association of PROC6 with mortality (PROSPERO registration number: CRD42023340215) after adjustment for age, sex and BMI, where available. In the IPD meta-analysis including sixteen cohorts of patients with different non-communicable chronic diseases (NCCDs) (N = 15,205) the estimated summary hazard ratio for 3-years all-cause mortality was 2.10 (95 % CI 1.75-2.52) for a 2-fold increase in PROC6, with some heterogeneity observed between the studies (I2=70 %). This meta-analysis is the first study documenting that fibroblast activities, as quantified by circulating endotrophin, are independently associated with mortality across a broad range of NCCDs. This indicates that, irrespective of disease, interstitial tissue remodeling, and consequently fibroblast activities, has a central role in adverse clinical outcomes, and should be considered with urgency from drug developers as a target to treat.
Subject(s)
Biomarkers , Humans , Chronic Disease , Biomarkers/blood , Collagen Type VI/blood , Collagen Type VI/metabolism , Collagen Type VI/genetics , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Peptide FragmentsABSTRACT
Fibrosis is a common feature of more than 50 different diseases and the cause of more than 35% of deaths worldwide, of which liver, kidney, skin, heart and, recently, lungs are receiving the most attention. Tissue changes, resulting in loss of organ function, are both a cause and consequence of disease and outcome. Fibrosis is caused by an excess deposition of extracellular matrix proteins, which over time results in impaired organ function and organ failure, and the pathways leading to increased fibroblast activation are many. This narrative review investigated the common denominator of fibrosis, fibroblasts, and the activation of fibroblasts, in response to excess energy consumption in liver, kidney, heart, skin and lung fibrosis. Fibroblasts are the main drivers of organ function loss in lung, liver, skin, heart and kidney disease. Fibroblast activation in response to excess energy consumption results in the overproduction of a range of collagens, of which types I, III and VI seem to be the essential drivers of disease progression. Fibroblast activation may be quantified in serum, enabling profiling and selection of patients. Activation of fibroblasts results in the overproduction of collagens, which deteriorates organ function. Patient profiling of fibroblast activities in serum, quantified as collagen production, may identify an organ death trajectory, better enabling identification of the right treatment for use in different metabolic interventions. As metabolically activated patients have highly elevated risk of kidney, liver and heart failure, it is essential to identify which organ to treat first and monitor organ status to correct treatment regimes. In direct alignment with this, it is essential to identify the right patients with the right organ deterioration trajectory for enrolment in clinical studies.
Subject(s)
Fibroblasts , Fibrosis , Metabolic Syndrome , Humans , Fibroblasts/metabolism , Metabolic Syndrome/metabolism , Sclerosis , Kidney Diseases/physiopathology , Collagen/metabolismABSTRACT
BACKGROUND: Sepsis is associated with high morbidity and mortality, primarily due to systemic inflammation-induced tissue damage, resulting organ failure, and impaired recovery. Regulated extracellular matrix (ECM) turnover is crucial for maintaining tissue homeostasis in health and in response to disease-related changes in the tissue microenvironment. Conversely, uncontrolled turnover can contribute to tissue damage. Systemic Inflammation is implicated to play a role in the regulation of ECM turnover, but the relationship between the two is largely unclear. METHODS: We performed an exploratory study in 10 healthy male volunteers who were intravenously challenged with 2 ng/kg lipopolysaccharide (LPS, derived from Escherichia coli) to induce systemic inflammation. Plasma samples were collected before (T0) and after (T 1 h, 3 h, 6 h and 24 h) the LPS challenge. Furthermore, plasma was collected from 43 patients with septic shock on day 1 of ICU admission. Circulating neo-epitopes of extracellular matrix turnover, including ECM degradation neo-epitopes of collagen type I (C1M), type III (C3M), type IV (C4Ma3), and type VI (C6M), elastin (ELP-3) and fibrin (X-FIB), as well as the ECM synthesis neo-epitopes of collagen type III (PRO-C3), collagen type IV (PRO-C4) and collagen type VI (PRO-C6) were measured by ELISA. Patient outcome data were obtained from electronic patient records. RESULTS: Twenty-four hours after LPS administration, all measured ECM turnover neo-epitopes, except ELP-3, were increased compared to baseline levels. In septic shock patients, concentrations of all measured ECM neo-epitopes were higher compared to healthy controls. In addition, concentrations of C6M, ELP-3 and X-FIB were higher in patients with septic shock who ultimately did not survive (N = 7) compared to those who recovered (N = 36). CONCLUSION: ECM turnover is induced in a model of systemic inflammation in healthy volunteers and was observed in patients with septic shock. Understanding interactions between systemic inflammation and ECM turnover may provide further insight into mechanisms underlying acute and persistent organ failure in sepsis.
Subject(s)
Sepsis , Shock, Septic , Humans , Male , Lipopolysaccharides , Extracellular Matrix , Epitopes , Escherichia coliABSTRACT
Neutrophil activation can release neutrophil extracellular traps (NETs) in acute inflammation. NETs result in the release of human neutrophil elastase (HNE) and calprotectin, where the former can degrade the latter and generate protein fragments associated with neutrophil activity. We investigated this in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) using the novel neoepitope biomarker CPa9-HNE, quantifying a specific HNE-mediated fragment of calprotectin in serum. CPa9-HNE was compared to total calprotectin. Initially, CPa9-HNE was measured in healthy (n = 39), COPD (n = 67), and IPF (n = 16) serum using a neoepitope-specific competitive enzyme-linked immunosorbent assay. Then, a head-to-head comparison of CPa9-HNE and total calprotectin, a non-neoepitope, was conducted in healthy (n = 19), COPD (n = 25), and IPF (n = 19) participants. CPa9-HNE levels were significantly increased in COPD (p < 0.0001) and IPF subjects (p = 0.0001) when compared to healthy participants. Additionally, CPa9-HNE distinguished IPF (p < 0.0001) and COPD (p < 0.0001) from healthy participants more effectively than total calprotectin for IPF (p = 0.0051) and COPD (p = 0.0069). Here, CPa9-HNE also distinguished IPF from COPD (p = 0.045) participants, which was not observed for total calprotectin (p = 0.98). Neutrophil activity was significantly higher, as assessed via serum CPa9-HNE, for COPD and IPF compared to healthy participants. Additionally, CPa9-HNE exceeded the ability of non-neoepitope calprotectin serum measurements to separate healthy from lung disease and even COPD from IPF participants, indicating that neutrophil activity is essential for both COPD and IPF.
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BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive extracellular matrix (ECM) remodeling, herein ECM degradation. Fibronectin (FN) is an important component of the ECM that is produced by multiple cell types, including fibroblasts. Extra domain B (EDB) is specific for a cellular FN isoform which is found in the ECM. We sought to develop a non-invasive test to investigate whether matrix metalloproteinase 8 (MMP-8) degradation of EDB in cellular FN results in a specific protein fragment that can be assessed serologically and if levels relate to pulmonary fibrosis. METHOD: Cellular FN was cleaved in vitro by MMP-8 and a protein fragment was identified by mass spectrometry. A monoclonal antibody (mAb) was generated, targeting a neo-epitope originating from EDB in cellular FN. Utilizing this mAb, a neo-epitope specific enzyme-linked immunosorbent assay (FN-EDB) was developed and technically validated. Serum FN-EDB was assessed in an IPF cohort (n = 98), registered at clinicaltrials.gov (NCT02818712), and in healthy controls (n = 35). RESULTS: The FN-EDB assay had high specificity for the MMP-8 degraded neo-epitope and was technically robust. FN-EDB serum levels were not influenced by age, sex, ethnicity, or BMI. Moreover, FN-EDB serum levels were significantly higher in IPF patients (median 31.38 [IQR 25.79-46.84] ng/mL) as compared to healthy controls (median 28.05 [IQR 21.58-33.88] ng/mL, p = 0.023). CONCLUSION: We developed the neo-epitope specific FN-EDB assay, a competitive ELISA, as a tool for serological assessment of MMP-8 mediated degradation of EDB in cellular FN. This study indicates that degradation of EDB in cellular FN is elevated in IPF and warrants further investigation.
Subject(s)
Pulmonary Fibrosis , Humans , Matrix Metalloproteinase 8 , Fibronectins/chemistry , Fibronectins/metabolism , Epitopes , Antibodies, Monoclonal , BiomarkersABSTRACT
Systemic Sclerosis (SSc) hallmark is skin fibrosis, but up to 80% of the patients have fibrotic involvement in the pulmonary system. Antifibrotic drugs which have failed in a general SSc population have now been approved in patients with SSc-associated interstitial lung disease (ILD). This indicates that the fibrotic progression and regulation of fibroblasts likely depend on local factors specific to the tissue type. This study investigated the difference between dermal and pulmonary fibroblasts in a fibrotic setting, mimicking the extracellular matrix. Primary healthy fibroblasts were grown in a crowded environment and stimulated with TGF-ß1 and PDGF-AB. The viability, morphology, migration capacity, extracellular matrix formation, and gene expression were assessed: TGF-ß1 only increased the viability in the dermal fibroblasts. PDGF-AB increased the migration capacity of dermal fibroblasts while the pulmonary fibroblasts fully migrated. The morphology of the fibroblasts was different without stimulation. TGF-ß1 increased the formation of type III collagen in pulmonary fibroblasts, while PDGF-AB increased it in dermal fibroblasts. The gene expression trend of type VI collagen was the opposite after PDGF-AB stimulation. The fibroblasts exhibit different response profiles to TGF-ß1 and PDGF-AB; this suggests that drivers of fibrosis are tissue-dependent, which needs to be considered in drug development.
Subject(s)
Scleroderma, Systemic , Transforming Growth Factor beta1 , Humans , Transforming Growth Factor beta1/pharmacology , Transforming Growth Factor beta1/metabolism , Cells, Cultured , Fibrosis , Lung/pathology , Scleroderma, Systemic/metabolism , Fibroblasts/metabolism , Skin/metabolismABSTRACT
BACKGROUND: Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone. METHODS: Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants. RESULTS: In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied. CONCLUSIONS: This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.
Subject(s)
Idiopathic Pulmonary Fibrosis , Pulmonary Fibrosis , Animals , Biomarkers , Bleomycin/toxicity , Collagen Type III/metabolism , Complement C3/pharmacology , Idiopathic Pulmonary Fibrosis/chemically induced , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Indoles , Lung/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/pathology , RatsABSTRACT
Cardiovascular diseases are common in patients with chronic obstructive pulmonary disease (COPD). Clot formation and resolution secondary to systemic inflammation may be a part of the explanation. The aim was to determine whether biomarkers of clot formation (products of von Willebrand Factor formation and activation) and clot resolution (product of fibrin degeneration) during COPD exacerbation predicted major cardiovascular events (MACE). The cohort was based on clinical data and biobank plasma samples from a trial including patients admitted with an acute exacerbation of COPD (CORTICO-COP). Neo-epitope biomarkers of formation and the activation of von Willebrand factor (VWF-N and V-WFA, respectively) and cross-linked fibrin degradation (X-FIB) were assessed using ELISAs in EDTA plasma at the time of acute admission, and analyzed for time-to-first MACE within 36 months, using multivariable Cox proportional hazards models. In total, 299/318 participants had samples available for analysis. The risk of MACE for patients in the upper quartile of each biomarker versus the lower quartile was: X-FIB: HR 0.98 (95% CI 0.65-1.48), VWF-N: HR 1.56 (95% CI 1.07-2.27), and VWF-A: HR 0.78 (95% CI 0.52-1.16). Thus, in COPD patients with an acute exacerbation, VWF-N was associated with future MACE and warrants further studies in a larger population.
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BACKGROUND: Asthma is a heterogeneous disease; therefore, biomarkers that can assist in the identification of subtypes and direct therapy are highly desirable. Asthma is a chronic inflammatory disease that leads to changes in the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) degradation causing fragments of type I collagen that is released into circulation. OBJECTIVE: Here, we asked if MMP-generated type I collagen (C1M) was associated with subtypes of asthma. METHODS: C1M was serologically assessed at baseline in the adult participants of the All Age Asthma study (ALLIANCE) (n = 233), and in The Prospective Epidemiological Risk Factor study (PERF) (n = 283). In addition, C1M was assessed in mice sensitized to ovalbumin (OVA) and challenged with OVA aerosol. C1M was evaluated in mice with and without acute neutrophilic inflammation provoked by poly(cytidylic-inosinic) acid and mice treated with CP17, a peptide inhibiting neutrophil accumulation. RESULTS: Serum C1M was significantly increased in asthmatics compared to healthy controls (p = 0.0005). We found the increased C1M levels in asthmatics were related to blood neutrophil and body mass index (BMI) in the ALLIANCE cohort, which was validated in the PERF cohort. When patients were stratified into obese (BMI > 30) asthmatics with high neutrophil levels and uncontrolled asthma, this group had a significant increase in C1M compared to normal-weight (BMI < 25) asthmatics with low neutrophil levels and controlled asthma (p = 0.0277). C1M was significantly elevated in OVA mice with acute neutrophilic inflammation compared to controls (P = 0.0002) and decreased in mice treated with an inhibitor of neutrophil infiltration (p = 0.047). CONCLUSION & CLINICAL RELEVANCE: C1M holds the potential to identify a subtype of asthma that relates to severity, obesity, and high neutrophils. These data suggest that C1M is linked to a subtype of overall inflammation, not only derived from the lung. The link between C1M and neutrophils were further validated in in vivo model. TRIAL REGISTRATION: (ALLIANCE, NCT02419274 ).
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BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease with limited treatment options. A phase 2 trial (NCT01766817) showed that twice-daily treatment with BMS-986020, a lysophosphatidic acid receptor 1 (LPA1) antagonist, significantly decreased the slope of forced vital capacity (FVC) decline over 26 weeks compared with placebo in patients with IPF. This analysis aimed to better understand the impact of LPA1 antagonism on extracellular matrix (ECM)-neoepitope biomarkers and lung function through a post hoc analysis of the phase 2 study, along with an in vitro fibrogenesis model. METHODS: Serum levels of nine ECM-neoepitope biomarkers were measured in patients with IPF. The association of biomarkers with baseline and change from baseline FVC and quantitative lung fibrosis as measured with high-resolution computed tomography, and differences between treatment arms using linear mixed models, were assessed. The Scar-in-a-Jar in vitro fibrogenesis model was used to further elucidate the antifibrotic mechanism of BMS-986020. RESULTS: In 140 patients with IPF, baseline ECM-neoepitope biomarker levels did not predict FVC progression but was significantly correlated with baseline FVC and lung fibrosis measurements. Most serum ECM-neoepitope biomarker levels were significantly reduced following BMS-986020 treatment compared with placebo, and several of the reductions correlated with FVC and/or lung fibrosis improvement. In the Scar-in-a-Jar in vitro model, BMS-986020 potently inhibited LPA1-induced fibrogenesis. CONCLUSIONS: BMS-986020 reduced serum ECM-neoepitope biomarkers, which were previously associated with IPF prognosis. In vitro, LPA promoted fibrogenesis, which was LPA1 dependent and inhibited by BMS-986020. Together these data elucidate a novel antifibrotic mechanism of action for pharmacological LPA1 blockade. Trial registration ClinicalTrials.gov identifier: NCT01766817; First posted: January 11, 2013; https://clinicaltrials.gov/ct2/show/NCT01766817 .
Subject(s)
Collagen/drug effects , Idiopathic Pulmonary Fibrosis/drug therapy , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Respiratory System Agents/administration & dosage , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Collagen/metabolism , Epitopes/blood , Female , Humans , Idiopathic Pulmonary Fibrosis/pathology , In Vitro Techniques , Male , Middle Aged , Models, Biological , Vital Capacity/drug effectsABSTRACT
BACKGROUND: Asthma 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). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The 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. RESULTS: Serum 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). CONCLUSION: C4Ma3 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.
Subject(s)
Antibodies, Anti-Idiotypic/therapeutic use , Aspergillosis, Allergic Bronchopulmonary , Asthma , Autoantigens/metabolism , Collagen Type IV/metabolism , Cystic Fibrosis , Adult , Animals , Asthma/drug therapy , Child , Humans , Mice , Omalizumab/therapeutic useABSTRACT
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-ß1 with or without treatment with an ALK5/TGF-ß1 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-ß1 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-ß1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-ß1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. CONCLUSIONS: TGF-ß1 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.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cicatrix/chemically induced , Cicatrix/metabolism , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Protein Kinase Inhibitors/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Biomarkers/metabolism , Cells, Cultured , Cicatrix/drug therapy , Collagen/antagonists & inhibitors , Collagen/metabolism , Drug Evaluation, Preclinical/methods , Extracellular Matrix/drug effects , Fibroblasts/drug effects , Fibronectins/antagonists & inhibitors , Fibronectins/metabolism , Fibrosis/chemically induced , Fibrosis/drug therapy , Fibrosis/metabolism , Humans , Indoles/antagonists & inhibitors , Indoles/metabolism , Protein Kinase Inhibitors/therapeutic use , Pyridones/antagonists & inhibitors , Pyridones/metabolism , Transforming Growth Factor beta1/toxicityABSTRACT
PURPOSE OF REVIEW: 'Biomarkers of remodeling' represent a loose collection of features referring to several biological adaptations of the lung to cope with stressing factors. In addition, remodel-'ing' infers a dynamic process that would require a spatiotemporal resolution. This review focuses on different aspects of remodeling in pediatric and adult care. RECENT FINDINGS: This review will cover aspects of pediatric remodeling, adult remodeling and techniques and procedures to adequately assess remodeling across different age spectra. In pediatrics, the onset and first features of remodeling are discussed and the continuation into adolescence is addressed. For adults, this review addresses predominant features of remodeling throughout the adult life span and whether there are currently interventions available to treat or reverse remodeling. SUMMARY: The term 'remodeling' is often referred to via biomarkers that reflect the endstage of a process, although it rather reflects a continuous process starting in childhood and progressing to all age-levels in patients with asthma. Hence, only few biomarkers or surrogates are able to 'capture' its spatiotemporal component, and hardly any are ready for routine use in clinical practice. Given the clinical impact of the remodeling processes, new biomarkers are needed to adequately treat patients with asthma and objectively monitor treatment response beyond symptom control and lung function.
Subject(s)
Airway Remodeling/physiology , Asthma , Biomarkers , Adult , Age Factors , Asthma/metabolism , Asthma/physiopathology , Child , Disease Progression , HumansABSTRACT
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, (nâ¯=â¯983) smoker (nâ¯=â¯205) and non-smoker controls (nâ¯=â¯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 (nâ¯=â¯980; 95% Cl 1.18-1.84; pâ¯<â¯0.0001), and comparable to the fibrinogen hazard ratio of 1.59 per SD (nâ¯=â¯983; 95% Cl 1.29-1.96; pâ¯=â¯0.0003). X-FIB (pâ¯<â¯0.001), fibrinogen (pâ¯<â¯0.0001) and PRO-FIB (pâ¯<â¯0.05) were significantly elevated in symptomatic COPD (mMRCâ¯≥â¯2) as compared to asymptomatic COPD. X-FIB was the only biomarker that was associated with emphysema (pâ¯<â¯0.001), and only plasma fibrinogen (pâ¯<â¯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.
Subject(s)
Fibrinogen/analysis , Pulmonary Disease, Chronic Obstructive/diagnosis , Biomarkers/blood , Cohort Studies , Forecasting , Humans , Pulmonary Disease, Chronic Obstructive/mortalityABSTRACT
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 < 0.0001)). Cox proportional-hazards regression adjusted for relevant confounders showed that high levels of plasma C4Ma3, but not TUM, were related to a higher risk of mortality (hazard ratio 5.12 (95% CI 2.28-11.50), P < 0.0001). High levels of plasma fibrinogen were not associated with all-cause mortality in this subpopulation, contradictory to published results. Whereas plasma C4Ma3 multiplied by fibrinogen showed to be related to a higher risk of mortality (hazard ratio 5.74 (95% CI 2.65-12.41), P < 0.0001). Plasma C4Ma3 levels were related to the number of hospitalizations due to COPD exacerbations in the year before study start (P = 0.0375). Fibrinogen levels were related to hospitalized exacerbations prior to study start (P = 0.0058) and were also related to future exacerbations (P < 0.0001). CONCLUSION: We compared herein fibrinogen, C4Ma3 and TUM as biomarkers for COPD prognosis. Fibrinogen was related to future exacerbation, whereas C4Ma3 and the combination of C4Ma3 with fibrinogen were superior to fibrinogen alone in predicting mortality. This pilot study suggests that the assessment of plasma C4Ma3 could be important for identifying COPD patients with a poor prognosis. TRIAL REGISTRATION: NCT00292552 , GSK Study No. SCO104960.
Subject(s)
Autoantigens/blood , Collagen Type IV/blood , Fibrinogen/metabolism , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/mortality , Aged , Biomarkers/blood , Cohort Studies , Female , Hospitalization/trends , Humans , Longitudinal Studies , Male , Middle Aged , Mortality/trends , Pilot Projects , Prognosis , Prospective Studies , Pulmonary Disease, Chronic Obstructive/diagnosisABSTRACT
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 < 0.0001) and 1.31 (95%CI 1.10-1.57, p = 0.0029), respectively. Assessing the elastin degradome demonstrated that specific elastin degradation fragments have potential utility as biomarkers identifying subtypes of COPD patients at risk of poor prognosis and supports further exploration in confirmatory studies.
Subject(s)
Biomarkers/metabolism , Elastin/genetics , Lung/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Cohort Studies , Disease Progression , Elastin/ultrastructure , Female , Humans , Lung/pathology , Male , Matrix Metalloproteinase 12/genetics , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 9/genetics , Middle Aged , Myeloblastin/genetics , Proportional Hazards Models , Proteolysis , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathologyABSTRACT
Chronic obstructive pulmonary disease (COPD) is characterized by high levels of protease activity leading to degradation of elastin followed by loss of elasticity of the lung and the development of emphysema. Elastin is an essential structural component of the lung parenchyma to support the expansion and recoil of the alveoli during breathing. The lung extracellular matrix is vulnerable to pathological structural changes upon upregulation of serine proteases, including cathepsin G (CG) and proteinase 3 (PR3). In this study, we explored the diagnostic features of elastin neo-epitopes generated by CG and PR3. Two novel competitive enzyme-linked immunosorbent assays (ELISA) measuring CG and PR3 generated elastin fragments (EL-CG and ELP-3 respectively) were developed for assessment in serum. Both assays were technically robust and biologically validated in serum from patients with COPD. Serological levels of both elastin fragments were significantly elevated in patients with COPD compared to healthy controls. These data suggest that EL-CG and ELP-3 may serve as plausible biologic markers of destructive changes in COPD.
Subject(s)
Cathepsin G/metabolism , Elastin/metabolism , Extracellular Matrix/metabolism , Lung/metabolism , Myeloblastin/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Aged , Elastin/biosynthesis , Elastin/blood , Enzyme-Linked Immunosorbent Assay , Female , Healthy Volunteers , Humans , Male , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/pathologyABSTRACT
A high level of extracellular matrix (ECM) turnover characterizes several lung diseases with fibrotic features. Type III collagen is one of the most abundant collagens in lung parenchyma, and cathepsins play a role in lung pathology, being responsible for tissue remodeling. In this study, we explore the diagnostic features of neo-epitope fragments of type III collagen generated by cathepsins that could reflect the pathological tissue turnover in patients with different diseases. A novel enzyme-linked immunosorbent assay (ELISA) measuring cathepsins B, L, S and K -generated type III collagen fragments (C3C) was developed for assessment in serum and plasma. The assay was biologically validated in serum from patients with chronic obstructive pulmonary disease (COPD). Serological levels of C3C were significantly elevated in patients with COPD compared to healthy controls (p = 0.0006). Levels of C3C in serum and heparin plasma of COPD patients had a highly significant correlation (R2 = 0.86, p<0.0001). The data suggests that the C3C fragment is elevated in patients with COPD compared to healthy controls.