Increased Collagen Turnover Is a Feature of Fibromuscular Dysplasia and Associated With Hypertrophic Radial Remodeling: A Pilot, Urine Proteomic Study

Fibromuscular dysplasia (FMD), a nonatherosclerotic, noninflammatory disease of medium-sized arteries, is an underdiagnosed disease. We investigated the urinary proteome and developed a classifier for discrimination of FMD from healthy controls and other diseases. We further hypothesized that urinary proteomics biomarkers may be associated with alterations in medium-sized, but not large artery geometry and mechanics. The study included 33 patients with mostly multifocal, renal FMD who underwent in depth arterial exploration using ultra-high frequency ultrasound. The cohort was separated in a training set of 23 patients with FMD from Belgium and an independent test set of 10 patients with FMD from Italy. For each set, controls matched 2:1 were selected from the Human Urinary Proteome Database. The specificity of the classifier was tested in 700 additional controls from general population studies, patients with chronic kidney disease (n=66) and coronary artery disease (n=31). Three hundred thirty-five urinary peptides, mostly related to collagen turnover, were identified in the training cohort and combined into a classifier. When applying in the test cohort, the area under the receiver operating characteristic curve was 1.00, 100% specificity at 100% sensitivity. The classifier maintained a high specificity in additional controls (98.3%), patients with chronic kidney (90.9%) and coronary artery (96.8%) diseases. Furthermore, in patients with FMD, the proteomic score was positively associated with radial wall thickness and wall cross-sectional area. In conclusion, a proteomic score has the potential to discriminate between patients with FMD and controls. If confirmed in a wider and more diverse cohort, these findings may pave the way for a noninvasive diagnostic test of FMD.

Effects of Alvelestat, an Oral Neutrophil Elastase Inhibitor, on Elevated Elastase and Collagen Turnover Biomarkers in Patients with Bronchiolitis Obliterans Syndrome after Hematopoietic Cell Transplantation

Introduction Bronchiolitis Obliterans Syndrome (BOS) is a rare but devastating complication of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) and is associated with a high morbidity and mortality. There is a dearth of treatment options for BOS and new strategies are needed. Airway neutrophilia is a hallmark of BOS, even in the absence of infection, and neutrophil elastase (NE) is an enzyme that has been implicated in the pathogenesis of BOS. We are conducting a phase 1b study of an oral NE inhibitor, alvelestat, in patients with BOS after HCT . Biomarkers, including the elastin breakdown peptides desmosine/isodesmosine (DES/IDES), and stimulated neutrophil elastase assess direct effect on NE activity. Neo-epitope by-products of collagen type 3 and 6 synthesis (PRO-C3 and PRO-C6) and degradation (C3M and C6M) are measured as biomarkers of fibrosis/tissue modelling Methods Patients age ≥18 years with BOS and chronic GVHD after HCT were recruited to the National Cancer Institute protocol (NCT02669251). This phase 1 study had 2 parts: 8-week intra-patient dose escalation period, followed by a continuation period that allowed for up to 6 months of treatment. Alvelestat was given orally starting at 60mg twice daily, increased every 2 weeks to 120mg twice daily, 180mg twice daily, and finally 240mg twice daily. Peripheral blood samples were collected at baseline and at the end of each dose-escalation stage. Plasma DES/IDES was measured by isotopic dilution liquid chromatography-tandem mass spectrometry (Huang et al Thorax 2012;67:502-508). Ex vivo zymosan stimulated neutrophil elastase activity was measured by ProteaseTag® immunoassay (ProAxsis Ltd, Northern Ireland). PRO-C3, PRO-C6, C3M and C6M were measured by competitive ELISA. (Nordic Biosciences, Denmark). Results are presented as Mean and Standard Error Mean (SEM). Results Between 2016 and 2018, 7 patients were enrolled (3 men and 4 women). Median FEV 1 after bronchodilator at time of enrollment was 44% predicted (range 38-74). All 7 patients were able to tolerate dose escalation of alvelestat up to the maximum dose 240mg twice daily. Preliminary clinical results were previously presented. DES/IDES was elevated at baseline (mean 0.464 (SEM 0.0508) ng/ml, with 6 of 7 subjects above the Upper Limit of Normal (ULN, 0.280 ng/ml)). Levels progressively declined during the dose escalation period to 0.380 (SEM 0.0419) ng/ml by week 8, representing a mean within subject % change from baseline (CFB) of -16.2% (SEM 6.794, Figure 1a) Ex vivo zymosan stimulated elastase activity also showed progressive decrease over the dose escalation period, with some subjects demonstrating 100% suppression (Figure 1b). Collagen synthesis as measured by PRO-C3 and PRO-C6 was increased above ULN at baseline and declined with alvelestat treatment (Figure 1c and 1d). There was no consistent change in collagen degradation biomarkers (C3M, C6M) There was consistency of a suppressive effect on biomarkers of elastase activity and collagen turnover in 6 of 7 treated patients, all of whom had improved or stable lung disease (ranging from change in FEV1 % predicted at end of treatment from +9% to -6%). Conclusion NE can damage lung tissue due to elastin breakdown, pro-inflammatory and pro-fibrotic effects (Sallenave J-M, J Leuk Biol 2015;98:137-139). This is the first evidence of elevated elastase activity as detected by elastin breakdown in patients with BOS and chronic GVHD. Treatment with the selective NE inhibitor, alvelestat was associated with progressive reduction of plasma desmosine levels over 8 weeks of within-subject dose escalation and reduction stimulated neutrophil elastase activity. The consistent suppression of elastase and of collagen synthesis/turnover biomarkers following alvelestat treatment is encouraging for its potential to impact progressive lung fibrosis in BOS and chronic GVHD. Disclosures Parkin: Mereo BioPharma Group: Current Employment. Moore: Mereo BioPharma Group: Current Employment. Pavletic: Center for Cancer Research: Research Funding; National Cancer Institute: Research Funding; National Institutes of Health: Research Funding; Celgene: Research Funding; Actelion: Research Funding; Eli Lilly: Research Funding; Pharmacyclics: Research Funding; Kadmon: Research Funding.

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

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

Collagen type III and VI remodeling biomarkers are associated with kidney fibrosis in lupus nephritis

Background: Lupus nephritis (LN) occurs in up to 40% of patients with systemic lupus erythematosus (SLE). Reliable biomarkers of kidney damage are needed to identify SLE patients at risk to develop LN in order to improve screening, treat earlier, and halt progression to kidney failure. Novel biomarkers of extracellular matrix remodeling were evaluated as markers of kidney fibrosis and disease activity in LN patients. Methods: Biomarkers of the interstitial collagen type III (PRO-C3) and type VI (PRO-C6) formation as well as of collagen type III (C3M) degradation were evaluated in the serum and urine of 40 patients with LN, 20 SLE patients without LN, 20 healthy controls and 10 biopsy controls (histological kidney inflammation/damage without SLE). Their association with histological markers of interstitial fibrosis and tubular atrophy, with inflammatory cell infiltration and with disease activity and chronicity in the LN patients was assessed. Results: Despite PRO-C3 (serum) and PRO-C6 (serum and urine) were significantly elevated in LN patients compared to healthy controls, they were not able to separate the LN from the SLE patients. C3M (urine) levels were not different in the LN group compared to the others. C3M (urine) strongly correlated and PRO-C6 (serum and urine) inversely correlated with kidney function (eGFR). The biomarkers of interstitial collagen turnover PRO-C6 (serum) and C3M (urine) correlated with histological markers of interstitial fibrosis, tubular atrophy, and monocyte infiltration. Conclusions: Non-invasive collagen turnover biomarkers are promising tools to identify SLE patients with kidney histological modifications.

Turnover of Type I and III Collagen Predicts Progression of Idiopathic Pulmonary Fibrosis

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

The Collagen-Based Medical Device MD-Tissue Acts as a Mechanical Scaffold Influencing Morpho-Functional Properties of Cultured Human Tenocytes

Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.

Collagen remodeling markers show differentiated expression in patients with ST- and non-ST elevation myocardial infarction

Background Following acute myocardial infarction (MI), the left ventricle undergoes molecular and extracellular matrix (ECM) changes. The ECM is a dynamic structure with a potential role in cardiac remodeling post-MI. Collagens are the major components of both cardiac and arterial ECM. Purpose We evaluated circulating levels of type I, IV and VI collagen fragments in two cohorts of patients with acute MI to investigate collagen turnover post-MI. The cohorts were Malmö AMI in elderly (MAMI-Y) and Assessing Platelet Activity in Coronary Heart Disease (APACHE). Methods Serum was collected from 190 patients from the discovery cohort (MAMI-Y: mean age 74, SD 10.8) at four timepoints: admission when MI, after 3–6 days, 6 weeks, 12 months; citrate plasma was collected from 142 patients from the validation cohort (APACHE: mean age 65, SD 11.6) at four timepoints: hospitalization, 3 days, 7–9 days, 6 months. The biomarkers of matrix metalloproteinase (MMP)-mediated degradation of type I collagen (C1M), MMP-mediated degradation of type IV collagen (C4M) and formation of type VI collagen (PRO-C6) were measured at all timepoints (immunosorbent assays). Differences in the markers at the different timepoints were calculated using repeated measures ANOVA. Results Circulating levels of the formation biomarker PRO-C6 significantly increased from baseline and remained high at all three following timepoints in both MAMI-Y and APACHE studies (all p<0.001). In contrast, the degradation biomarkers C1M and C4M showed a similar pattern of an initial increase 3 days post-MI followed by a decrease over time, with C1M in MAMI-Y and both C1M and C4M in APACHE having returned to baseline level by the final timepoint. Circulating baseline levels of PRO-C6 correlated with age (r=0.397, p<0.0001 in MAMI-Y, r=0.427, p<0.0001 in APACHE). Categorizing the subjects into ST elevation MI (STEMI; MAMI-Y: N=67, APACHE: N=71) or non-ST elevation MI (NSTEMI; MAMI-Y: N=130, APACHE: N=52) revealed that a correlation with age remained in both subgroups (r=0.443, p<0.0001 and r=0.325, p<0.0001, respectively, in MAMI-Y, and r=0.516, p<0.0001 and r=0.316, p=0.023, respectively, in APACHE). Moreover, PRO-C6 was elevated in STEMI patients that had previously experienced an MI in both cohorts (MAMI-Y: p=0.017, APACHE: p=0.016). C1M and C4M levels were not different in patients with prior MI in any of the cohorts. No association was found between any biomarker and gender or diabetes. Echocardiography showed a correlation between baseline levels of C1M and ejection fraction (r=0.228, p=0.023) in the whole MAMI-Y cohort and among NSTEMI, but not among STEMI, subjects (r=0.337, p=0.004). Conclusions We observed changes in circulating fragments reflecting collagen turnover in the acute phase post-MI, more pronounced in STEMI patients. This may indicate that STEMI patients have more active collagen remodeling than NSTEMI patients and may have more altered left ventricle function and remodeling. Funding Acknowledgement Type of funding source: Other. Main funding source(s): This work was supported by the Danish Research Foundation “den danske forskningsfond”, the Innovation foundation (Innovationsfonden), Swedish Research Council, Swedish Heart and Lung Foundation, Swedish Society for Medical Research, Swedish Society of Medicine, the Crafoord Foundation, the Åke Wiberg foundation and the Stroke foundation.