Jyotshna Mandal

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.AimTo determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.ResultsIPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.ConclusionOur data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts

Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases.

Vasoactive Intestinal Peptide for Diagnosing Exacerbation in Chronic Obstructive Pulmonary Disease

Background: Vasoactive intestinal peptide (VIP) is the most abundant neuropeptide in the lung. VIP has been linked to pulmonary arterial hypertension and hypoxia. Objectives: We aimed to assess circulating VIP levels at exacerbation and at stable chronic obstructive pulmonary disease (COPD) and to evaluate the diagnostic performance in a well-characterized cohort of COPD patients. Methods: The nested cohort study included patients with Global Initiative for Chronic Obstructive Lung Disease stage II-IV. Patients were examined at stable state and at acute exacerbation of COPD (AE-COPD), and dedicated serum was collected at both conditions. Serum VIP levels were determined by enzyme-linked immunosorbent assay. Diagnostic accuracy was analyzed by receiver operating characteristic curve and area under the curve (AUC). Results: Patients with acute exacerbation (n = 120) and stable COPD (n = 163) had similar characteristics at baseline. Serum VIP levels did not correlate with oxygen saturation at rest (p = 0.722) or at exercise (p = 0.168). Serum VIP levels were significantly higher at AE-COPD (130.25 pg/ml, 95% CI 112.19-151.83) as compared to stable COPD (40.07 pg/ml, 95% CI 37.13-43.96, p < 0.001). The association of increased serum VIP with AE-COPD remained significant after propensity score matching (p < 0.001). Analysis of the Youden index indicated the optimal serum VIP cutoff value as 56.6 pg/ml. The probability of AE-COPD was very low if serum VIP was ≤35 pg/ml (sensitivity >90%) and very high if serum VIP was ≥88 pg/ml (specificity >90%). Serum VIP levels presented a robust performance to diagnose AE-COPD (AUC 0.849, 95% CI 0.779-0.899). Conclusions: Increased serum VIP levels are associated with AE-COPD.

Longitudinal Measurement of Serum Vascular Endothelial Growth Factor in Patients with Chronic Obstructive Pulmonary Disease

Background: Impaired vascular endothelial growth factor (VEGF) signaling causes emphysema in animal models. In chronic obstructive pulmonary disease (COPD) patients, alterations in VEGF tissue expression have been observed. We hypothesize that circulating VEGF may be a biomarker to phenotype COPD patients. Objective: The aim of this study was to investigate VEGF serum levels in stable and exacerbated COPD. Methods: VEGF serum levels as well as parameters of short- and long-term outcome were assessed and analyzed in two COPD cohorts [PROMISE, n = 117; ProCOLD (PC), n = 191]. Results: VEGF serum levels at stable COPD were neither related to forced expiratory volume in 1 s nor to the Modified Medical Research Council dyspnea score, 6-min walking distance or BODE index. There was no association between single VEGF levels and COPD exacerbation frequency or mortality at 1 and 2 years of follow-up. In PC an increase in VEGF over time (ΔVEGF) was associated with the exacerbation frequency as well as the 1- and 2-year hospitalization rate (p = 0.046, 0.009 and 0.006, respectively). Furthermore, in PC ΔVEGF was associated with 1- and 2-year survival (p = 0.009 and 0.041, respectively). Conclusions: Single serum VEGF levels, at stable and exacerbated COPD, were not associated with clinically significant outcomes in COPD. Conversely, the VEGF course seems related to COPD prognosis.

IFNΛ3/4 locus polymorphisms and IFNΛ3 circulating levels are associated with COPD severity and outcomes

BackgroundInterferon lambdas (IFNLs) have important anti-viral/bacterial and immunomodulatory functions in the respiratory tract. How do IFNLs impact COPD and its exacerbations?MethodsFive hundred twenty eight patients were recruited in a prospective observational multicentre cohort (PROMISE) study. The genetic polymorphisms (rs8099917 and rs12979860) within the IFNL3/4 gene region and circulating levels of IFNL3 in COPD patients were determined and associated with disease activity and outcome during a median follow-up of 24 months.ResultsThe GG genotype significantly influenced severe exacerbation rate (42 vs. 23%; p = 0.032) and time to severe exacerbation (HR = 2.260; p = 0.012). Compared to the TT or TG genotypes, the GG genotype was associated with severe dyspnoea (modified medical research council score ≥ median 3; 22 vs 42%, p = 0.030). The CC genotype of the rs12979860 SNP was associated with a poorer prognosis (body mass index, airflow obstruction, dyspnea and exercise capacity index ≥ median 4; 46 vs. 36% TC vs. 20.5% TT; p = 0.031). Patients with stable COPD and at exacerbation had significantly lower circulating IFNL3 compared to healthy controls (p < 0.001 and p < 0.001, respectively). Circulating IFNL3 correlated to post-bronchodilator FEV1%predicted and the tissue maturation biomarker Pro-collagen 3.ConclusionIFNL3/4 polymorphisms and circulating IFNL3 may be associated with disease activity and outcomes in COPD.Trial registrationClinical Trial registration http://www.isrctn.com/ identifier ISRCTN99586989 on 16 April 2008.

Constitutive high expression of protein arginine methyltransferase 1 in asthmatic airway smooth muscle cells is caused by reduced microRNA-19a expression and leads to enhanced remodeling

Background In asthma remodeling airway smooth muscle cells (ASMCs) contribute to airway wall thickness through increased proliferation, migration, and extracellular matrix deposition. Previously, we described that protein arginine methyltransferase 1 (PRMT1) participates in airway remodeling in pulmonary inflammation in E3 rats. Objective We sought to define the asthma‐specific regulatory mechanism of PRMT1 in human ASMCs. Methods ASMCs from healthy subjects and asthmatic patients were activated with platelet‐derived growth factor (PDGF)–BB. PRMT1 was localized by means of immunohistochemistry in human lung tissue sections and by means of immunofluorescence in isolated ASMCs. PRMT1 activity was suppressed by the pan‐PRMT inhibitor AMI‐1, signal transducer and activator of transcription 1 (STAT1) was suppressed by small interfering RNA, and extracellular signal‐regulated kinase (ERK) 1/2 mitogen‐activated protein kinase (MAPK) was suppressed by PD98059. MicroRNAs (miRs) were assessed by using real‐time quantitative PCR and regulated by miR mimics or inhibitors. Results PRMT1 expression was significantly increased in lung tissue sections and in isolated ASMCs of patients with severe asthma. PDGF‐BB significantly increased PRMT1 expression through ERK1/2 MAPK and STAT1 signaling in control ASMCs, whereas in ASMCs from asthmatic patients, these proteins were constitutively expressed. ASMCs from asthmatic patients had reduced miR‐19a expression, causing upregulation of ERK1/2 MAPK, STAT1, and PRMT1. Inhibition of PRMT1 abrogated collagen type I and fibronectin deposition, cell proliferation, and migration of ASMCs from asthmatic patients. Conclusions PRMT1 is a central regulator of tissue remodeling in ASMCs from asthmatic patients through the pathway: PDGF‐BB–miR‐19a–ERK1/2 MAPK and STAT1. Low miR‐19a expression in ASMCs from asthmatic patients is the key event that results in constitutive increased PRMT1 expression and remodeling. Therefore PRMT1 is an attractive target to limit airway wall remodeling in asthmatic patients. Graphical abstract Figure. No Caption available.

LSC Abstract – Irisin stimulates hyaluronic acid secretion in COPD: A new role for irisin associated with disease progression and severity

Irisin is a recently identified hormone secreted by skeletal muscle cells and has been proposed to mediate the beneficial effects of exercise. However, there is a high controversy for the physiological role and potential therapeutic value of irisin. Hyaluronic acid (HA) is an extracellular molecule that plays a key role in airway remodeling in COPD. The aim of our study was to investigate if irisin affects HA secretion in COPD and if this is associated to disease progression and severity. We used primary cultures of airway smooth muscle cells (ASMC) and fibroblasts from patients with COPD and skeletal muscle cells (SkMC) as controls. We observed that irisin stimulates significantly (p 10 PY, included in the PROMISE cohort. The primary outcome of the study was exacerbation and/or death. Median observation time was 24 months. We observed a significant correlation (rho=0.142, p=0.002) between serum levels of irisin and HA. Irisin was associated significantly (p=0.004) with the number of severe exacerbations per year (OR 0.134 95% CI 0.051-0.269) while HA was associated significantly (p These results indicate that irisin stimulates HA secretion, in a cell-specific way, and this is associated with COPD progression and severity. These molecules may be proved to be valuable systemic biomarkers in clinical follow up of COPD.

Circulating vasoactive intestinal peptide for diagnosis of exacerbation of COPD

Background: Vasoactive Intestinal Peptide (VIP) is an abundant neuropeptide in the lung, which has been linked to pulmonary arterial hypertension and hypoxia in COPD. We hypothesize that circulating VIP is increased at exacerbation as compared to stable COPD. Methods: In a nested cohort study, 283 patients (mean age 66.31±11.38) with GOLD II-IV (mean post BD FEV1 %pred 47.8±16.37) were examined at stable state and at exacerbation. Serum VIP level, from dedicated frozen samples was determined by ELISA. Diagnostic accuracy was analyzed by receiver-operating characteristic (ROC) curve and area under the curve (AUC). Results: Patients in the stable and exacerbated groups had similar demographics, except for the BORG score (p 90%) and very high if serum VIP was ≥88pg/ml (specificity >90%). Diagnostic performance improved by using a combination of clinical and lung function variables. Conclusion: Increased serum VIP levels are associated with exacerbation of COPD.

Disease specific effects of adrenomedullin in COPD and asthma derived human bronchial epithelial cells

Background: Serum levels of Pro-Adrenomedullin (Pro-ADM) were associated with reduced survival and exacerbations in COPD patients. ADM signals through receptor activity modifying protein family (RAMP) -1, -2 and -3 and calcitonin like receptor (CRLR). Aims/objectives: We investigated: (i) ADM, CRLR, RAMP-1, -2, and -3 expression in human lung tissue and isolated primary bronchial epithelial cells of asthma (n=12), COPD patients (n=12) and controls, (n=11) and (ii) effect of ADM on CXCL1, CXCL5 and IL-6, IL-8, IL-10, and TNF-α secretion. Methods: ADM and its receptors expression were analyzed by immune-histochemistry and western blotting. ADM (10 -6 M)-induced cytokine secretion was determined by ELISAs (0, 24, and 48 hours). Results: ADM was similarly expressed in healthy and diseased tissues. CRLR expression was higher in sub-epithelial mesenchymal cells than in bronchial epithelial cells. RAMP-1, -2 and -3 were expressed only by sub-epithelial cells. These expression patterns were confirmed in isolated airway epithelial and mesenchymal cells. ADM induced the secretion of CXCL1 only in asthmatics by 2.4 times (48 hours). CXCL5 expression was also up-regulated in asthmatic cells by 3.8 times (48 hours). IL-6 secretion was increased by ADM in asthma and control cells by 9 times (24 hours). IL-8 secretion was up-regulated only in COPD by 2.2 times (48 hours). ADM did not modify the secretion of TNF-α and IL-10. Conclusion: Our results suggest a cell type specific but not a disease specific expression pattern of ADM and its receptors in the human airways. In COPD cells, but not in asthma, IL-8 was increased by ADM. In asthma, ADM increased the secretion of all pro-angiogenic cytokines.