Increased pulmonary Wnt (wingless/integrated)-signaling in patients with sarcoidosis

SummaryBackgroundSarcoidosis is an inflammatory multisystemic granulomatous disease of unknown aetiology commonly affecting the lungs, and pulmonary fibrosis often develops in chronic sarcoidosis. It has been suggested that Wnt (Wingless/integrated)-signaling has a role in inflammatory and fibrotic processes in the lungs, but its role in sarcoidosis has not been investigated. We hypothesised that Wnts secreted from T cells or other inflammatory cells have a role in the pathogenesis of sarcoidosis.MethodsBrush biopsies and bronchoalveolar lavage (BAL) were obtained through bronchoscopy from healthy controls (n = 18) and patients with sarcoidosis (n = 48). Semi-quantitative RT-PCR, electrophoretic mobility shift assay (EMSA) and immunocytochemistry were performed to analyse Wnt expression and activation of the Wnt-signal transducer β-catenin.ResultsAltered expression of Wnt5A, Wnt7A and Wnt7B mRNA in BAL cells was observed, as well as an increased activation of β-catenin, measured by EMSA and confirmed with immunocytochemistry, in resident lung cells from patients with sarcoidosis. More pronounced changes in Wnt expression were seen with advancing disease stage. Thus, by three independent methods, we have found evidence of increased pulmonary Wnt-activation in sarcoidosis.ConclusionsIn the lungs of patients with sarcoidosis there is a previously unappreciated increased Wnt-signal activation that could contribute to the inflammatory processes

miRNA-mRNA-protein dysregulated network in COPD in women

Rationale: Chronic obstructive pulmonary disease (COPD) is a complex disease caused by a multitude of underlying mechanisms, and molecular mechanistic modeling of COPD, especially at a multi-molecular level, is needed to facilitate the development of molecular diagnostic and prognostic tools and efficacious treatments.Objectives: To investigate the miRNA-mRNA-protein dysregulated network to facilitate prediction of biomarkers and disease subnetwork in COPD in women.Measurements and Results: Three omics data blocks (mRNA, miRNA, and protein) collected from BAL cells from female current-smoker COPD patients, smokers with normal lung function, and healthy never-smokers were integrated with miRNA-mRNA-protein regulatory networks to construct a COPD-specific dysregulated network. Furthermore, downstream network topology, literature annotation, and functional enrichment analysis identified both known and novel disease-related biomarkers and pathways. Both abnormal regulations in miRNA-induced mRNA transcription and protein translation repression play roles in COPD. Finally, the let-7-AIFM1-FKBP1A pathway is highlighted in COPD pathology.Conclusion: For the first time, a comprehensive miRNA-mRNA-protein dysregulated network of primary immune cells from the lung related to COPD in females was constructed to elucidate specific biomarkers and disease pathways. The multi-omics network provides a new molecular insight from a multi-molecular aspect and highlights dysregulated interactions. The highlighted let-7-AIFM1-FKBP1A pathway also indicates new hypotheses of COPD pathology.Peer reviewe

Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin-lysoPA axis in COPD.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and a leading cause of mortality and morbidity worldwide. The aim of this study was to investigate the sex dependency of circulating metabolic profiles in COPD.Serum from healthy never-smokers (healthy), smokers with normal lung function (smokers), and smokers with COPD (COPD; Global Initiative for Chronic Obstructive Lung Disease stages I-II/A-B) from the Karolinska COSMIC cohort (n=116) was analysed using our nontargeted liquid chromatography-high resolution mass spectrometry metabolomics platform.Pathway analyses revealed that several altered metabolites are involved in oxidative stress. Supervised multivariate modelling showed significant classification of smokers from COPD (p=2.8×10-7). Sex stratification indicated that the separation was driven by females (p=2.4×10-7) relative to males (p=4.0×10-4). Significantly altered metabolites were confirmed quantitatively using targeted metabolomics. Multivariate modelling of targeted metabolomics data confirmed enhanced metabolic dysregulation in females with COPD (p=3.0×10-3) relative to males (p=0.10). The autotaxin products lysoPA (16:0) and lysoPA (18:2) correlated with lung function (forced expiratory volume in 1 s) in males with COPD (r=0.86; p<0.0001), but not females (r=0.44; p=0.15), potentially related to observed dysregulation of the miR-29 family in the lung.These findings highlight the role of oxidative stress in COPD, and suggest that sex-enhanced dysregulation in oxidative stress, and potentially the autotaxin-lysoPA axis, are associated with disease mechanisms and/or prevalence

Assessing Recent Smoking Status by Measuring Exhaled Carbon Monoxide Levels

The main expectations of applying proteomics technologies to clinical questions are the discovery of disease related biomarkers. Despite technological advancement to increase proteome coverage and depth to meet these expectations the number of generated biomarkers for clinical use is small. One of the reasons is that found potential biomarkers often are false discoveries. Small sample sizes, in combination with patient sample heterogeneity increase the risk of false discoveries. To be able to extract relevant biological information from such data, high demands are put on the experimental design and the use of sensitive and quantitatively accurate technologies. The overall aim of this thesis was to apply quantitative proteomics methods for biomarker discovery in clinical samples. A method for reducing bias by controlling for individual variation in smoking habits is described in paper I. The aim of the method was objective assessment of recent smoking in clinical studies on inflammatory responses. In paper II, the proteome of alveolar macrophages obtained from smoking subjects with and without the inflammatory lung disease chronic obstructive pulmonary disease (COPD) were quantified by two-dimensional gel-electrophoresis (2-DE). A gender focused analysis showed protein level differences within the female group, with down-regulation of lysosomal pathway and up-regulation of oxidative pathway in COPD patients. Paper III, a mass spectrometry based proteomics analysis of tumour samples, contributes to the molecular understanding of vulvar squamous cell carcinoma (VSCC) and we identified a high risk patient subgroup of HPV-negative tumours based on the expression of four proteins, further suggesting that this subgroup is characterized by an altered ubiquitin-proteasome signalling pathway. Paper III describes a data analysis workflow for the extraction of biological information from quantitative mass spectrometry based proteomics data. High patient-to-patient tumour proteome variability was addressed by using pathway profiling on individual tumour data, followed by comparison of pathway association ranks in a multivariate analysis. We show that pathway data on individual tumour level can detect subpopulations of patients and identify pathways of specific importance in pre-defined clinical groups by the use of multivariate statistics. In paper IV, the potentials and limits of quantitative mass spectrometry on clinical samples was evaluated by defining the quantitative accuracy of isobaric labels and label-free quantification. Quantification by isobaric labels in combination with pI pre-fractionation showed a lower limit of quantification (LOQ) than a label-free analysis without pI pre-fractionation, and 6-plex TMT were more sensitive than 8-plex iTRAQ. Precursor mixing measured by isolation interference (MS1 interference) is more linked to the quantitative accuracy of isobaric labels than reporter ion interference (MS2 interference). Based on that we could define recommendations for how much isolation interference that can be accepted; in our data <30% isolation interference had little effect the quantitative accuracy. In conclusion, getting biological knowledge from proteomics studies requires a careful study design, control of possible confounding factors and the use of clinical data to identify disease subtypes. Further, to be able to draw conclusions from the data, the analysis requires accurate quantitative data and robust statistical tools to detect significant protein alterations. Methods around these issues are developed and discussed in this thesis

Asthmatics Exhibit Altered Oxylipin Profiles Compared to Healthy Individuals after Subway Air Exposure

Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications.This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air.Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information.Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E(2) (PGE(2)). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change.Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study

Abstract Background Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren’s syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown. Methods Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16). Results A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio. Conclusions These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease

Evaluation of α-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of a-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of highthroughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against a-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresisgels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity