Oxidative stress dependent microRNA-34a activation via PI3Kα reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells

Sirtuin-1 (SIRT1) and SIRT6, NAD(+)-dependent Class III protein deacetylases, are putative anti-aging enzymes, down-regulated in patients with chronic obstructive pulmonary disease (COPD), which is characterized by the accelerated ageing of the lung and associated with increased oxidative stress. Here, we show that oxidative stress (hydrogen peroxide) selectively elevates microRNA-34a (miR-34a) but not the related miR-34b/c, with concomitant reduction of SIRT1/-6 in bronchial epithelial cells (BEAS2B), which was also observed in peripheral lung samples from patients with COPD. Over-expression of a miR-34a mimic caused a significant reduction in both mRNA and protein of SIRT1/-6, whereas inhibition of miR-34a (antagomir) increased these sirtuins. Induction of miR-34a expression with H2O2 was phosphoinositide-3-kinase (PI3K) dependent as it was associated with PI3Kα activation as well as phosphatase and tensin homolog (PTEN) reduction. Importantly, miR-34a antagomirs increased SIRT1/-6 mRNA levels, whilst decreasing markers of cellular senescence in airway epithelial cells from COPD patients, suggesting that this process is reversible. Other sirtuin isoforms were not affected by miR-34a. Our data indicate that miR-34a is induced by oxidative stress via PI3K signaling, and orchestrates ageing responses under oxidative stress, therefore highlighting miR-34a as a new therapeutic target and biomarker in COPD and other oxidative stress-driven aging diseases

Sirt1 expression is associated with CD31 expression in blood cells from patients with chronic obstructive pulmonary disease

Background: Cigarette smoke induced oxidative stress has been shown to reduce silent information regulator 1 (Sirt1) levels in lung tissue from smokers and patients with COPD patients. Sirt1 is known to inhibit endothelial senescence and may play a protective role in vascular cells. Endothelial progenitor cells (EPCs) are mobilized into circulation under various pathophysiological conditions, and are thought to play an important role in tissue repair in chronic obstructive lung disease (COPD). Therefore, Sirt1 and EPC-associated mRNAs were measured in blood samples from patients with COPD and from cultured CD34+ progenitor cells to examine whether these genes are associated with COPD development. Methods: This study included 358 patients with a smoking history of more than 10 pack-years. RNA was extracted from blood samples and from CD34+ progenitor cells treated with cigarette smoke extract (CSE), followed by assessment of CD31, CD34, Sirt1 mRNA, miR-34a, and miR-126-3p expression by real-time RT-PCR. Results: The expression of CD31, CD34, Sirt1 mRNAs, and miR-126-3p decreased and that of miR-34a increased in moderate COPD compared with that in control smokers. However, no significant differences in these genes were observed in blood cells from patients with severe COPD compared with those in control smokers. CSE significantly decreased Sirt1 and increased miR-34a expression in cultured progenitor cells. Conclusion: Sirt1 expression in blood cells from patients with COPD could be a biomarker for disease stability in patients with moderate COPD. MiR-34a may participate in apoptosis and/or senescence of EPCs in smokers. Decreased expression of CD31, CD34, and miR-126-3p potentially represents decreased numbers of EPCs in blood cell from patients with COPD

Integrative transcriptomics in smoking related lung diseases

Chronic lung diseases including Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF) and lung cancer are major causes of morbidity and mortality in the United States due to high incidence and limited therapeutic options. In order to address this critical issue, I have leveraged RNA sequencing and integrative genomics to define disease-associated transcriptomic changes which could be potentially targeted to lead to new therapeutics. We sequenced the lung transcriptome of subjects with IPF (n=19), emphysema (n=19, a subtype of COPD), or neither (n=20). The expression levels of 1770 genes differed between IPF and control lung, and 220 genes differed between emphysema and control lung (p<0.001). Upregulated genes in both emphysema and IPF were enriched for the p53/hypoxia pathway. These results were validated by immunohistochemistry of select p53/hypoxia proteins and by GSEA analysis of independent expression microarray experiments. To identify regulatory events, I constructed an integrative miRNA target prediction and anticorrelation miRNA-mRNA network, which highlighted several miRNA whose expression levels were the opposite of genes differentially expressed in both IPF and emphysema. MiR-96 was a highly connected hub in this network and was subsequently overexpressed in cell lines to validate several potential regulatory connections. Building upon these successful experiments, I next sought to define gene expression changes and the miRNA-mRNA regulatory network in never smoker lung cancer. Large and small RNA was sequenced from matched lung adenocarcinoma tumor and adjacent normal lung tissue obtained from 22 subjects (8 never, 14 current and former smokers). I identified 120 genes whose expression was modified uniquely in never smoker lung tumors. Using a repository of gene-expression profiles associated with small bioactive molecules, several compounds which counter the never smoker tumor signature were identified in silico. Leveraging differential expression information, I again constructed an mRNA-miRNA regulatory network, and subsequently identified a potential never smoker oncomir has-mir-424 and its transcription factor target FOXP2. In this thesis, I have identified genes, pathways and the miRNA-mRNA regulatory network that is altered in COPD, IPF, and lung adenocarcinoma among never smokers. My findings may ultimately lead to improved treatment options by identifying targetable pathways, regulators, and therapeutic drug candidates.2017-02-01T00:00:00

Genetics Association and Epigenetic Changes in COPD

Genome-wide association studies (GWASs) have successfully identified susceptibility loci associated with COPD. The genes mapped on these loci, eg The FAM13A gene (family with sequence similarity 13, member A), provide a new approach to understand the COPD pathology. Furthermore, heavy smoking is reported to correlate with altered methylation and epigenetic changes of multiple genes in small airway cells. These changes have been shown to be associated with the severity of COPD. It is likely that smoking-induced changes in epigenetic control of gene expression result in genetically vulnerable individual’s results in reduced tissue repair, tissue damage and persistent inflammation associated with COPD pathophysiology

Extracellular vesicles and chronic obstructive pulmonary disease (COPD): a systematic review

Background: Chronic Obstructive Pulmonary Disease (COPD) is a common inflammatory disease of the airways characterized by irreversible airflow limitation, ranking the third highest cause of death worldwide. Extracellular vesicles (EVs) are important intercellular communication mediators released by cells into their extracellular environment with the capacity to transfer biological signals. EVs involved in COPD hold great potential to understand disease pathogenesis and identify important biomarkers. This systematic review aims to examine all available research on EVs in the pathogenesis and diagnosis of COPD to identify existing knowledge and support further research within the field. Methods: Publications were searched using PubMed and EMBASE with the search terms (Exosomes or extracellular vesicles or microvesicles or microparticles or ectosomes) AND (chronic obstructive pulmonary disease or COPD or emphysema or bronchitis). Results: Initial search yielded 512 papers of which 142 were manually selected for review and 43 were eligible for analyses. The studies were divided into groups according to the role of EVs in pathogenesis, EV origin and cargo, their role in COPD exacerbations and their diagnostic utility. EVs were found to be involved in the mechanism of pathogenesis of COPD, derived from various cell types, as well as containing modified levels of miRNAs. EVs also varied according to the pathophysiological status of disease, therefore presenting a possible method for COPD diagnosis and progress monitoring. Conclusion: The current findings show the limited but good quality research looking at the role of EVs in COPD, demonstrating the need for more studies to better define and provide further insight into the functional characteristics of EV in COPD pathogenesis

COPD phenotypes and machine learning cluster analysis : A systematic review and future research agenda

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or ot-for-profit sectors.Peer reviewedPostprin

Quantification of microRNA expression in single cells using microfluidics

Chronic obstructive pulmonary disease (COPD) is a lung condition characterised by progressive airflow limitation in part due to narrowing and fibrosis of small airways. COPD is associated with cellular senescence which is driven by stressors such as oxidative stress. MicroRNA-21 (miR-21) and microRNA-34a (miR-34a) are upregulated in COPD, however their regulatory role in COPD pathogenesis or in response to oxidative stress remains unclear. To better understand the role of miR-21 and miR-34a in COPD, a microfluidic platform was developed to quantify miR-21 and miR-34a molecules in single cells. Sandwich hybridisation assay was optimised and integrated into microfluidic chambers for single cell miRNA detection. The sensitivity was demonstrated by quantifying levels of miRNA in nasal cells and fluid. Levels of miR-21 were varied in nasal cells and fluid within and between individuals. Levels of miR-21 and miR-34a were increased in small airway epithelial cells (SAEC) and fibroblasts (SAF) from COPD subjects compared to non-smokers, and were varied within and between subjects. MiR-21 and miR-34a were detected simultaneously from the same cell using a multiplex assay which showed a positive correlation between miR-21 and miR-34a expressed in SAEC and SAF. Baseline gene expression of miR-21 and miR-34a targets differed in SAEC and SAF. Altered levels of miR-21 and miR-34a influenced their targets mRNA and protein levels, however the effect was different in COPD cells compared to healthy cells. MiR-21 and miR-34a levels were elevated in response to oxidative stress, while their target gene expression was reduced and senescent markers were increased. This study demonstrated that a microfluidic platform can be developed to quantify single miRNA molecules in single cells to determine cell-to-cell variation of miRNAs within cell populations. MiR-21 and miR-34a are crucial regulators in COPD and may have a protective role in response to oxidative stress, however further investigation is required.Open Acces

Systems biology approaches to investigate mechanisms of obstructive lung diseases

Both asthma and chronic obstructive pulmonary disease (COPD) are obstructive lung diseases with a large impact on global health, causing 400 000 and 3 million deaths respectively each year. The numbers may be underestimated, since COPD often contributes to death without being registered as the cause of death. There is currently no therapeutic cure for either of these diseases, only symptomatic relief. One problem is that the available therapeutics do not always work, since the diseases present a range of clinical phenotypes with possibly different endotypes, so-called umbrella diseases. The aim of this thesis was to study asthma, COPD caused by smoking, and obstructive lung disease related to preterm birth using systems biology approaches. This includes studying several analytical platforms in a range of compartments in order to subphenotype the patients into subgroups and elucidate the related mechanisms. Identifying these endotypes increases the possibility of finding effective therapeutics for all patient groups. Obstructive lung diseases are often studied by collecting bronchoalveolar lavage (BAL) and epithelial cells from the lungs during bronchoscopy. This procedure is invasive, and costly which is why the cohorts studied are often small. Subgrouping results in even smaller sample sizes, which decreases the power of statistical analysis. Using multivariate analysis is a means of increasing power by taking all variables into account. A workflow for performing the multivariate method orthogonal projections to latent structures discriminant analysis (OPLS-DA) to compare groups one by one in small sample sizes was developed using the programming language R, and was formatted into an R package entitled roplspvs. The roplspvs package performs OPLS modeling using the package ropls in R, including variable selection to extract the variables driving the separation the most. As OPLS models are prone to overfitting, the significance of the models was investigated thoroughly using permutations. Using roplspvs on small sample sizes, it was shown that permutations performed before variable selection (termed “sans v.s.”) and permutations including the variable selection step (termed “over v.s.”) are better suited to estimate the level of model statistics achieved by random than permutations post variable selection. An example of running the package was shown using a publicly available metabolomics dataset. The roplspvs packages, along with the commercially available software SIMCA and univariate statistics, were then applied to investigate alterations between groups in a range of projects, including three clinical cohorts of asthma, COPD and BPD, as well as a project investigating the degradation of proteins in the processing of blood samples prior to biobanking the evaluate protein stability. COPD in smokers and ex-smokers was studied by investigating the miRNA content of small extracellular vesicles (EVs) using OPLS modeling as well as univariate analysis, with the finding that COPD gave highly altered miRNA content of small EVs compared to healthy subjects. After stratifying the analysis by gender, potential alterations compared to smokers were identified in males with significant p[CV-ANOVA]=0.05 and p[permutations over variable selection]=0.12, but permutations sans variable selection were highly insignificant. The alterations were connected to potentially affected pathways through pathway analysis of genes regulated by the altered miRNA. Pathway affected by COPD and smoking were mainly connected to cell- growth and death with the p53-pathway mostly altered, while the less pronounced miRNA alterations related to COPD alone was connected to degradation through autophagy and proteolysis. Premature birth has been connected to lung obstruction in adults who developed bronchopulmonary disease (BPD) during the neonatal period. To characterize T-cells in adults with a history of BPD, FACS analysis was performed on BAL cells. Univariate analysis showed increased levels of CD8+ T-cells, and decreased levels of CD4+ T-cells in subjects with BPD. Applying OPLS and stratifying the analysis by gender, it was indicated that the alterations were mostly driven by females. Asthmatic subjects were subphenotyped into clusters using four platforms from blood and urine into phenotypic groups, which were studied using OPLS models to compare the groups. Clinical features were extracted that separated a large portion of the groups. Finally, the metabolome of urine was used to separate asthmatics into severe and mild asthma, stratifying the analysis by oral corticosteroids (OCS). It was found that carnitines, which were the strongest drivers for separating the groups, were not affected by OCS use. Using roplspvs, it was shown that the levels of carnitines were strongly affected by gender, with higher levels in males than in females. In conclusion, it was shown that OPLS models can be used to investigate cohorts consisting of small sample sizes, and that permutation procedures including variable selection efficiently test the significance of the models. Subgroups of COPD and asthmatic subjects were compared, showing alterations in miRNA levels, metabolome, and lymphocyte composition, as well as in clinical data connected to potential endotypes with separate disease mechanisms. Stratifications by gender supported earlier findings that gender has a strong effect on obstructive lung diseases. Together with further analysis on the cohorts in this study using other platforms, this is a step towards finding candidates for diagnostics and therapeutics

Understanding shared pathogenesis between chronic obstructive pulmonary disease (COPD) and lung cancer by means of cell specific genomics

Introduction: COPD (Chronic Obstructive Pulmonary Disease) and lung cancer are related conditions associated with inflammation. Relatively little focus has been given to the endothelium, through which inflammatory cells transmigrate to reach the lung. We sought to determine if coding and non-coding alterations in pulmonary endothelium exist in COPD and lung cancer. Methods: Patients with and without COPD undergoing thoracic surgery were recruited. Pulmonary Endothelial Cells were isolated from lung and tumour and extracted RNA (ribonucleic acid) used for miRNA (micro-RNA) and mRNA (messenger RNA) microarrays. Ingenuity pathway analysis (IPA) was also carried out. Results: 2071 genes and 43 miRNAs were significantly upregulated in COPD. 4 targets were validated by quantitative polymerase chain reaction, of which miR-181b-3p was chosen for functional validation. Another target, miR-429, was also increased in lung tumour. Several cancer-related pathways such as transforming growth factor- β were altered in the IPA. There was significantly reduced tube formation and endothelial sprouting in Human umbilical vein endothelial cells transfected with miR-181b-3p, consistent with an effect on angiogenesis. Conclusions: Upregulation of miR-181b-3p reduces tube formation and sprouting by endothelial cells. This might be significant in the development of emphysema as lung vasculature is important in the structural maintenance of alveoli