Systems medicine approach unravels MMP2 and NOTCH3 as key mediators of cigarette smoke-induced airway remodelling in COPD

Abstract

Background and aim: Cigarette smoking is known to cause airway remodelling leading to loss of lung plasticity, a key feature of chronic obstructive pulmonary disease (COPD). Despite the availability of several disease management approaches, an effective cure is elusive due to a lack of clear molecular insight into COPD pathogenesis. Thus, utilizing bioinformatics tools, this study aimed to identify crucial hub genes in COPD pathogenesis and validate them using in-vitro experiments and COPD patient samples.

Study methodology: In-silico identification of molecular interactions was analysed using bioinformatics tools like String, GEO datasets, CTD, Genecards, Disgenet, Opentargets, and Cytoscape. Airway epithelial cells (AECs) were exposed to different concentrations of cigarette smoke extract (CSE), followed by assessments of fibrosis and EMT-related parameters and markers using cellular and molecular biology techniques such as the MTT assay, AO/EtBr assay, trypan blue assay, the migration and invasion assays, morphological analysis, immunoblotting, immunocytochemistry, and RT-qPCR. Further, key genes expression and cytokines profile were assessed in PBMCs and plasma from COPD patients and healthy volunteers via RT-qPCR and ELISA, respectively.

Key findings: Four online databases (CTD, Genecards, Opentargets, and Disgenet) and a clinical dataset from the Gene Expression Omnibus were utilized to identify upregulated differentially expressed genes (DEGs). Subsequently, ten hub genes for COPD were identified using MCODE and cytohubba indices of Cytoscape, of which NOTCH3 and matrix metalloprotease (MMP) 2 were selected for further validation owing to their crucial role in COPD. CSE exposure of AECs caused alteration in cellular morphology, induced fibrous phenotype, upregulation of fibrosis and EMT markers, and increased expression of NOTCH3 and MMP2. Furthermore, chemical inhibition of MMP2 downregulated NOTCH3, suggesting NOTCH pathway upregulation by CSE-induced MMP2 activation. Inhibition of either MMP2 or NOTCH3 reversed CSE-induced fibrotic or EMT-related changes in AECs. PBMCs derived from COPD patients showed modulation of NOTCH3 and MMP2. JAG1, a NOTCH ligand, and many inflammatory markers were also significantly upregulated in COPD patient samples compared to healthy volunteers.

Significance: Our multi-level holistic approach, combining in-silico and in-vitro studies elucidated that MMP2 and NOTCH3 could be key mediators in CSE-induced airway epithelial cell remodelling, which was also confirmed through COPD patients' sample analysis. We, thus, identify MMP2 and NOTCH3 as important gene targets for controlling CS-induced COPD pathophysiology.

Keywords: Airway remodelling; Bioinformatics; COPD; Cigarette smoke; JAG1; MMP2; NOTCH3.