The Effects Of Cigarette Smoke Extract And Transforming Growth Factor-β₁on Human Airway Smooth Muscle Cells From Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by airflowlimitation that is usually progressive[1]. It’s a leading cause of morbidity and mortalityworldwide and results in an economic and social burden that is both substantial andincreasing[2,3]. In COPD a number of changes occur in the lungs, namely thedevelopment of persistent inflammation and irreversible airflow limitation[1]. Smallairway remodeling is considered to have the greatest influence on airflow limitation[4-6].The small airway remodeling in COPD consists of folded mucosa, thickening ofbasement membrane and deposition of extracellular matrix proteins (ECM), as well asincreased airway smooth muscle (ASM) mass, especially in severe COPD[4,5,7]. TheECM is a complex structured network of secreted macromolecules and preteolyticenzymes that provide the basis of cell-cell and cell-matrix interactions. In the lung,ECM components fundamentally influence the structure and function of airways.Studies with histologically stained airway tissues have shown the specific ECM proteinsare altered in the airways of patients with COPD[8,9].Cigarette somking is recognized as a main risk factor for the development ofCOPD. Most studies address the paradigm that the aetiology of COPD is cigarettesmoke induced inflammation leading to tissue damage, however some studies suggestedthat airway remodeling maybe induced independently of inflammation[10]. Other studieshave found that fibroblast from patients with COPD produce an excessive amount offibronectin and perlecan or reduced proteoglycans (decorin and biglycan) in response tocigarette smoke extract (CSE), in comparison to cells from people without COPD[10,11].However, it remains largely unknown how human ASM cells from subjects with orwithout COPD respond to CSE.Transforming growth factor-β1(TGF-β1) is a pro-fibrotic cytokine, which isincreased in several forms of acute and chronic adult lung diseases such as COPD,asthma and pulmonary fibrosis[12-15]. It is considered to play a crucial role in thepathogenesis of tissue fibrosis, stimulating the production of fibronectin, perlecan andvarious collagens[16-18]. Fibulin-1(FBLN-1) is a secreted glycoprotein that is associatedwith ECM formation and rebuilding. It is known that FBLN-1is associated with variousECM proteins such as fibronectin (FN), nidogen-1, and laminin-1[19-22]. However, therole of FBLN-1in the aetiology and pathology of airway tissue fibrosis is unclear. In this study we hypothesized that ASM cells from people with COPD wouldresponse to stimulations of CSE or TGF-β1intrinsically differently as compaired toASM cells from people without COPD, specifically in the production of pro-inflammatory meidators and factors relating to airway remodeling as well as cellularbehaviors. Futhermore, we hypothesized that the pro-fibrotic cytokine TGF-β1would upregulate the deposition of FBLN-1, with greater production observed in cells frompeople with COPD in comparison to those from people without COPD.To test these hypotheses, we stimulated the in vitro cultured ASM cells frompeople with or without COPD using CSE or TGF-β1. Then we examined the geneexpression of ECM and adhesion related molecules. Based on the gene expressionanalysis, we selected some and matrix metalloproteinase (MMP) proteins andsubsequently measured the protein production of the collagens and MMPs in responseto the CSE or TGF-β1stimulation. We also tested the effect of CSE on cell attachmentand wound healing. Finally, we investigated the effect of TGF-β1(pro-fibrotic factor)on the airway fibrosis. The main findings are the following:1) CSE and TGF-β1generally upregulated expression of genes associated withECM and adhesion molecules, but CSE and TGF-β1caused differential effects on thesegenes especially in COPD group. Specifically, collagen genes were upregulated to agreater extent in COPD than non-COPD ASM cells by5%CSE or TGF-β1whereas theMMPs genes were upregulated to a greater extent in non-COPD than COPD ASM cells.However, when stimulated at a higher concentration of CSE (10%), the increased extentof MMPs genes was greater in COPD than non-COPD ASM cells. Even though5%CSE and TGF-β1upregulated greater extent of collagen genes in COPD group andgreater extent of MMPs genes in non-COPD group, TGF-β1upregulated higher foldthen5%CSE for collagen genes and5%CSE up-regulated higher fold then TGF-β1forMMPs genes.2) The results of MMPs ELISA shown that CSE but not TGF-β1increased theproduction of MMP-1, and there is siganificant difference in the CSE-induced changeof MMP-1between COPD and non-COPD ASM cells. Neither CSE nor TGF-β1hadeffects on the production of MMP-2even thought the production of MMP-2is higherthan MMP-1,-3and-10as ELISA rusltes indicated. MMP-3and-10had the similarchange pattern after stimulated by CSE or TGF-β1. The higher concentration of CSEincreased the production of MMP-3and-10in both COPD and non-COPD ASM cellsbut TGF-β1only increased the production of MMP-3and-10in COPD ASM cells. 3) CSE or TGF-β1increased the deposition of COL8A1only in COPD ASM cells,while the expression of COL8A1appeared to be different between COPD and non-COPD airway tissues.4) CSE decreased the ability for attachment and wound healing of the COPD ASMcells but not of the non-COPD ASM cells. In contrast, TGF-β1increased the attachmentof COPD ASM cells.5) TGF-β1decreased the release of soluble FBLN-1but increased the deposition ofFBLN-1, thus promoting the process for soluble FBLN-1to incorporate into ECM.Altogether, these findings demonstrate that CSE stimulation could increase theproduction of several MMPs, especially the MMP-1(collagenase) in ASM cells fromCOPD patients, and TGF-β1could promote the soluble FBLN-1to incorporate intoECM and thus implicated the role of FBLN-1in airway fibrosis. Furthermore, COL8A1was found to express differently between the COPD and non-COPD airway tissues,suggesting an important role of COL8A1in airway remodeling. These provide newevidence as regards the contribution of cigarette smoking to the pathogenesis of COPD,notably by influencing the degration function of MMPs in airway remodeling andultimately leading to airflow limitation.