Negative pressure wound therapy is a widely used treatment for chronic, nonhealing wounds. Surprisingly, few studies have systematically evaluated the cellular and molecular effects of negative pressure treatment on human skin. In addition, no study to date has directly compared recently available single‐use negative pressure modalities to traditional negative pressure devices in a controlled setting. Here we developed a novel large‐scale ex vivo human skin culture system to effectively evaluate the efficacy of two different negative pressure wound therapy modalities. Single‐use and traditional negative pressure devices were applied to human ex vivo wounded skin sheets cultured over a period of 48 hours. Cellular tissue response to therapy was evaluated via a combination of histological analysis and transcriptional profiling, in samples collected from the wound edge, skin adjacent to the wound, and an extended skin region. Single‐use negative pressure wound therapy caused less damage to wound edge tissue than traditional application, demonstrated by improved skin barrier, reduced dermal‐epidermal junction disruption and a dampened damage response. Transcriptional profiling confirmed significantly less activation of multiple pro‐inflammatory markers in wound edge skin treated with single‐use vs traditional negative pressure therapy. These findings may help to explain the greater efficacy of sNPWT in the clinic, while offering a noninvasive system to develop improved NPWT‐based therapies.
BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive, incurable scarring disease of the lungs with a prognosis worse than most cancers. Pathologically, IPF is characterised by upregulation of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1), activation of fibroblasts, and deposition of collagen in the alveolar interstitium. Recent evidence has highlighted the role of senescent type 2 alveolar epithelial cells in the pathogenesis of IPF. In a clinical trial, a treatment regimen containing a corticosteroid drug accelerated pulmonary fibrosis leading to more hospitalizations and deaths, particularly in patients with telomere shortening which drives cellular senescence.AimTo investigate the potential pro-fibrotic actions of corticosteroids on lung epithelial cells in vitro, including effects on cellular senescence and interactions with TGF-β1.MethodsThe synthetic glucocorticoid dexamethasone (DEX) was incubated with A549 and BEAS-2B human lung epithelial cells in the presence or absence of TGF-β1. Cellular senescence was assessed by morphology, senescence-associated beta-galactosidase (SA β-Gal) expression, and qPCR for transcription of senescence-associated molecular markers. Conditioned media were screened for growth factors and cytokines and cultured with human lung fibroblasts. An IPF lung tissue RNA array dataset was re-analysed with a focus on senescence markers.ResultsDEX induced senescence in lung epithelial cells associated with increased p21 (CDKN1A) expression independently of p16 (CDKN2A) or p53 (TP53). DEX amplified upregulation of the pro-fibrotic mediator serpin E1/plasminogen activator inhibitor-1 (PAI-1) in the presence of TGF-β1. The senescence-associated secretory phenotype from lung epithelial cells treated with DEX plus TGF-β1-treated contained increased concentrations of GM-CSF and IL-6 and when incubated with primary human lung fibroblasts there were trends to increased senescence and production of fibrosis markers. Upregulation of senescence markers was demonstrated by analysis of an IPF transcriptomic dataset.DiscussionDEX induces senescence in lung epithelial cell lines in vitro and interacts with TGF-β1 to amplify production of the pro-fibrotic mediator serpin E1 (PAI-1). This may be a mechanism by which corticosteroids promote pulmonary fibrosis in susceptible individuals. Serpin E1/PAI-1 is a potential druggable target in pulmonary fibrosis.
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