Latha Satish1,2, Alexander Evdokiou1, Eleni Geletu1, Jennifer M Hahn1, Dorothy M Supp1,3. 1. Research Department, Shriners Hospitals for Children-Cincinnati, Cincinnati, OH 45229 USA. 2. Department of Pathology & Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45229, USA, and. 3. Department of Surgery, University of Cincinnati, Cincinnati, OH 45229, USA.
Abstract
BACKGROUND: Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. METHODS: Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-β1; 10 ng/mL), with or without pirfenidone (400 μg/mL), for an additional 24 h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. RESULTS: We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000 μg/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000 μg/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-β1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-β1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400 μg/mL) in both normal and keloid keratinocytes. CONCLUSIONS: For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence. Published by Oxford University Press 2020.
BACKGROUND: Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. METHODS: Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-β1; 10 ng/mL), with or without pirfenidone (400 μg/mL), for an additional 24 h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. RESULTS: We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000 μg/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000 μg/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-β1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-β1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400 μg/mL) in both normal and keloid keratinocytes. CONCLUSIONS: For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence. Published by Oxford University Press 2020.