18th Annual Meeting of the Wound Healing Society SAWC/WHS Joint Meeting

Abstract

Introduction: The oral mucosa is distinct in that it demonstrates preferential healing with little/no scarring. In skin/internal organs, repair by scarring can lead to fibrous tissue accumulation, resulting in disability and organ dysfunction. Fibroblast to myofibroblast differentiation plays an important role in scarring and is associated with an accumulation of the matrix polysaccharide hyaluronan (HA). This study aims to understand the role of HA in influencing scarring by comparing its metabolism in fibroblasts with a non-scarring phenotype (oral mucosal fibroblasts; OMF) to patient-matched dermal fibroblasts (DF). Methods: OMF and DF (n=4) differentiation to myofibroblasts was induced by TGFb1-stimulation and assessed by a-smooth muscle actin expression (QPCR/immunocytochemistry). A range of techniques were used to asses HA generation, assembly, HA Synthase (HAS) expression, Smad-signalling and proliferation including metabolic radio-labelling, column chromatography, western-blots, 3H-thymidine incorporation and microarray analysis. HA synthesis was inhibited using 4-methyl-umbelliferone depletion of UDP-glucuronic acid and siRNA technology was used to down-regulate Smad3 expression. Results: The fibroblast populations demonstrated distinct differences in their TGFb1-responses. DF readily differentiated to myofibroblasts and demonstrated a proliferative response to TGFb1, whereas OMF were resistant to differentiation and demonstrated an anti-proliferative response to TGFb1. In DF, differentiation was associated with increased HA generation, HA pericellular coat assembly and increased HAS1&2 transcription. In contrast, resistance to differentiation in OMF was associated with reduced HA generation and inability to induce pericellular coat assembly or HAS1&2 transcription. Inhibiting HA synthesis in DF significantly attenuated differentiation, induced an anti-proliferative response to TGFb1 and attenuated Smad3-signalling. IL- 1b stimulation in DF induced HA synthesis and HAS1&2 transcription, however did not induce pericellular coat assembly or differentiation, suggesting that HA coat assembly is specifically required for differentiation. Both fibroblast populations demonstrated Smad3 phosphorylation; however, downregulation of Smad3 (siRNA) led to loss of the TGFb1-mediated proliferative response in DF whereas it led to loss of the TGFb1-mediated anti-proliferative response in OMF. Discussion: Both fibroblast phenotypes respond to TGFb1. However, the levels of HA generated by the cells influences the outcome of this response such that increased HA facilitates TGFb1-mediated proliferation and differentiation. This represents an important target for future anti-scarring research.