M. Fahrenholtz1, X. Wang1, H. Li1, Y. Dhamija1, P. Duann1, M. Rae1, K. Grande-Allen2, S. Keswani1, S. Balaji1 1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Rice University,Bioengineering,Houston, TX, USA
Introduction: Mid gestation fetus heals cutaneous wounds without scar and its anti-fibrotic phenotype is defined by negligible resting tension and distinct extracellular matrix (ECM) with elevated levels of hyaluronan (HA) produced by fetal fibroblasts. In contrast, adult skin is characterized by relatively low levels of HA, and much higher resting tension. Biomechanical tension induces a pro-fibrotic phenotype in fibroblasts which is characterized by increased inflammatory cytokine and ECM production, which leads to the formation of scar tissue in vivo. The role of HA production by fibroblasts under biomechanical stretch has not been fully examined. We hypothesize that higher mechanical tension will alter HA metabolism of fibroblasts via differential regulation of HA synthases (HAS1-3) and hyaluronidases (Hyal1-2).
Methods: Primary murine adult dermal fibroblasts (AFb) were cultured on collagen-coated silicone membranes +/-10% static strain. AFb expression of HA synthesis(HAS1-2), remodeling(HYAL1-2), and receptor(CD44) genes, as well as phenotype(ASMA) were assessed by qPCR. Total HA production was measured by ELISA. Data is presented as mean+/-SD, n=3/group, p values by ANOVA with post-hoc Tukey HSD.
Results: Mechanical tension induced differential HA gene regulation in AFb, with significantly increased HAS1 gene expression (1.67+/-1.03 vs 5.13+/-0.70, p < 0.01) and decreased HAS2 expression (0.92+/-0.10 vs 0.063+/-0.009, p < 0.01), but no change in HAS3 expression after 24h application of stretch, as compared to unstretched condition. Both Hyal1 and Hyal2 were down-regulated under tension (1.72+/-0.84 vs 0.17+/-0.10, p < 0.05, and 1.64+/-0.70 vs 0.13+/-0.05, p < 0.01, respectively). Total HA quantification at 24 h showed no significant influence of stretch on AFb production of HA, despite differential regulation of HA synthesis and HA remodeling genes, indicating a need to assess additional time points and hyaluronidase activity. ASMA gene expression was not influenced by tension at 24h. Tension downregulated CD44 gene expression (1.18+/-0.32 vs 0.33+/-0.08, p < 0.05), which may influence AFb ability to interact with HA produced under mechanical stretch.
Conclusion: Our data suggest that biomechanical forces have a significant role in influencing the dermal fibroblasts’ cell-matrix interaction and their regulation of the ECM-specifically HA. Understanding the time course of these effects on fibroblast responses to tension, especially under the influence of exogenous wound factors and chemokine imbalance, may yield novel therapeutic interventions to promote anti-fibrotic healing. Understanding the contribution of mechanical environment via morphological and phenotypic alterations may yield novel therapeutic targets in recapitulating fetal regenerative healing in postnatal tissues.