H. V. Vangapandu1, N. Templeman1, D. Colchado1, A. Blum1, H. Li1, X. Wang1, E. Steen1, P. Bollyky2, S. Keswani1, M. Robertson1, C. Coarfa1, S. Balaji1 1Baylor College Of Medicine,Dept Of Surgery,Houston, TX, USA 2Stanford University,Palo Alto, CA, USA
Introduction: Wound responses involve fibroblast(FB)-mediated scar formation potentiated by mechanical tension. There is significant heterogeneity in how different people scar from similar injuries. Exosomes play an important role in cell communication which governs scarring. It is unknown if heterogeneous scarring is attributable to differences between FBs, how they respond to tension, and their intercellular communication. We hypothesize that there are differences in FB responses to tension that contribute to scar heterogeneity via exosomes.
Methods: Skin and paired scar tissue was obtained from women with C-section scars who underwent abdominoplasty. Scars were classified as "low" or "high" based on VSS (<3 vs. >6). Fetal skin was evaluated as non-scarring control. Sections were histologically stained (H&E; Trichrome). FBs were isolated from normal skin and scar, cultured on silicone membranes +/-10% static strain (24h), and evaluated for differences in proliferation(Ki67), fibrogenic potential(aSMA, fibrosis array) and genes implicated in exosome biogenesis(RAB27a-b;SMPD3). Exosomes from different scar phenotypes were analyzed(size-Zetasizer; NGS), and adoptively transferred into SCID murine skin wounds, and wound repair was evaluated. p-values by ANOVA; (n=3/group).
Results: High-scarring patient scars had thicker collagen bundles with interstitial FBs in dermis. These FBs also had a significantly higher proliferation rate(p<0.05) in vitro as compared to low and non-scarring FBs. PCR-array results showed that twice as many fibrotic genes were altered in scarFB vs skinFB in high-scar patients as compared to low-scar patients. Rab27a-b and SMPD3 expression was different in non-, low- vs. high-scarring patients skinFB. In low-scar patients, gene expression was similar between normal skinFB and scarFB, where as in the high-scar patients, Rab27a and SMPD3 levels were lower in the scarFB as compared to skinFB. Size distribution profile of exosomes was similar in skin and scarFB of low-scar patients. High-scar FBs produced exosomes larger in size and greater in abundance (p<0.05). NGS showed that there are baseline differences in small ncRNA of different FB-derived exosomes. While tension induced differential changes in a-SMA, Rab27a-b and SMPD and exosome size and cargo profiles in skinFB of different patients, notably, tension-induced expression of the normal skinFB was similar to its paired scarFB baseline phenotype. Lastly, non-scarring FB-derived exosomes resulted in regenerative wound phenotype in SCID dorsal wounds, whereas low and high-scar FB derived exosomes induced a corollary severity in scarring.
Conclusion: Our findings suggest that fibroblasts from different scarring phenotypes respond differentially to tension, including changes in exosome production that could influence fibrogenic phenotype and maintain the scar memory. Mining these exosome profiles will lead to novel scar therapeutics.