Mechanical Tension Influences The Regulatory Landscape Of MSC-derived Exosomes During Wound Healing
Hima Vangapandu1, Matthew Robertson1, Daniel Colchado1, Natalie Templeman1, Hui Li1, Yao Ning1, Alexander Blum1, Paul Bollyky2, Sundeep Keswani1, Cristian Coarfa1, Swathi Balaji1.
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Palo Alto, CA, USA.
Mesenchymal stem cells (MSCs) have a huge therapeutic potential in wound healing. While it is known that the microenvironment affects the MSC secretome, the role of mechanical tension on the extracellular vesicles, namely exosomes, released by MSCs, is not known. We hypothesize that tension regulates MSC exosome production and influences wound healing via paracrine effects on dermal fibroblasts. Human MSCs were cultured on silicone membranes +/-10% tonic strain for 24h and analyzed for phenotypic changes (morphology; alpha-SMA; fibrosis PCR-array). Exosomes were isolated and their size (zetasizer), protein levels(BCA Assay), surface markers(CD63,HSP70,CD9), and genomic cargo(Next-Gen Sequencing(NGS)) were analyzed. Human dermal fibroblasts(hdFB) were treated with MSC-derived exosomes from +/- tension, and changes in gene expression and migration assay were evaluated. p-values by ANOVA;(n=3/group).Tension resulted in the loss of the characteristic morphology of the MSC spindle shape and increased alpha-SMA staining. There was a significant change in ~30/77(>2-fold) fibrotic/inflammation genes with tension. There was an increase in exosomal size distribution and protein cargo under tension (p<0.05). NGS of the exosomes revealed that the abundance of their tRNA increased, whereas the miRNAs and lincRNAs were reduced under tension. KEGG gene pathway analysis of down regulated miRNAs showed enrichment of intracellular/extracellular wound healing processes. The top MSC exosomal-miRs that were downregulated in response to tension corresponded to Hippo(growth/homeostasis), Ras, PI3K(proliferation) and AMPK (energy sensing) signaling. Measurement of the gene expression implicated in wound healing within these pathways revealed that YAP1, GSK3B and PPP2R1A decreased in hdFB upon treatment with MSC-derived exosomes under tension. Interestingly, MSC-derived exosomes under tension increased hdFB migration in a scratch wound assay (p<0.05). Mechanical tension induces a fibrogenic and inflammatory phenotype in MSCs. Understanding how bioactive cargo in MSC exosomes can be regulated by tension and can influence fibroblast behavior will contribute to the development of exosome-based clinical therapies for wound healing and fibrosis.
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