Abstract
Purpose :
Numerous reports have shown that extracellular vesicles, also known as exosomes, contain "essential" proteins reflective of the cytosolic environment, including cytoskeletal, heat shock, metabolic, and ribosomal proteins. However, the role of growth factors encapsulated in exosomes on corneal wound healing remains unknown. The objective of this study was to evaluate the protein content and functionality of exosomes isolated from corneal epithelium pre- and post-wounding to determine if exosomal cargo is altered following injury that may contribute to myofibroblast differentiation.
Methods :
Confluent corneal epithelial layers were generated in vitro using the corneal human cell line, HCE-TJ, and wounded via scratch. Secreted extracellular vesicles were isolated from unwounded and wounded epithelium using ultracentrifugation approaches followed by proteomic analysis by LC/MS-MS. We further assessed the effects of epithelial-derived exosomes compared to epithelial-conditioned media post-wounding on human corneal fibroblasts (HCFs) and evaluated protein expression of the myofibroblast marker, α-SMA, by IHC and Western blot.
Results :
In isolated exosomes, we identified a broad distribution of over 556 proteins detected by proteomics. ECM-associated proteins were the most abundant class identified in the HCE-derived samples, including fibronectin, thrombospondin, laminin, and collagen. Heat-shock proteins also contributed to the dominant class of proteins, as well as endocytic/vesicle-trafficking proteins, such as the Rab proteins and dynein. Protein expression varied little between unwounded and wounded epithelium. Importantly, very few growth factors were identified in secreted exosomes, suggesting that many of the factors secreted by the corneal epithelium in response to injury, such as TGF-β, PDGF, IL-1, and TNF-α, are likely secreted as soluble proteins. We identified slight increases in α-SMA expression in HCFs following stimulation with wounded epithelial-exosomes and conditioned media, consistent with myofibroblast differentiation.
Conclusions :
Exosomal protein content pre- and post-wounding was high in heat-shock, ECM-associated, and vesicle-trafficking proteins. Myofibroblast differentiation following epithelial wounding may be promoted by factors secreted from the epithelium (both free-floating and encapsulated within exosomes).
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.