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Jennifer A Tran, Xiaoqing Q Guo, Sriniwas Sriram, Audrey E K Hutcheon, James D Zieske; Role of epithelial cell-derived exosomes in corneal wound healing. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4899.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies from our lab and others have demonstrated that wound-healing epithelium is essential in the stimulation of stromal fibrosis and myofibroblast formation. Exosomes are functional cell-derived nanovesicles that have become the subject of increasing interest in recent years since they may play a role in protein, mRNA, and miRNA transport, as well as wound repair. In this study, we treated differentially cultured human corneal fibroblasts (HCF) with epithelial cell-derived exosomes to determine their effect on HCF morphology and potential to stimulate fibrosis.
Exosomes were extracted from epithelial cell line culture media, ultracentrifuged at >100,000 x g (Beckman Coulter) for 1 hour, labeled with the lipophilic fluorescent dye (PKH26: Sigma-Aldrich), washed, and applied to HCF cultured in basic medium (BM: EMEM + 10% FBS) with the following conditions for up to 7 days: 1) Control: BM only, 2) T1: BM + 2ng/ml TGF-β1, and 3) T3: BM + 2ng/ml TGF-β3. HCF cell morphology and exosomal uptake patterns were visualized 2, 24, and 48 hours post-exosomal application with an inverted fluorescent microscope.
Control HCF revealed exosomal uptake patterns consistent with elongated, fibroblast-like morphologies, while those with T1 or T3 treatment had uptake patterns characterized as shorter and more contracted, similar to myofibroblasts in cell culture. In addition, HCF samples demonstrated time-dependent uptake of exosomes, whereby the amount of particles taken up by HCFs seemed to increase with time, with the brightest staining appearing at 48 hours post-exosomal application.
These results show that exosomes can be purified from epithelial cells and HCF can take up molecular information from epithelial cells via exosomal communication. This is the first time this has been demonstrated in corneal cells, thus providing further insight into how the different corneal layers may interact in vivo. Further studies will be done to elucidate the roles exosomes may play in corneal wound healing.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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