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Kyuyeon Han, Jin-Hong Chang, Dimitri T Azar; Proteomics-based characterization of the influence of MMP14 on the protein content of corneal fibroblast-derived exosomes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):945.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effect of metalloproteinase 14 (MMP14) on exosomal proteins contained within corneal fibroblast-derived exosomes.
Exosomes were isolated via precipitation and serial ultracentrifugation from wild-type (WT) and MMP14 exon4-deficient (Δexon4)corneal fibroblasts. Theamounts of secreted exosomes and their size distribution were evaluated by nano tracking analysis.The presence of exosomal marker protein was determined by western blotting. The protein content of WT fibroblast-derived and MMP14 exon4-deficient fibroblast-derived exosomes was compared via Coomassie staining of SDS-PAGE gels. Profiling of different proteins was achieved by LC-MS/MS proteomics. Flow cytometric analysis was used to study the fusion of exosomes to human umbilical vein endothelial cells (HUVECs).
Exosomes from WT and Δexon4fibroblastshad a similar size distribution and morphology. WT corneal fibroblasts secreted higher numbers of exosomes than Δexon4 fibroblasts. ΔExon4 fibroblast-derived exosomes contained more exosomal proteins than WT fibroblast-derived exosomes. Proteomics analysis identified 155 proteins uniquely found in WT fibroblast-derived exosomes and 63 proteins uniquely found Δexon4 fibroblast-derived exosomes. WT fibroblast-derived exosomes had a lower fusion rate to HUVECs than Δexon4 fibroblast-derived exosomes.
The unique compositions of the exosomes according to the presence of MMP14 suggest that MMP14 expression influences the protein composition of corneal fibroblast-derived exosomes and may thereby regulate the fusion of exosomes to endothelial cells, ultimately affecting angiogenesis within the cornea.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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