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Jingwen Cai, Mariam Lotfy Khaled, Yutao Liu; Proteomics Analysis of LOXL1-containing Exosomes Secreted by Len Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4910. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Exfoliation syndrome (XFS) is the most common cause of secondary glaucoma. XFS is characteristic with the accumulation of exfoliation material in the anterior chamber including lens. Several environmental factors including TGF-b, IL-6, hyperhomocysteine, and oxidative stress, may contribute to the pathogenesis of XFS. We aimed to study how these environmental factors affect the proteomic content of exosomes secreted by lens epithelial cells.
Lens epithelial cell line B3 reached 80% confluence before changing to serum-free condition media at different conditions, non-treated control, TGF-b (10ng/ml), IL-6 (50ng/ml), hyperhomocysteine (500mM), and H2O2 (250mM). Exosomes were isolated using the Total Exosome Isolation Reagent from Invitrogen. The isolated exosomes were characterized using nanoparticle tracking analysis (NTA) with ZetaView for their size and particle concentration. Exosomes was freeze-dry and lysed for proteomics analysis using mass spectrometry after trypsin digestion. Lists of measured peptide masses were used to identify the protein components in the isolated exosomes. Extracellular proteins including LOXL1 and its interaction partners were examined. Levels of LOXL1 protein in cellular lysate from B3 cells under the four treatment conditions were examined using ELISA assay.
NTA data indicated the isolated exosomes with a typical size distribution (50-150 nm). Proteomics analysis indicated the presence of several exosome protein markers, including CD44, CD81, FLOT1, and HSPA8. While LOXL1 protein was not actively secreted under normal condition, IL-6 treatment increased secretion of LOXL1 into exosomes. Other extracellular proteins[l1] , such as EFEMP1 and DCN, were increased under different treatments. IL-6 and TGF-β treatments promoted the secretion of FBLN5 and RELN, while hyperhomocysteine and oxidative stress induced the secretion of FBLN1, LTBP1, and BMP1. Secretion of fibronectin was dramatically increased with IL-6 treatment. [l2] The results from the ELISA assay for LOXL1 indicated that production of LOXL1 significantly increased under treatment of IL-6 and H2O2.
We have characterized the protein components in secreted exosomes from lens epithelial cells under the influence of XFS-related environmental factors. This study will advance our understanding of the molecular mechanism of XFS.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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