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Jingwen Cai, Yan Yu, Yutao Liu; The effect of XFS-related factors on secreted extracellular vesicles from lung fibroblasts. Invest. Ophthalmol. Vis. Sci. 2020;61(7):5017.
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Exfoliation syndrome (XFS) is characterized with exfoliation material deposition in the eye and other organs, including lung. We aimed to determine the impact of XFS-related factors (TGFβ1, IL6, & glucose oxidase) on extracellular vesicles (EVs) derived from lung fibroblasts.
The lung fibroblast IMR90 cells were treated with TGFβ1 (10ng/ml), IL6 (50ng/ml), or glucose oxidase (GO) (8mU/ml) in FBS-free media for up to 48 hours. EVs were isolated using differential ultracentrifugation of 110,000g X 70 minutes twice. The resuspended EVs were analyzed with ZetaView and transmission electron microscopy (TEM) for particle size, concentration and morphology. The EVs from 48-hour treatment were used for mass spectrometry-based proteomic analysis (n=6) to identify differentially expressed (DE) proteins. Cellular lysates in 48-hour treated groups were also subjected to proteomics analysis (n=3) to identify DE cellular proteins under each treatment. The statistical analyses were performed using student t-test.
EVs in all groups showed similar size range (100-150nm) and round, cup-shaped morphology under TEM. After 48-hour, all treated groups produced significantly more EVs than control groups. Proteomic analysis detected >30 exosome markers in each sample including CD63, CD81 and HSPA8. With TGFβ1 treatment, 45 EV DE proteins were enriched in focal adhesion, response to stress, and ECM. With IL6 treatment, 29 EV DE proteins were enriched in vesicle-mediated transport, lipid binding, and cytoskeleton regulation. With GO treatment, 13 DE proteins were enriched in response to wounding, collagen fibril organization, and cellular adhesion. The IPA identified TGFβ1, IL6R, and IGF2BP1 as upstream regulators of DE proteins in TGFβ1, IL6, and GO groups, respectively, further validating the relationship between treatment and EV DE proteins. Moreover, the DE proteins identified from cell lysates were enriched in similar classifications of EV DE proteins, such as focal adhesion and ECM-receptor interaction with TGFβ1 treatment, cytoskeletal protein binding with IL6 treatment, and extracellular organization with GO treatment, indicating the response of EVs to cellular changes.
XFS-related factors promoted EV release from lung fibroblasts with specific enriched proteins. These EVs may contribute to the ECM remodeling and serve as biomarkers for XFS.
This is a 2020 ARVO Annual Meeting abstract.
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