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. 2023:3:1257737.
doi: 10.3389/fopht.2023.1257737. Epub 2023 Oct 6.

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells

Affiliations

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells

Fiona S McDonnell et al. Front Ophthalmol (Lausanne). 2023.

Abstract

Introduction: Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes.

Methods: EVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting.

Results: Results showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronectin and EDIL3 were significantly less abundant in glaucomatous EVs (<0.3 fold change across all groups) compared to non-glaucomatous EVs.

Discussion: Overall, these data establish that ECM materials are prominent proteomic cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma.

Keywords: conventional outflow pathway; extracellular matrix; extracellular vesicles; glaucoma; trabecular meshwork.

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Conflict of interest statement

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Properties of EVs isolated from glaucomatous and non-glaucomatous TM cells. (A) Concentration of EVs isolated from TM cells (106 particles/mL) as measured by nanoparticle tracking analysis (B) Diameter of EVs isolated from TM cells as measured by nanoparticle tracking analysis. (C) Western blots showing EV markers CD9 and TSG101 in fractions 5-8 and 9-10, and negative EV control markers Calnexin and Albumin in cell lysate samples only.
Figure 2
Figure 2
Venn diagrams summarizing the proteomic datasets. Top panel: all verified proteins from NTM and GTM EVs. Bottom panel: top 100 most abundant proteins in each dataset for further analysis. NTM, Normal Trabecular Meshwork; GTM, Glaucomatous Trabecular Meshwork.
Figure 3
Figure 3
Analysis of proteomic dataset by protein class showing differences in ECM proteins between EV subpopulations. (A–C) Non-glaucomatous TM EVs. (A) Pooled fractions 5–10; (B) pooled fractions 5–8; and (C) pooled fractions 9 and 10. (D–F) Glaucomatous TM EVs. (D) Pooled fractions 5–10; (E) pooled fractions 5–8; and (F) pooled fractions 9 and 10.
Figure 4
Figure 4
Differences in the expression of candidate ECM proteins between non-glaucomatous and glaucomatous EVs. Fibronectin is decreased in glaucomatous EVs compared with non-glaucomatous EVs. EDIL3 is decreased in glaucomatous EVs compared with non-glaucomatous EVs. The band intensity was normalized to the number of particles in each sample. *p < 0.05, **p < 0.01, Student’s t-test. F, fractions.

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References

    1. Quigley HA. Neuronal death in glaucoma. Prog Retin Eye Res (1999) 18(1):39–57. doi: 10.1016/S1350-9462(98)00014-7 - DOI - PubMed
    1. AGIS Investigators . The advanced glaucoma intervention study (AGIS): 7. the relationship between control of intraocular pressure and visual field deterioration. Am J Ophthalmol (2000) 130(4):429–40. doi: 10.1016/s0002-9394(00)00538-9 - DOI - PubMed
    1. Bill A. Basic physiology of the drainage of aqueous humor. Exp Eye Res (1977) 25 Suppl:291–304. doi: 10.1016/S0014-4835(77)80025-0 - DOI - PubMed
    1. Bradley JM, Kelley MJ, Zhu X, Anderssohn AM, Alexander JP, Acott TS. Effects of mechanical stretching on trabecular matrix metalloproteinases. Invest Ophthalmol Vis Sci (2001) 42(7):1505–13. doi: 10.1167/iovs.06-0875 - DOI - PubMed
    1. Acott TS, Kelley MJ. Extracellular matrix in the trabecular meshwork. Exp Eye Res (2008) 86(4):543–61. doi: 10.1016/j.exer.2008.01.013 - DOI - PMC - PubMed