Abstract
Purpose :
We have previously reported that elevated levels of GFAP and vimentin in the vitreous humor (VH) from patients with proliferative vitreoretinopathy (PVR) were significantly correlated with PVR severity. To explore how intracellular structural proteins such as GFAP and vimentin, hallmarks of reactive gliosis, were released into the VH during the development of PVR, we performed proteome analysis of exosomes isolated from VH in PVR patients.
Methods :
Exosomes were isolated from undiluted VH from patients with PVR(N=4), Macular hole (MH; N=5), or epiretinal membrane (ERM; N=5) using differential ultracentrifugation. We confirmed their size, morphology, and exosome markers using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and an exosome detection antibody array. Tryptic fragment sequencing of exosome contained proteins was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) and a Thermo Lumos Fusion tribrid Orbitrap mass spectrometer. The pathway analysis of MS data was performed using Ingenuity Pathway Analysis (IPA).
Results :
We recovered a range of 1.5-4.0 x 109 exosomes per vitreous sample (500 μL), characterized by their size (peak ~131 nm), morphology, and positive exosome markers including FLOT1, ICAM, ALIX, EpCAM, ANXA5, TSG101, CD81 and CD63 (Figure 1). We observed a large pool of GFAP and vimentin in VH of PVR patients was contained in the vitreal exosomes. We also identified 627 proteins from vitreal exosomes (533, 431, and 318 in PVR, MH, and ERM, respectively). Of the 94 exosomal proteins related to “reactive gliosis” and “gliosis of retina,” 42 (45%) exosomal proteins were only present in PVR (Figure 2A). The gliosis-related exosomal proteins in PVR were also connected with key pathways related to PVR development, including inflammation, epithelial-mesenchymal transition, tissue healing, cellular proliferation, and growth of connective tissue (Figure 2B).
Conclusions :
Our results suggest that vimentin and GFAP were released into the VH in a controlled fashion by exosomes in association with PVR development and the exosome mediated reactive gliosis may play a key role in PVR formation.
This is a 2021 ARVO Annual Meeting abstract.