Proteomic Insight into the Role of Exosomes in Proliferative Vitreoretinopathy Development

Abstract

Purpose: To characterize vitreous humor (VH) exosomes and to explore their role in the development of proliferative vitreoretinopathy (PVR) using mass spectrometry-based proteome profiling. Methods: Exosomes were isolated from undiluted VH from patients with retinal detachment (RD) with various stages of PVR (n = 9), macular hole (MH; n = 5), or epiretinal membrane (ERM; n = 5) using differential ultracentrifugation. The exosomal size, morphology, and exosome markers were analyzed using a nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and an exosome detection antibody array. The 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 the MS data was performed. Results: The number of exosome particles were significantly increased only in the RD with severe PVR group compared with the control groups and the RD without PVR or with mild PVR groups. Of 724 exosome proteins identified, 382 were differentially expressed (DE) and 176 were uniquely present in PVR. Both DE proteins and exosome proteins that were only present in PVR were enriched in proteins associated with previously known key pathways related to PVR development, including reactive retinal gliosis, pathologic cellular proliferation, inflammation, growth of connective tissues, and epithelial mesenchymal transition (EMT). The SPP1, CLU, VCAN, COL2A1, and SEMA7A that are significantly upregulated in PVR were related to the tissue remodeling. Conclusions: Exosomes may play a key role in mediating tissue remodeling along with a complex set of pathways involved in PVR development.

Dynamics of MicroRNA Secreted via Extracellular Vesicles During the Maturation of Embryonic Stem Cell-Derived Retinal Pigment Epithelium

“…This observation suggests a potentially biologically active status of cells in terms of sEV secretion during mid-stage maturation (28-39 days in culture), which aligns with peak therapeutic efficacy in hESC-RPE cell transplantation therapy, as opposed to earlier or later stages (Davis et al, 2017). X Notably, our previous research demonstrated higher EV particle numbers in vitreous samples from proliferative vitreoretinopathy (PVR) patients compared to non-PVR patients, supporting numbers of EV secretion is regulated either in physiologic or pathologic conditions (Nair et al,2022). 40 Despite differences in particle numbers, other characterizations of EVs, including size, morphology, and comprehensive EV profiles of the three groups of hESC-RPE-sEV, were comparable.…”

Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy

“…Human embryonic stem cells (WA09; WiCell Research Institute, Madison, WI, USA) were spontaneously differentiated into RPE cells as previously described. 24 Briefly, colonies of hESCs were manually passaged and seeded onto tissue culture plates coated with Matrigel hESC-Qualified Matrix (#354277; Corning, Corning, NY, USA), and they received biweekly medium exchanges of XVIVO-10 culture medium (#(BE) BP04-743Q; Lonza, Walkersville, MD, USA) for approximately 3 months. Pigmented regions were then manually isolated, expanded for two passages, and cryopreserved at 2 to 5 days postseed as a cellular suspension using CryoStor10 cryopreservation medium (#210102; BioLife Solutions, Bothell, WA, USA).…”

“…EV separation using dUC was completed as previously shown. 10 , 24 Varying input volumes of CCM (5 mL, 15 mL, 20 mL, and 30 mL) were centrifuged at 25,000 × rpm (64,000 × g ) for 60 minutes at 4°C in a Beckman Coulter Optima LE-80K (Brea, CA, USA) ultracentrifuge with rotor SW55 Ti. Then the supernatant was subjected to ultracentrifugation at 41,000 × rpm (173,000 × g ) for 120 minutes at 4°C.…”

Intraocular RGD-Engineered Exosomes and Active Targeting of Choroidal Neovascularization (CNV)

“…The retinas from 4–6-week-old wild-type C57BL/6J mice were extracted and digested using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) according to the manufacturer’s protocol. Exosomes were recovered using differential ultracentrifugation as previously described [ 26 ]. In brief, the supernatant received from the digestion process was mixed with cold HBSS and centrifuged at 25,000× RPM for 60 min at 4 °C in a Beckman Coulter Optima L80-XP ultracentrifuge with rotor SW60 Ti.…”