Analysis of extracellular vesicles in vitreous samples

Abstract

 

Purpose

 

Extracellular vesicle (EV) secretion represents an evolutionally conserved feature of living cells. EVs are known to transfer protein and RNA cargos between cells placing EV analysis into the mainstream of biomedical research. The assessment of EVs may provide insight into the pathomechanism of various disorders. Furthermore, they may not only serve as potential novel biomarkers, but also therapeutic targets and vehicles. EVs were previously shown to participate in angiogenesis, tissue remodeling and tissue regeneration. In this work we aimed at first time characterization of protein and RNA profiles of EVs isolated from human vitreous samples.

 

Methods

 

We analyzed EVs from vitreous fluid collected during vitrectomy from patients with proliferative diabetic retinopathy and primary rhegmatogenous retinal detachment. EVs were isolated using differential centrifugation, and were visualized by transmission electron microscopy (TEM). Size histograms of EV preparations were determined by a resistive pulse sensing approach (qNano). Cellular origin of EVs was determined by flow cytometry (FC). Protein and RNA profiles of EVs were analyzed by mass spectrometry (MS) and bioanalyzer assay (Agilent).

 

Results

 

TEM clearly shows various populations of EVs in the vitreous fluid (Figure 1). Peak EV size was around 150 nm in diameter. The presence of EVs in vitreous fluid was also confirmed using FC based on annexin V binding. Most EVs in the vitreous fluid were derived from platelets and endothelial cells. MS revealed classical EV-associated proteins including actin, actin-binding proteins (e.g. ankyrin), tubulin, clusterin, heat shock proteins and enzymes. However, we also identified eye-specific proteins in EVs including retinal dehydrogenase, retinol binding protein and lens specific proteins (lensin, crystallin etc.). Most importantly RNA profiling has revealed that miRNA molecules were present in vitreous-fluid-derived EVs in very high amounts (Figure 2).

 

Conclusions

 

In this work we successfully isolated and characterized EVs from vitreous fluid, and demonstrated the presence of miRNAs in these structures. Demonstration of angiogenesis-inducing miRNAs in vitreous fluid EVs may lead to identification of new biomarkers or novel therapeutic targets in eye disorders.

 

 

Figure 1. EVs visualized by TEM. Magnification is 30,000x

 

 

Figure 2. RNA profile from EVs showing mostly small RNA molecules.

 

Keywords: 763 vitreous