Human aqueous humor exosomes

Abstract

Aqueous humor (AH) is a dynamic intraocular fluid that supports the vitality of tissues that regulate intraocular pressure. We recently discovered that extracellular nanovesicles called exosomes are a major constituent of AH. Exosomes function in extracellular communication and contain proteins and small RNA. Our goal was to characterize the physical properties of AH exosomes and their exosomal RNA (esRNA) content. We isolated exosomes from human AH collected during cataract surgery from five patients using serial ultracentrifugation. We measured the size and concentration of AH exosomes in solution using nanoparticle tracking analysis. We found a single population of vesicles having a mean size of 121 ± 11 nm in the unprocessed AH. Data show that centrifugation does not significantly affect the mean particle size (121 ± 11 nm versus 124 ± 21 nm), but does impact the final number of exosomes in solution (87% loss from the unprocessed AH; n = 5). We extracted esRNA from the pooled human AH samples using miRCURY RNA isolation kit from Exiqon. The quality of extracted esRNA was evaluated using Agilent Bioanalyzer 2100 and was used to generate a sequencing library for small RNA sequencing with Illumina MiSeq sequencer. More than 10 different miRNAs were identified; abundant species included miR-486-5p, miR-204, and miR-184. We found that the majority of extracellular vesicles in the AH were in the exosome size range, suggesting that miRNAs housed within exosomes may function in communication between AH inflow and outflow tissues.

Keywords: Aqueous humor; Exosomal RNA; Exosome; Glaucoma; miRNA.

Figure 1

Human aqueous humor contains a single nanovesicle population with a size characteristic of exosomes. A) Flow chart detailing our experimental design. Each aqueous humor sample from a single patient was equally split 3 ways and processed as shown. Specifically, AH samples were centrifuged at low speed (10,000×g for 30min at 4°C in a Beckman TLS-55 rotor) to pellet cell debris and other large contaminants. This cleared supernatant was then centrifuged at high speed (100,000×g for 60min at 4°C in the same rotor) to pellet extracellular vesicles. This vesicle pellet was re-suspended in PBS and centrifuged again at high speed to remove any contaminants. Size and concentration of the vesicle populations in these three portions were measured by nanoparticle tracking analysis. B) A histogram showing the vesicle size versus concentration resulting from the vesicle isolation steps (mean of 5 individual samples).

Figure 2

Characterization of exosomal RNA (esRNA) extracted from purified nanovesicles from fresh human aqueous humor. The esRNA was evaluated using Agilent Bioanalyzer 2100 with Agilent RNA 6000 Pico Kit. A) Shows the RNA ladder and B) displays extracted esRNA from one of the human aqueous humor samples. The size of extracted esRNA was less than 200 nucleotides (nt), mostly around 25 nt. C) shows the miRNAs that were identified via small RNA sequencing using Illumina MiSeq sequencing system. The height of each bar indicated the number of sequence reads with perfect match to mature miRNA.