Abstract
Purpose :
Extracellular vesicles (EV) are detectable in aqueous humor (AH) from adult patients in previous studies. However, the presence of EVs in AH from pediatric diseased eyes has not been explored. Also, the expression profile of exosomal tetraspanins (CD9, CD63 and CD81) on AH EVs remains unclear. Herein, we measure the concentration of the pediatric diseased eye-derived AH EVs and analyze their size distribution and the phenotypic expression levels of exosomal tetraspanins of AH EV at single vesicle resolution.
Methods :
AH samples were obtained from 3 congenital cataract, 3 congenital glaucoma and 5 retinoblastoma (RB) eyes. Plasma samples were obtained from 2 RB patients. Unprocessed, enrichment-free 10uL AH samples or 1uL plasma samples were subjected to Nanoparticle Tracking Analysis (NTA) (Nanosight NS300, Malvern Panalytical) for size distribution and concentration, and to Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100, Nanoview Biosciences), for interferometric sizing and fluorescent-based immunophenotyping of exosomal marker expression (CD81, CD9, and CD63, including CD41a, which detects platelet derived vesicles).
Results :
By NTA, the concentration of AH EVs is within 2.65E+09-1.58E+10 particles/mL with a major population in a mean modal size of 93.3 ± 19.8 nm across diseases in contrast to the 1.55E+11-2.56E+12 particles/mL with a mean modal size of 60.2 ± 2.1 nm detected in plasma samples. SP-IRIS analysis revealed that CD9+, CD63+ and CD81+ EVs were detected across all AH samples using the Exoview R100 platform. Platelet-derived CD41a+ EVs could only be detected in the plasma EVs. In addition, we identified an enrichment of a single-positive CD63+ EV population in AH across all disease groups (50.9% and 13.6% CD63+ EV among all fluorescent positive EVs in AH and plasma, respectively) suggesting a unique phenotypic exosomal tetraspanin expression pattern in AH.
Conclusions :
Exosome size ranged-EVs are readily detectable in unprocessed AH with a dominant mono-CD63+ EV in AH regardless of pediatric eye disease types. These novel findings uncover a new path on AH exosomal biomarker research focusing on CD63+ EVs.
This is a 2021 ARVO Annual Meeting abstract.