Abstract
Purpose :
A single point mutation (R345W) in fibulin-3 causes an early-onset macular degeneration. The purpose of current study was to study the effects of R345W-fibulin-3 expression on RPE cell polarity. As emerging evidence suggests that exosomes and exosomal microRNAs play roles in intercellular communication, our next goal was to define the exosome cargo in RPE cells expressing WT-fibulin-3 versus RPE cells expressing R345W-fibulin-3.
Methods :
Trans-epithelial electrical resistance (TER) and zonula occludens-1 (ZO-1) expression were monitored in primary RPE cells to study the effects of R345W mutation on RPE cell polarity. Changes in the expression of RPE signature genes were studied by performing RT-PCR. Exosomes were isolated from ARPE-19 cell culture media and primary human RPE cell culture media by density gradient ultracentrifugation and analyzed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita). Small RNA-seq libraries were generated by using NEXTflex Small RNA Sequencing Kit v3 (Bioo Scientific), followed by deep sequencing (Illumina HiSeq 2500).
Results :
The expression of R345W-fibulin-3 reduced polarity and attenuated differentiation in primary RPE cells, as evidenced by decreased TER, reduced ZO-1 expression, and down-regulated RPE signature gene expression. Pathway analysis revealed that exosomes derived from ARPE-19 cells overexpressing WT-fibulin-3 contained critical members of sonic hedgehog signaling (Shh) and ciliary tip components. In contrast, exosomes derived from ARPE-19 cells overexpressing R345W-fibulin-3 contained lysosome components. miRNA-204 and miRNA-211 were enriched in exosomes derived from primary RPE cells expressing WT-fibulin-3.
Conclusions :
Our data indicate that exosomes derived from RPE cells expressing WT-fibulin-3 contain Shh signaling and miRNA species, respectively, that are critical for the development and/or maintenance of RPE cell polarity and differentiation, whereas mutant fibulin-3 leads to reduced polarity and less differentiation in RPE cells by loss of exosome-mediated hedgehog and miRNA204/211 paracrine signaling.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.