Abstract
Purpose :
To investigate the role of misfolded proteins in retinal pigment epithelial (RPE) dysfunction. The effects of R345W-Fibulin-3 expression on RPE cell phenotype and extracellular vesicle (EV) content were studied.
Methods :
Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type or R345W-Fibulin-3 or tag only as control. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of the growth factor, vascular endothelial growth factor (VEGF), was measured by ELISA assays. Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells. Conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by migration assay. EVs were isolated from ARPE-19 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 :
In primary RPE cells expressing R345W-Fibulin-3, ZO-1 distribution was discontinuous and TER values were significantly lower compared to RPE cells expressing WT-Fibulin-3 (p<0.01). VEGF secretion was attenuated in basal, but not apical media, whereas Fibulin-3 secretion was reduced in both the apical and basal directions in the R345W-Fibulin-3 cells (p<0.01). All of the RPE signature genes measured were downregulated while genes associated with EMT were upregulated in the R345W-Fibulin-3 cells (p<0.01). Migration assays revealed a higher recovery rate in the ARPE-19 cells overexpressing R345W-Fibulin-3, compared to controls (p<0.01). Proteomic studies identified abundant EMT-promoting proteins in EVs derived from the R345W-Fibulin-3 cells. Moreover, miRNA-204/211, known to play a critical role in promoting RPE differentiation, are highly expressed in terminally differentiated primary RPE cells compared to controls.
Conclusions :
Our data suggest that expression of the R345W-Fibulin-3 point mutation promotes EMT and alters EV content in RPE cells.
This is a 2020 ARVO Annual Meeting abstract.