June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The R345W-Fibulin-3 point mutation induces RPE cell epithelial-mesenchymal transition and alters the content of their extracellular vesicles
Author Affiliations & Notes
  • Mi Zhou
    Penn State Hershey Medical Center, Hershey, Pennsylvania, United States
  • Stephanie Grillo
    Penn State Hershey Medical Center, Hershey, Pennsylvania, United States
  • Sarah Weber
    Penn State Hershey Medical Center, Hershey, Pennsylvania, United States
  • Yuanjun Zhao
    Penn State Hershey Medical Center, Hershey, Pennsylvania, United States
  • Han Chen
    Microscopy Imaging Facility, Penn State Hershey Medical Center, Pennsylvania, United States
  • John Hulleman
    Ophthalmology, UT Southwestern Medical Center, Texas, United States
  • Jeffrey Sundstrom
    Penn State Hershey Medical Center, Hershey, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Mi Zhou, None; Stephanie Grillo, None; Sarah Weber, None; Yuanjun Zhao, None; Han Chen, None; John Hulleman, None; Jeffrey Sundstrom, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1502. doi:
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      Mi Zhou, Stephanie Grillo, Sarah Weber, Yuanjun Zhao, Han Chen, John Hulleman, Jeffrey Sundstrom; The R345W-Fibulin-3 point mutation induces RPE cell epithelial-mesenchymal transition and alters the content of their extracellular vesicles. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1502.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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