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Updated: Dec 15, 2025
Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells
Published on: February 10, 2021
Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells
Mi Zhou1, Sarah R Weber1, Yuanjun Zhao1
1Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, PA, United States.
Purpose:
To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied.
Methods:
Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay.
Results:
Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT.
Conclusions:
The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.
Insights
Misfolded Fibulin-3 (R345W-Fibulin-3) disrupts retinal pigment epithelial (RPE) cell function and barrier integrity. This protein mutation promotes an epithelial-mesenchymal transition (EMT) in RPE cells.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Protein misfolding is implicated in various cellular dysfunctions.
- Retinal pigment epithelial (RPE) cells are crucial for retinal health.
- Fibulin-3 is a key protein in extracellular matrix and cellular processes.
Purpose of the Study:
- To investigate the impact of mutant R345W-Fibulin-3 on RPE cell phenotype.
- To understand the role of protein misfolding in RPE cell dysfunction.
Main Methods:
- Primary RPE cells were cultured and infected with lentivirus to express wild-type (WT) or R345W-Fibulin-3.
- Assessed barrier function via zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER).
- Measured vascular endothelial growth factor (VEGF) secretion using ELISA and evaluated differentiation and epithelial-mesenchymal transition (EMT) markers via qPCR and migration assays.
Main Results:
- RPE cells expressing R345W-Fibulin-3 showed disrupted ZO-1 distribution and lower TER compared to WT.
- VEGF secretion was altered, and Fibulin-3 secretion was reduced in mutant cells.
- RPE signature genes were downregulated, EMT-associated genes upregulated, and migration assays indicated faster recovery in mutant RPE cells.
Conclusions:
- Expression of R345W-Fibulin-3 promotes EMT in RPE cells.
- Protein misfolding of Fibulin-3 contributes to RPE cell dysfunction and altered phenotype.
