Extracellular Vesicle -Mediated Epithelial to Mesenchymal Transition as a Disease Mechanism in Retinal Pigment Epithelia
Open Access
- Author:
- Zhou, Mi
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 19, 2022
- Committee Members:
- Xuemei Huang, Outside Unit Member
Jeffrey Sundstrom, Chair & Dissertation Advisor
Nicholas Buchkovich, Major Field Member
John Wills, Major Field Member
James Connor, Major Field Member
Patricia Grigson Kennedy, Outside Field Member
Ralph Keil, Program Head/Chair - Keywords:
- RPE
EMT
Unfolded Protein Response
Fibulin-3
TGF-beta
Extracellular Vesicles - Abstract:
- Retinal pigment epithelial (RPE) cells play a central role in maintaining the health and functional integrity of both photoreceptors and the choroid. This functional unit is required for maintaining proper visual function. Numerous retinal degenerative diseases are initiated by RPE dysfunction, including several inherited retinal degenerations and age-related macular degeneration (AMD). However, the molecular mechanisms underlying RPE dysfunction in retinal degeneration remain largely unknown. 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. 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. Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345WFibulin-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. The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells. Extracellular vesicles (EVs) play a critical role in cell-cell communication and modulate cellular differentiation. While RPE cells have been shown to secrete EV, the potential role of EVs in regulating RPE differentiation has not been studied. We next investigate the size, cargo, and function of extracellular vesicles (EVs) derived from RPE cells expressing WT-Fibulin-3 compared to RPE cells expressing the R345W-Fibulin-3 mutation, and determine the role of these EVs in RPE cell dysfunction. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media of ARPE-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) were performed to study the morphology of the EVs. The amount and size distribution of EVs were determined by Nanoparticle Tracking Analysis (NTA). EV protein concentrations were quantified using the DCTM Protein Assay (Bio-Rad). EV markers were validated by conducting Western blot analysis. EV cargo were analyzed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita). The EV-associated transforming growth factor beta 1 (TGF-β1) protein was measured by enzyme-linked immunosorbent assay (ELISA). EV incubated with trypsin were conducted to determine the orientation of WT-Fibulin-3 and R345W-Fibulin-3 in EV. EV uptake was investigated by using PKH67-labeled vesicles and was analyzed by confocal imaging. The EV transplant study was conducted, and migration ability was evaluated in ARPE-19 cells with or without exposure to EVs by conducting scratch assays. Pan-TGF-β-neutralizing antibody was used to determine whether EVs derived from RPE cells induce EMT via TGF-β signaling. mRNA expression levels of EMT markers were measured after EV treatment using RT-PCR. TEM imaging revealed concave-appearing vesicles, and Cryo-EM imaging showed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm. Imaging also indicated a greater number of small EVs (~30 nm) in the R345W group compared to the WT group. This result was further confirmed by NTA showing that, in the R345W group, the particle size distributions were smaller than those of the WT-ARPE-EV. There were no significant differences in EV protein concentrations per EV between WT and R345W groups. Pathway analysis revealed that primary cilia and sonic hedgehog (SHH) pathways were found to be 3- to 5-fold more abundant in EVs derived from WT ARPE-19 cells. In contrast, EMT drivers, lysosome components, and ribosome components were 3- to 7-fold more abundant in EVs secreted from R345W ARPE-19 cells. Subsequent studies revealed enhanced content of TGF-β1 associated with R345W-ARPE-EVs compared to WT-ARPE-EVs. Fibulin-3 can be digested with a low concentration of trypsin, but not EGFR, Flotillin-1, or ALIX, indicating that Fibulin-3 is outside of EV rather than within them. There were no significant differences in EV uptake between WT and mutant groups. Critically, EV transplant studies showed that treatment of recipient RPE cells with R345W-ARPE-EV was sufficient to induce an enhanced migration ability and elevated EMT marker expression in RPE cells. The effects were significantly inhibited after the addition of pan-TGF-β-neutralizing antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size, cargo, and function of EVs. Notably, EVs derived from RPE cells expressing R345W-Fibulin-3 are sufficient to enhance the rate of wound healing closure and elevate EMT marker expression in untransfected RPE cells.