Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells

ABSTRACTPurposePrevious studies in our lab found that expression of R345W-Fibulin-3 induces retinal pigment epithelial (RPE) cells to undergo epithelial-mesenchymal transition (EMT). The purpose of the current study was to investigate the size, cargo and function of extracellular vesicles (EVs) derived from RPE cells expressing wild-type (WT)-Fibulin-3 compared to RPE cells expressing the R345W-Fibulin-3 mutation, and to determine the role of these EVs in regulating RPE cell dysfunction.MethodsARPE-19 cells were infected with luciferase-tagged wild-type Fibulin-3 (WT)- or luciferase-tagged R345W-Fibulin-3 (mutant) using lentivirus. 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 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). 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.ResultsTEM imaging revealed concave-appearing vesicles, and cryo-EM imaging showed spherical vesicles with two subpopulations of EVs: a small group with diameters around 30nm and a large group with diameters around 100nm. Imaging also indicated a greater number of small EVs (~30 nm) in the mutant group compared to the WT group. This result was further confirmed by NTA showing that, in the mutant group, the particle size distributions were smaller than those of the WT EVs. There were no significant differences in EV protein concentrations per EV between WT and mutant groups. Proteomic studies showed that EVs derived from ARPE-19 cells expressing WT-Fibulin-3 contain critical members of sonic hedgehog signaling (SHH) signaling and ciliary tip components, whereas EVs derived from RPE cells expressing R345W-Fibulin-3 contain EMT mediators, including TGF-β-induced protein (TGFBI), vimentin, and mothers against decapentaplegic homolog 4 (SMAD4), indicating that the EV cargo reflects the phenotypic status of their parental cells. Subsequent studies revealed enhanced activity of TGF-β1 associated with mutant EVs compared to WT EVs. Critically, EV transplant studies showed that treatment of recipient RPE cells with mutant RPE cell-derived EVs was sufficient to induce an enhanced migration ability and elevated EMT marker expression of RPE cells.ConclusionsThe expression of R345W-Fibulin-3 alters the size, cargo and autocrine function of EVs. Notably, EVs derived from RPE cells expressing R345W-Fibulin-3 are sufficient to induce EMT in uninfected RPE cells.

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Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Cellular Physiology and Biochemistry ◽

10.1159/000489246 ◽

2018 ◽

Vol 46 (4) ◽

pp. 1704-1715 ◽

Cited By ~ 6

Author(s):

Yao Zhang ◽

Dongwan Zhao ◽

Shuai Yang ◽

Haipei Yao ◽

Min Li ◽

...

Keyword(s):

Retinal Pigment Epithelial ◽

Epithelial Mesenchymal Transition ◽

Retinal Pigment ◽

Protective Effects ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Pigment Epithelial ◽

Experimental Pvr ◽

Tgf Β1

Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent.

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Long noncoding RNA ERLR mediates epithelial-mesenchymal transition of retinal pigment epithelial cells and promotes experimental proliferative vitreoretinopathy

Cell Death and Differentiation ◽

10.1038/s41418-021-00756-5 ◽

2021 ◽

Author(s):

Shuai Yang ◽

Hui Li ◽

Haipei Yao ◽

Yao Zhang ◽

Huiqian Bao ◽

...

Keyword(s):

Long Noncoding Rna ◽

Proliferative Vitreoretinopathy ◽

Noncoding Rna ◽

Retinal Pigment Epithelial ◽

Epithelial Mesenchymal Transition ◽

Retinal Pigment ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Pigment Epithelial ◽

Tgf Β1

AbstractProliferative vitreoretinopathy (PVR) is a disease that causes severe blindness and is characterized by the formation of contractile fibrotic subretinal or epiretinal membranes. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a hallmark of PVR. This work aims to examine the role of a long noncoding RNA (lncRNA) named EMT-related lncRNA in RPE (ERLR, LINC01705-201 (ENST00000438158.1)) in PVR and to explore the underlying mechanisms. In this study, we found that ERLR is upregulated in RPE cells stimulated with transforming growth factor (TGF)-β1 as detected by lncRNA microarray and RT-PCR. Further studies characterized full-length ERLR and confirmed that it is mainly expressed in the cytoplasm. In vitro, silencing ERLR in RPE cells attenuated TGF-β1-induced EMT, whereas overexpressing ERLR directly triggered EMT in RPE cells. In vivo, inhibiting ERLR in RPE cells reduced the ability of cells to induce experimental PVR. Mechanistically, chromatin immunoprecipitation (ChIP) assays indicated that the transcription factor TCF4 directly binds to the promoter region of ERLR and promotes its transcription. ERLR mediates EMT by directly binding to MYH9 protein and increasing its stability. TCF4 and MYH9 also mediate TGF-β1-induced EMT in RPE cells. Furthermore, ERLR is also significantly increased in RPE cells incubated with vitreous PVR samples. In clinical samples of PVR membranes, ERLR was detected through fluorescent in situ hybridization (FISH) and colocalized with the RPE marker pancytokeratin (pan-CK). These results indicated that lncRNA ERLR is involved in TGF-β1-induced EMT of human RPE cells and that it is involved in PVR. This finding provides new insights into the mechanism and treatment of PVR.

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GEP100/ARF6 regulates VEGFR2 signaling to facilitate high-glucose-induced epithelial-mesenchymal transition and cell permeability in retinal pigment epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00312.2018 ◽

2019 ◽

Vol 316 (6) ◽

pp. C782-C791 ◽

Cited By ~ 1

Author(s):

Zhi-Peng You ◽

Shan-Shan Chen ◽

Zhong-Yi Yang ◽

Shu-Rong Li ◽

Fan Xiong ◽

...

Keyword(s):

Cell Migration ◽

High Glucose ◽

Retinal Pigment Epithelial ◽

Epithelial Mesenchymal Transition ◽

Retinal Pigment ◽

Cell Permeability ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Pigment Epithelial ◽

Vegfr2 Signaling

Cell permeability and epithelial-mesenchymal transition (EMT) were found to be enhanced in diabetic retinopathy, and the aim of this study was to investigate the underlying mechanism. ARPE-19 cell line or primary retinal pigment epithelial (RPE) cells were cultured under high or normal glucose conditions. Specific shRNAs were employed to knock down ADP-ribosylation factor 6 (ARF6), GEP100, or VEGF receptor 2 (VEGFR2) in ARPE-19 or primary RPE cells. Cell migration ability was measured using Transwell assay. Western blotting was used to measure indicated protein levels. RPE cells treated with high glucose showed increased cell migration, paracellular permeability, EMT, and expression of VEGF. Knockdown of VEGFR2 inhibited the high-glucose-induced effects on RPE cells via inactivation of ARF6 and MAPK pathways. Knockdown ARF6 or GEP100 led to inhibition of high-glucose-induced effects via inactivation of VEGFR2 pathway. Knockdown of ARF6, but not GEP100, decreased high-glucose-induced internalization of VEGFR2. High-glucose enhances EMT and cell permeability of RPE cells through activation of VEGFR2 and ARF6/GEP100 pathways, which form a positive feedback loop to maximize the activation of VEGF/VEGFR2 signaling.

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