Exosomes mediate an epithelial-mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy

Abstract

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß-2) was used to induce epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co-cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT-promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR-543 was found in exosomes from EMTed RPE cells, and miR-543-enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.

Keywords: epithelial-mesenchymal transition; proliferative vitreoretinopathy; retinal pigment epithelium.

FIGURE 1

EMT induction in ARPE‐19 cells and characterization of isolated exosomes (A) Western blot analysis. ARPE‐19 cells were treated with 10 ng/mL TGFβ2 for 48 h. The protein expression of E‐cadherin, α‐SMA and fibronectin was detected by Western blot. Relative protein expression (normalized to β‐actin) was quantified in the Western blots based on grey scale values. The data are presented as the mean ± SEM. n = 3. Statistical significance was analysed by two‐tailed Student's t test. *P < .05; (B) Real‐time quantitative PCR analysis. ARPE‐19 cells were treated with 10 ng/mL TGFβ2 for 48 h. The mRNA expression levels of EMT‐related proteins were detected with real‐time quantitative PCR. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by two‐tailed Student's t test *P < .05; (C) Immunofluorescence analysis of EMT‐related proteins in ARPE‐19 cells. After treatment with TGFβ2 for 48 h, EMT‐related proteins were detected using appropriate antibodies. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 μm; (D) ALIX, CD63, CD9 and CD81 (common exosomal markers) immunoblots of exosomes derived from normal and EMTed ARPE‐19 cells. (E) NTA analysis of exosomes from normal and EMTed ARPE‐19 cells. (F) TEM analysis of exosomes from normal and EMTed ARPE‐19 cells. Scale bar: 100 nm. EXO‐N ARPE, exosome derived from normal ARPE‐19 cells; EXO‐EMTed ARPE, exosome derived from EMTed ARPE‐19 cells; N: control condition

FIGURE 2

Exosomes from EMTed ARPE‐19 cells induce EMT in recipient ARPE‐19 cells. (A) Western blot analysis. Recipient ARPE‐19 cells were incubated with 100 μg/mL exosomes from EMTed ARPE‐19 cells for 48 h. The protein expression of E‐cadherin, α‐SMA and fibronectin was detected by Western blot. Relative protein expression (normalized to β‐actin) was quantified in Western blots based on grey scale values. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; (B) Real‐time quantitative PCR analysis. Recipient ARPE‐19 cells were incubated with 100 μg/mL exosomes from EMTed ARPE‐19 cells for 48 h. The mRNA expression levels of EMT‐related proteins were detected with real‐time quantitative PCR. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; (C) Immunofluorescence analysis of EMT‐related proteins in ARPE‐19 cells. After incubation with 100 μg/mL exosomes from EMTed ARPE‐19 cells for 48 h, EMT‐related proteins were detected using appropriate antibodies. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 μm. EXO‐N ARPE, exosome derived from normal ARPE‐19 cells; EXO‐EMTed ARPE, exosome derived from EMTed ARPE‐19 cells; N, control condition

FIGURE 3

Co‐culture of normal and EMTed ARPE‐19 cells. A, Western blot analysis. Recipient ARPE‐19 cells were co‐cultured with EMTed ARPE‐19 cells with or without GW4869 for 48 h. The protein expression of E‐cadherin, α‐SMA and fibronectin was detected by Western blot. Relative protein expression (normalized to β‐actin) was quantified in Western blots based on grey scale values. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; B, Real‐time quantitative PCR analysis. Recipient ARPE‐19 cells were co‐cultured with EMTed ARPE‐19 cells with or without GW4869 for 48 h. The mRNA expression levels of EMT‐related proteins were detected with real‐time quantitative PCR. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; (C) Immunofluorescence analysis of EMT‐related proteins in recipient ARPE‐19 cells. After co‐culturing with EMTed ARPE‐19 cells for 48 h, EMT‐related proteins were detected using appropriate antibodies. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 μm

FIGURE 4

Detection of exosome uptake by recipient ARPE‐19 cells. Recipient ARPE‐19 cells that had been incubated for 24 h with CM‐Dil‐labelled exosomes are depicted (CM‐Dil in red, PKH in green, DAPI in blue). Scale bar: 25 μm. Images shown on the right were magnified from the white boxes shown in images on the left. Profile analysis was performed using Image J software

FIGURE 5

Exosomal microRNA sequencing and exosomal miR‐543 promote EMT of ARPE‐19 cells. A, Heat map of differentially expressed exosomal miRNA from normal and EMTed ARPE‐19 cells. B, Real‐time quantitative PCR verification of miRNA‐seq. Exosomal miR‐10a‐5P, miR‐543 and miR‐323a‐3p expression in normal and EMTed ARPE‐19 cells was detected by RT‐qPCR. n = 3, Statistical significance was analysed by two‐tailed Student's t test. *P < .05; (C) Real‐time quantitative PCR analysis of miR‐543 in recipient ARPE‐19 cells. ARPE‐19 cells were incubated with 100 μg/mL exosome loaded with miR‐543 mimics or miRNA mimics control for 3, 12, 24 and 48 h, and the cellular miR‐543 levels were detected with RT‐qPCR. Statistical significance was analysed by one‐way ANOVA. D, Western blot analysis. Recipient ARPE‐19 cells were incubated with 100 μg/mL exosomes loading with miR‐543 mimics or miRNA mimics control for 48 h. The protein expression of E‐cadherin, α‐SMA and fibronectin was detected with Western Blot. Relative protein expression (normalized to β actin) was quantified in the Western blots based on their grey scale values. The data are presented as the mean ± SEM. n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; E, Real‐time quantitative PCR analysis. Recipient ARPE‐19 cells were incubated with 100 μg/mL exosomes loaded with miR‐543 mimics or miRNA mimics control for 48 h. The mRNA expression levels of EMT‐related proteins were detected with real‐time quantitative PCR. The data are presented as the mean ± SEM, n = 3. Statistical significance was analysed by one‐way ANOVA. *P < .05; F, Immunofluorescence analysis of EMT‐related proteins in ARPE‐19 cells. After incubation with 100 μg/mL exosomes loaded with miR‐543 mimics or miRNA mimics control for 48 h, EMT‐related proteins were detected using appropriate antibodies. Nuclei were stained with DAPI. The slides were examined by confocal microscopy. Original magnification: 630×, oil. Scale bar: 10 μm. EXO‐N ARPE: exosome derived from normal ARPE‐19 cells；EXO‐EMTed ARPE: exosome derived from EMTed ARPE‐19 cells; EXO‐miR‐543: exosome loaded with miR‐543 mimics; EXO‐ MOCK miR: exosome loaded with miRNA mimics control

FIGURE 6

Mechanism of EMT cascade mediated by exosome. In the PVR microenvironment, EMT allows RPE cells to transdifferentiate into mesenchymal cells and secrete exosomes containing pro‐EMT ingredients, including miR‐543. These pathologic exosomes are adopted by healthy RPE cells and a second round of RPE EMT starts. This EMT cascade causes more and more RPE cells to transdifferentiate into myofibroblasts, participate in the formation of PVR membrane, and eventually leads to the rapid progression of PVR