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表达 R345W-Fibulin-3 的视网膜色素上皮细胞的细胞外囊泡诱导受体细胞发生上皮-间充质转化。

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

机构信息

Department of Ophthalmology, Penn State Hershey College of Medicine, Hershey, Pennsylvania, USA.

Bioinformatics Core, Biomedical Research Core Facilities, University of Michigan Medical School, Ann Arbor, Michigan, USA.

出版信息

J Extracell Vesicles. 2023 Oct;12(10):e12373. doi: 10.1002/jev2.12373.

DOI:10.1002/jev2.12373
PMID:37855063
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10585439/
Abstract

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. 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 by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFβ was determined by pre-incubation of EVs with a pan-TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFβ-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV-bound TGFβ plays a critical role in the induction of EMT in RPE cells.

摘要

我们之前已经表明，R345W-Fibulin-3 的表达会诱导视网膜色素上皮 (RPE) 细胞发生上皮-间充质转化 (EMT)。本研究的目的是确定表达 R345W-Fibulin-3 突变的 RPE 细胞衍生的细胞外囊泡 (EVs) 是否足以诱导受体细胞发生 EMT。使用慢病毒感染 ARPE-19 细胞使其表达荧光素酶标记的野生型 (WT)-Fibulin-3 或荧光素酶标记的 R345W-Fibulin-3 (R345W)。通过超速离心或密度梯度超速离心从培养基中分离 EVs。使用透射电子显微镜和冷冻电子显微镜研究 EV 的形态。通过纳米颗粒跟踪分析 (NTA) 确定 EV 的大小分布。使用基于 LC-MS/MS 的蛋白质组学分析 EV 货物。通过酶联免疫吸附测定法测量 EV 相关的转化生长因子 β1 (TGFβ1) 蛋白。通过用 WT- 或 R345W-EVs 处理受体细胞来评估 EV 刺激 RPE 迁移的能力。通过用泛 TGFβ 阻断抗体或 IgG 对照预先孵育 EVs 来确定 EV 结合的 TGFβ 的作用。EM 成像显示出具有两种 EV 亚群的球形囊泡：一组直径约为 30nm，另一组直径超过 100nm，这通过 NTA 分析得到了证实。通路分析显示，R345W-EVs 中 sonic hedgehog 通路的成员较少，而 EMT 驱动因子则更丰富。此外，与对照相比，R345W-EVs 中 TGFβ1 的浓度更高。重要的是，用 R345W-EVs 处理足以增加受体细胞中 EMT 标志物的表达以及细胞迁移。用泛 TGFβ 中和抗体预先孵育 EVs 可显著抑制这种 EV 增加的细胞迁移。总之，R345W-Fibulin-3 的表达改变了 EV 的大小和货物，足以以 TGFβ 依赖的方式增强细胞迁移率。这些结果表明，EV 结合的 TGFβ 在 RPE 细胞 EMT 的诱导中发挥关键作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46a6/10585439/0cb4031deb24/JEV2-12-12373-g001.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46a6/10585439/b7bfc5c7490a/JEV2-12-12373-g007.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46a6/10585439/15bd37c2f618/JEV2-12-12373-g005.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/46a6/10585439/0cb4031deb24/JEV2-12-12373-g001.jpg

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