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.

中文翻译：

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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上皮-间质转化

我们之前已经证明，R345W-Fibulin-3 的表达会诱导视网膜色素上皮 (RPE) 细胞发生上皮间质转化 (EMT)。当前研究的目的是确定源自表达 R345W-Fibulin-3 突变的 RPE 细胞的细胞外囊泡 (EV) 是否足以诱导受体细胞中的 EMT。使用慢病毒，用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) 感染 ARPE-19 细胞。通过超速离心或密度梯度超速离心从培养基中分离出 EV。采用透射电子显微镜和低温电子显微镜来研究电动汽车的形态。EV 的尺寸分布通过纳米颗粒跟踪分析（NTA）确定。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 相关转化生长因子 β1 (TGFβ1) 蛋白。通过用 WT-或 R345W-EV 处理受体细胞来评估 EV 刺激 RPE 迁移的能力。EV 结合的 TGFβ 的作用通过将 EV 与泛 TGFβ 阻断抗体或 IgG 对照预孵育来确定。电镜成像显示球形囊泡具有两个 EV 亚群：一组直径约为 30 nm，一组直径超过 100 nm，经 NTA 分析证实。通路分析显示，R345W-EV 中 sonic hedgehog 通路的成员较少，而 EMT 驱动程序较丰富。此外，与对照相比，R345W-EV 具有更高浓度的 TGFβ1。至关重要的是，R345W-EV 处理足以增加 EMT 标志物表达以及受体细胞中的细胞迁移。EV 与泛 TGFβ 中和抗体预孵育可显着抑制 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，这足以以 TGFβ 依赖性方式提高细胞迁移率。这些结果表明，EV 结合的 TGFβ 在 RPE 细胞 EMT 的诱导中发挥着关键作用。