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

Author:

Zhou Mi1,Zhao Yuanjun1,Weber Sarah R.1,Gates Christopher2,Carruthers Nicholas J.2,Chen Han3,Liu Xiaoming4,Wang Hong‐Gang4,Ford Michael5,Swulius Matthew T.6,Barber Alistair J.1,Grillo Stephanie L.1,Sundstrom Jeffrey M.1ORCID

Affiliation:

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

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

3. Microscopy Imaging Core Facility Penn State Hershey College of Medicine Hershey Pennsylvania USA

4. Department of Pediatrics, Division of Hematology and Oncology Penn State Hershey College of Medicine Hershey Pennsylvania USA

5. MS Bioworks LLC Ann Arbor Michigan USA

6. Department of Biochemistry and Molecular Biology Penn State Hershey College of Medicine Hershey Pennsylvania USA

Abstract

AbstractWe 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.

Publisher

Wiley

Subject

Cell Biology,Histology

Cited by 2 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3