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Exosomes from bone marrow-derived mesenchymal stem cells facilitate corneal wound healing via regulating the p44/42 MAPK pathway

December 2022
Graefe's Archive for Clinical and Experimental Ophthalmology 261(2)

Authors:

PurposeThis study was aimed at exploring the function of Exosomes isolated from bone marrow-derived mesenchymal stem cells (BMSC-Exos) in corneal wound healing and at revealing the underlying mechanisms involving the p44/42 mitogen-activated protein kinase (MAPK) pathway.Methods The isolated BMSC-Exos were identified by transmission electron microscopy, Western blot, and nanoparticle tracking analysis. After coculture with BMSC-Exos, the proliferation and migration of human corneal epithelial cells (HCEs) were evaluated. The protein expression of p-MEK/MEK and p44/42 MAPK was detected by Western blot. A mouse model of alkali-burned cornea was established via NaOH exposure. After injection with BMSC-Exos, the pathological changes and expression of α-SMA (a fibrosis marker) and CD31 (a vascularization marker) in corneal tissues were detected.ResultsBMSC-Exos enhanced the proliferation and migration of HCEs in a dose-dependent manner. The p44/42 MAPK pathway was activated by the treatment of BMSC-Exos, and its blocking using U0126 partially abrogated the effects of BMSC-Exos on promoting the proliferation and migration of HCEs. In vivo, the injection of BMSC-Exos facilitated the remission of the pathological changes (inflammation) and weakened the upregulation of α-SMA (fibrosis) and CD31 (vascularization) in corneal tissues of mice with alkali-burn injury.ConclusionBMSC-Exos promoted the proliferation and migration of HCEs via activating the p44/42 MAPK pathway in vitro and also inhibited alkali burn-induced inflammation, fibrosis, and vascularization in corneal tissues in vivo. BMSC-Exos may be promising resources for promoting corneal wound healing.

Identification of BMSC-Exos and internalization in HCEs. A The images of BMSC-Exos under TEM. B Western blot of CD63 and TSG101 in BMSCs and BMSC-Exos. C The volume distribution of BMSC-Exos was detected by NTA. D The internalization of PKH67-labeled BMSC-Exos in HCEs was observed under a microscope

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BMSC-Exos enhanced the proliferation and migration of HCEs in a dose-dependent manner. A The viability of HCEs was measured by CCK-8 assay. B The proliferation of HCEs was determined by EdU staining. C The migration of HCEs was examined by wound healing assay. D The migration of HCEs was examined by trans-well assay. *P < 0.05 and **P < 0.01 vs. Ctrl

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BMSC-Exos upregulated p-MEK/MEK and p44/42 MAPK in HCEs. The expression of p-MEK/MEK and p44/42 MAPK in HCEs was detected by Western blot. *P < 0.05 and **P < 0.01 vs. Ctrl

…

Inhibition of the p44/42 MAPK pathway partially eliminated the effects of BMSC-Exos on the behaviors of HCEs. A The viability of HCEs was measured by CCK-8 assay. B The proliferation of HCEs was determined by EdU staining. C The migration of HCEs was examined by wound healing assay. D The migration of HCEs was examined by trans-well assay. HCEs were treated with U0126 (a MEK1/2 inhibitor) and/or BMSC-Exos. *P < 0.05 and **P < 0.01 vs. control; #P < 0.05 and ##P < 0.01 vs. Exos

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BMSC-Exos alleviated pathological injury of corneal tissues in a mouse model of alkali-burned cornea. A The histological changes were observed by HE staining. B The expression of α-SMA and CD31 in corneal tissues was detected by IF and quantified using IOD. **P < 0.01 vs. PBS-7; ##P < 0.01 vs. PBS-14

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https://doi.org/10.1007/s00417-022-05956-4

BASIC SCIENCE

Exosomes frombone marrow‑derived mesenchymal stem cells

facilitate corneal wound healing viaregulating thep44/42 MAPK

pathway

JinZhou1· YuanyuanDing2· YongqiangZhang3· DehuiZheng1· LifengYan1· MengxiangGuo1· YaniMao1·

LihongYang1

Received: 21 June 2022 / Revised: 4 December 2022 / Accepted: 21 December 2022

Abstract

Purpose This study was aimed at exploring the function of Exosomes isolated from bone marrow-derived mesenchymal stem

cells (BMSC-Exos) in corneal wound healing and at revealing the underlying mechanisms involving the p44/42 mitogen-

activated protein kinase (MAPK) pathway.

Methods The isolated BMSC-Exos were identiﬁed by transmission electron microscopy, Western blot, and nanoparticle

tracking analysis. After coculture with BMSC-Exos, the proliferation and migration of human corneal epithelial cells (HCEs)

were evaluated. The protein expression of p-MEK/MEK and p44/42 MAPK was detected by Western blot. A mouse model

of alkali-burned cornea was established via NaOH exposure. After injection with BMSC-Exos, the pathological changes and

expression of α-SMA (a ﬁbrosis marker) and CD31 (a vascularization marker) in corneal tissues were detected.

Results BMSC-Exos enhanced the proliferation and migration of HCEs in a dose-dependent manner. The p44/42 MAPK pathway was

activated by the treatment of BMSC-Exos, and its blocking using U0126 partially abrogated the eﬀects of BMSC-Exos on promoting the

proliferation and migration of HCEs. In vivo, the injection of BMSC-Exos facilitated the remission of the pathological changes (inﬂam-

mation) and weakened the upregulation of α-SMA (ﬁbrosis) and CD31 (vascularization) in corneal tissues of mice with alkali-burn injury.

Conclusion BMSC-Exos promoted the proliferation and migration of HCEs via activating the p44/42 MAPK pathway in

vitro and also inhibited alkali burn-induced inﬂammation, ﬁbrosis, and vascularization in corneal tissues in vivo. BMSC-

Exos may be promising resources for promoting corneal wound healing.

Keywords Bone marrow-derived mesenchymal stem cell· Exosome· Human corneal epithelial cell· Corneal wound healing

Key messages

BMSC-Exos exhibit therapeutic potential in corneal diseases.

BMSC-Exos promote the proliferation and migration of HCEs.

BMSC-Exos activate the p44/42 MAPK pathway in HCEs.

BMSC-Exos facilitate corneal wound healing in mice.

Jin Zhou and Yuanyuan Ding contributed equally.

* Jin Zhou

jinzhou5188@163.com

1 Department ofOphthalmology, Guangzhou Women

andChildren’s Medical Center, No. 9, Jinsui Road, Tianhe

District, GuangzhouCity510623, China

2 Department ofOphthalmology, Nanfang Hospital, Southern

Medical University, GuangzhouCity510515, China

3 Department ofOphthalmology, Beijing Children’s Hospital

East Branch, BeijingCity100002, China

/ Published online: 28 December 2022

Graefe's Archive for Clinical and Experimental Ophthalmology (2023) 261:723–734

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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