TLR9 agonist suppresses choroidal neovascularization by restricting endothelial cell motility via ERK/c-Jun pathway

Youjian Li; Kepeng Ou; Yuwei Wang; Liying Luo; Zhongzhu Chen; Jiahui Wu

doi:10.1016/j.mvr.2022.104338

TLR9 agonist suppresses choroidal neovascularization by restricting endothelial cell motility via ERK/c-Jun pathway

Microvasc Res. 2022 May:141:104338. doi: 10.1016/j.mvr.2022.104338. Epub 2022 Feb 9.

Authors

Youjian Li¹, Kepeng Ou¹, Yuwei Wang², Liying Luo², Zhongzhu Chen¹, Jiahui Wu³

Affiliations

¹ College of Pharmacy, National & Local Joint Engineering Research Center of Targeted and Innovative Therapeutics, IATTI, Chongqing University of Arts and Sciences, Chongqing, China.
² Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, National Clinical Research Center for Eye Diseases, Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai, China.
³ Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Engineering Center for Precise Diagnosis and Treatment of Eye Diseases, Shanghai Engineering Center for Visual Science and Photomedicine, National Clinical Research Center for Eye Diseases, Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai, China. Electronic address: jianeywu628@msn.com.

PMID: 35150733
DOI: 10.1016/j.mvr.2022.104338

Abstract

Introduction: Choroidal neovascularization (CNV) is the feature of neovascular age-related macular degeneration (AMD). It has been demonstrated that inflammation plays a key role in the development of CNV. Here we aim to investigate how TLR9 agonist (CpG-ODN), one of the key regulators of inflammatory responses, suppresses CNV in vivo.

Materials and methods: The cell viability was assessed by MTT and EdU test after CpG-ODN treatment. Endothelial cells gap assay, tube formation assay and transwell assay were practiced to observe how CpG-ODN affected the endothelial cells functions. The choroidal explants and laser-induced CNV model were built to investigate how CpG-ODN suppressed angiogenesis. The ERK and c-Jun expression were evaluated to assess if CpG-ODN affected cell proliferation. Flow cytometry and qPCR was practiced to observe how CpG-ODN regulated cell proliferation.

Results: Our data showed that CpG-ODN not only reduced CNV area in vivo, but also decreased the RPE damage. CpG-ODN inhibited endothelial cells from migration and forming tubes, while the effect was not toxic. EdU test and MTT test suggested that CpG-ODN inhibited endothelial cells proliferation. CpG-ODN significantly increased protein expression of phosphorylated c-Jun but reduced phosphorylated ERK in HUVECs, which was confirmed in ERK transfected 293T cells. JNK inhibitor abolished the suppression of endothelial cells migration and tube formation by CpG-ODN. The findings were also in agreement with the observation in CpG-ODN treated CNV eyes in vivo. The flow cytometry and qPCR data revealed that the suppression of cell motility by CpG-ODN was achieved by arresting endothelial cells cell cycle at G0/G1 phase.

Conclusions: Our study demonstrated that CpG-ODN suppressed endothelial cell motility by restricting the cell cycle progression at G0/G1 phase, the effect of which was achieved by interacting with ERK/c-Jun pathways.

Keywords: Cell proliferation; Choroidal neovascularization; CpG-ODN; TLR9 agonist.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Choroid / blood supply
Choroidal Neovascularization* / metabolism
Choroidal Neovascularization* / prevention & control
Endothelial Cells / metabolism
Humans
MAP Kinase Signaling System
Toll-Like Receptor 9 / metabolism

Substances

TLR9 protein, human
Toll-Like Receptor 9