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

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.