Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2

Background/Aims: Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Kruppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. Methods: We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. Results: Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2. Conclusions: Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future.