IGFBP4 suppresses EndMT to maintain corneal endothelial cell characteristics through down-regulating WNT2 to inhibit Wnt2/β-catenin signaling pathway

Abstract Objective: It is aimed to investigate the mechanism of endothelial-mesenchymal transition (EndMT), which is a significant limiting factor in the culture of corneal endothelial cells (CECs). Methods: The primary rabbits corneal endothelial cells (RCECs) at passage 0 (P0) and passage 3 (P3) were subjected to Illumina high-throughput RNA sequencing, leading to the identification of EndMT-related genes and signaling pathways. Target genes IGFBP4 and WNT2 were selected for validation, with observation indicators including EndMT markers, α-Smooth muscle actin（α-SMA) and vimentin, tight junction protein ZO-1(ZO-1) and aquaporin-1(AQP-1), as well as molecules related to Wnt2/β-catenin signaling pathway. Results: The results of high-throughput RNA sequencing suggest a potential association between the Wnt pathway and EndMT. Overexpression of IGFBP4 or knockdown of WNT2 in RCECs, the levels of α-SMA, vimentin, ZO-1 and AQP-1 were significantly reduced, as well as molecules related to Wnt2/β-catenin signaling pathway, such as Frizzled, Dvl, and p-β-catenin and p-TCF-4. On the contrary, when IGFBP4 is knocked down or WNT2 is overexpressed, the opposite results are obtained. Moreover, the IP experimental results provide evidence of the interaction between WNT2 and IGFBP4. Conclusion: In the in vitro culture of RCECs, the downregulation of IGFBP4 could activate Wnt/β- catenin signaling pathway and induce the occurrence of EndMT.