IGFBP4 suppresses EndMT and preserves rabbit corneal endothelial cell phenotypes by inhibiting the Wnt2/β-catenin signaling pathway.

Endothelial-mesenchymal transition (EndMT) represents a major limiting factor in the in vitro expansion of corneal endothelial cells (CECs). This study aims to identify the key genes and signaling pathways driving EndMT in rabbit CECs (RCECs) and to explore targeted interventions to preserve cellular phenotype, ultimately improving in vitro culture protocols. During serial passaging, primary RCECs underwent significant EndMT, characterized by progressive cellular enlargement and the acquisition of a fibroblast-like morphology. Comparative transcriptome analysis via high-throughput RNA sequencing of passage 0 (P0) and passage 3 (P3) RCECs revealed significant differential expression of EndMT-associated genes. Specifically, bioinformatic analyses (GO/KEGG) highlighted the downregulation of IGFBP4 and the upregulation of WNT components, implicating Wnt pathway activation in the EndMT process. Functionally, overexpressing IGFBP4 or knocking down WNT2 in RCECs substantially decreased the expression of EndMT markers (α-SMA and vimentin) as well as core components of the Wnt2/β-catenin pathway (Frizzled, Dvl, p-β-catenin, and p-TCF-4). Conversely, opposite genetic manipulations yielded elevated expression levels. Collectively, our findings demonstrate that IGFBP4 effectively suppresses EndMT and maintains the functional CEC phenotype by inhibiting the Wnt2/β-catenin signaling pathway.