Pax6 Directly Regulates the Raver2/sFlt-1 Expression in Corneal Epithelial Cells to Maintain the Cornea's Avascular Privilege.

PURPOSE: This study aimed to elucidate how Pax6 directly regulates Raver2 and sFlt-1 expression in corneal epithelial cells to maintain the cornea's avascular privilege during normal development. METHODS: The expression levels of Pax6, Raver2, and sFlt-1 in both the mouse cornea and human corneal epithelial cell line (HCE-T) were evaluated. Changes in Raver2 and sFlt-1 expression following Pax6 knockdown were also assessed. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), electrophoresis mobility shift assay (EMSA), and dual-luciferase reporter (DLR) assay were conducted to ascertain the regulatory relationship between Pax6 and Raver2. The impact on vascular endothelial cell proliferation, migration, and tube formation was examined after coculturing with siPax6-HCE-T conditioned culture medium. Additionally, corneal neovascularization (CoNV) was monitored following corneal epithelial scraping or injection of the AAV-siPax6 vector into the limbus. RESULTS: Pax6, Raver2, and sFlt-1 were highly expressed in both HCE-T cells and normal mouse corneal epithelial layer. Interference with Pax6 expression resulted in decreased levels of Raver2 and sFlt-1 both in vivo and in vitro. TFBS1 and TFBS2 in the Raver2 promoter region were identified as potential Pax6 binding sites. ChIP-qPCR and EMSA assays confirmed the direct interaction between Pax6 and Raver2. The DLR assay demonstrated that Pax6 binding to the TFBS1 and TFBS2 regulated Raver2 expression. Vascular endothelial cell proliferation, migration, and tube formation were enhanced when cocultured with siPax6-HCE-T conditioned culture medium. CoNV progressed after corneal epithelial scraping and AAV-siPax6 vector injection. CONCLUSIONS: Pax6 directly regulates Raver2/sFlt-1 expression in corneal epithelial cells, thus preserving the cornea's avascular privilege during normal development.