Abstract
Purpose :
FGF2 signaling can induce endothelial mesenchymal transition (EMT) in the human corneal endothelium that leads to cell proliferation and fibrosis. We have identified SNA1 as a direct target of FGF2 signaling, and the elucidation of SNAI1 signaling could help identify potential therapeutic targets for patients with vision loss due to endothelial dysfunction.
Methods :
Expression of SNAI1, SNAI2, ZEB1, ZEB2, COL1A1, COL1A2, FN1, VIM, CDK2, CCNE, CCND and COL8A2 were analyzed by immunoblotting and semi-quantitative reverse transcription PCR in human corneal endothelial cells (CECs). Knockdown of SNAI1, ZEB1 and CDK2 was done using siRNA transfection. Cell proliferation assay in ex vivo human corneal endothelium was performed using by anti-phosphohistone H3 immunostaining.
Results :
FGF2 stimulation alters cell morphology and induces expression of EMT-related genes including SNAI1, SNAI2, ZEB1 and ZEB2 in human CECs. This leads to increased expression of fibronectin, vimentin and COL1 expression in vitro and ex vivo. siRNA knockdown of SNAI1 showed that SNAI1 regulates ZEB1, an important mediator of EMT, and CDK2 and CCNE. Decreased expression of fibronectin, vimentin and COL1 were detected by siRNA knockdown of ZEB1, but not by knockdown of CDK2. Ex vivo immunostaining further showed that SNAI1 is key regulator for cell proliferation induced by FGF2 in human CECs.
Conclusions :
These data suggest that SNAI1 promotes fibrosis and proliferation in human CECs through ZEB1 and CDK2, and the 2 downstream targets of SNAI1 signal through independent pathways.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.