Injury induces endothelial to mesenchymal transition in the mouse corneal endothelium in vivo via FGF2

PURPOSE: To determine whether the mouse corneal endothelium enters endothelial to mesenchymal transition (EndoMT) following surgical injury in vivo. METHODS: The corneal endothelium in anesthetized mice was surgically injured in vivo under direct visualization. The secretion of interleukin-1 beta (IL-1β) and fibroblast growth factor 2 (FGF2) into the aqueous humor was analyzed with western blotting. The expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, Ccne1, and Cdh1 was analyzed with semiquantitative RT–PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. Col8a2 was used as a corneal endothelial marker, and Keratocan (Ktcn) was used as a stromal marker. β-actin was used as a loading control. RESULTS: Sequential expression of IL-1β and FGF2 was detected in the aqueous humor after surgical injury. FGF2 treatment induced expression of endothelial to mesenchymal transition–related genes including Snai1, and Zeb1 in the mouse ex vivo corneal endothelium. This led to increased expression of Col1a1, Col1a2, Fn1, and Vim and suppression of Cdh1 in a time-dependent manner. Expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced FGF2 expression in the in vivo mouse corneal endothelium. The injury-dependent expression of FGF2, Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 and the suppression of Cdh1 were inhibited by siRNA knockdown of FGF in the mouse corneal endothelium in vivo. Moreover, siRNA knockdown of FGF2 inhibited the formation of the injury-dependent retrocorneal membrane in the in vivo mouse corneal endothelium. CONCLUSIONS: These findings suggest that after surgical injury, FGF2 induces the expression of EndoMT-related genes Snai1, Zeb1, Col1a1, Col1a2, Fn1, Vim, Cdk2, and Ccne1 in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.