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 , , , , , , , , , and was analyzed with semiquantitative RT-PCR in the mouse corneal endothelium ex vivo and in vivo. Knockdown of FGF2 was done using siRNA. was used as a corneal endothelial marker, and Keratocan () was used as a stromal marker. βactin 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 , and in the mouse ex vivo corneal endothelium. This led to increased expression of , , , and and suppression of in a time-dependent manner. Expression of , , , , , , , , and was completely abolished by FGF2 siRNA knockdown in the mouse corneal endothelium ex vivo. Surgical injury induced expression in the in vivo mouse corneal endothelium. The injury-dependent expression of , , , , , , , , and and the suppression of 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 , , , , , , , and in the mouse corneal endothelium in vivo, similar to the human corneal endothelium ex vivo.