Zeb1 Mediates Injury-Induced Mesenchymal Transition in the Mouse Corneal Endothelium In Vivo

To investigate the role of Zeb1 in regulating fibrosis in endothelial-mesenchymal transition (MT) in the cornea. siRNA knockdown of Zeb1 inhibits FGF2-induced MT in the corneal endothelium. Endothelial cells that undergo MT show increased expression of endothelial-MT-associated genes, including COL1, fibronectin, and vimentin, and decreased expression of E-cadherin, leading to fibrous retrocorneal membrane formation.

To address potential off-target effects of siRNA knockdown, Zeb1^flox/flox:UBC-CreERT2 mouse was generated to allow for spatiotemporal control of Zeb1 targeting and studying the role of Zeb1 in the corneal endothelium in vivo.

Intracameral injection of 4-hydroxytamoxifen inhibited Fgf2-induced expression of Zeb1 mRNA and protein in the corneal endothelium of Zeb1^flox/flox:UBC-CreERT2 mouse. Fgf2 and surgical injury-dependent expression of MT-related genes and suppression of E-cadherin were inhibited by conditional targeting of Zeb1 in the mouse corneal endothelium in vivo. Surgical injury led to corneal edema with a central corneal thickness of 189.0 ± 14.6 µm (injury) vs 92.3 ± 2.8 µm (control), and injury-induced edema was significantly attenuated by Zeb1 targeting in the corneal endothelium with a central corneal thickness of 182.2 + 15.5 µm (injury) vs 106.8 ± 11.1 µm (injury + 4-hydroxytamoxifen), F = 120.9, P < .00001. Moreover, conditional targeting of Zeb1 also inhibited injury-dependent retrocorneal membrane formation in the mouse corneal endothelium in vivo.

These results suggest that ZEB1 signaling could be targeted for inhibiting retrocorneal membrane formation and anterior segment fibrosis and could be leveraged to treat certain forms of corneal blindness without relying on transplantation.