The Hippo signaling pathway regulates corneal endothelial regeneration

To explore the role of the Hippo signaling pathway in the regeneration of corneal endothelial cells (CECs) and to evaluate the pro-regenerative potential of pharmacological inhibition of this pathway across species.

Corneal endothelial wound healing models were established in rabbits and mice in vivo. Hippo pathway activity was assessed by immunofluorescence for YAP localization and Western blot for phosphorylated MST1/2, LATS1/2, and YAP. Primary rabbit, monkey, and human CECs, and injury models in mouse, rabbit and monkey were used to evaluate the effect of YAP activation/inhibition on CEC regeneration. Cell proliferation was assessed by EdU labeling and Ki67 immunofluorescence staining. Recovery of corneal opacity and edema was evaluated by slit-lamp examination and anterior segment optical coherence tomography. CEC healing was observed by alizarin red staining. Long-term durability was assessed 12 months after drug withdrawal in monkeys.

The Hippo pathway was downregulated and YAP was activated during CEC wound healing in rabbit and mouse. Knockdown of Yap1 and pharmacological inhibition of YAP suppressed CEC wound healing in rodents in both in vitro and in vivo models. XMU-MP-1, a small molecular inhibitor of Hippo signaling pathway, could promote the corneal endothelial regeneration in rodents and non-human primate in vivo models. Monkey CECs treated with XMU-MP-1 for a short term could maintain structural integrity and function for at least 12 months.

The Hippo pathway regulates corneal endothelial regeneration among various species as a conserved signal, which may serves as a novel target for non-invasive treatment of corneal endothelium decompensation.