Activated Hedgehog signaling in keratocytes leads to stromal stiffness and impairs corneal regeneration

The repair of corneal injuries remains a major challenge in clinical practice. Impaired corneal wound healing is closely associated with aberrantly activated stromal keratocytes and disorganized extracellular matrix. Here, we identify aberrant Hedgehog signaling in corneal keratocytes as a key driver of defective wound repair. In adult mice, Hedgehog signaling is suppressed in quiescent keratocytes but is pathologically reactivated following chemical injury, correlating with impaired repair. Keratocyte-specific Hedgehog activation via Ptch1 ablation disrupted corneal wound healing after epithelial scraping-a process that would normally resolve seamlessly under physiological conditions. Mechanistically, Hedgehog activation induced stromal thinning and stiffening through disorganized collagen fibrils. Transcriptomics analysis revealed keratocyte transdifferentiation into fibroblast-like phenotypes, accompanied by downregulation of extracellular matrix genes. Hedgehog-mediated stromal stiffening suppressed YAP activity in the overlying epithelium via Hippo pathway activation, blocking epithelial differentiation-a defect that was reversed by Hippo inhibition (XMU-MP-1). In chemical injury models, genetic Smo deletion or pharmacological Gli1/2 inhibition (GANT61) restored stromal architecture, normalized collagen organization, and rescued epithelial differentiation defects. These findings establish Hedgehog signaling in keratocytes as a critical regulator of stromal-epithelial crosstalk and highlight its targeted inhibition as a potential therapeutic strategy to restore corneal transparency and repair fidelity after injury.