CGRP/TSP1 Signaling Dampens Corneal Inflammation and Fibrosis by Targeting A2M During Corneal Stromal Wound Healing.

PURPOSE: To investigate the specific role of thrombospondin-1 (TSP1), encoded by the Thbs1 gene, in corneal stromal wound healing and to elucidate its underlying mechanism. METHODS: A corneal stromal injury model was established by removing the epithelium and superficial stroma. We assessed Thbs1 expression using single-cell sequencing and compared the corneal healing responses between wild-type and Thbs1-deficient (Thbs1-/-) mice. Bulk RNA sequencing was conducted to identify key downstream targets of Thbs1. The regulation of TSP1 by calcitonin gene-related peptide (CGRP) was investigated through intervening CGRP signaling in human telomerase-immortalized keratocytes and murine corneas. RESULTS: Thbs1 was highly expressed in corneal stroma and upregulated following injury. Thbs1-/- mice exhibited more severe corneal lesions than wild-type controls, with elevated corneal opacity scores and augmented inflammation and fibrosis markers. Transcriptomic analysis identified A2m (encoding α2-macroglobulin [A2M]) as a key downstream target of Thbs1. A2m expression also increased after injury, peaking later than Thbs1. Topical depletion of A2m in wild-type mice recapitulated the exacerbated corneal phenotype observed in Thbs1-/- mice. Exogenous CGRP robustly induced both TSP1 and A2M expression in vitro and in vivo, but the CGRP receptor antagonist BIBN4096 suppressed their expression and concurrently exacerbated corneal lesions. TSP1 and A2M were similarly upregulated in patients with corneal scarring from alkali burn or fungal infection. CONCLUSIONS: Our findings indicate that TSP1 and A2M induction represents a conserved stress-protective response to corneal stromal injury. The CGRP/TSP1/A2M signaling axis is critical for modulating stromal inflammation and fibrosis, highlighting its therapeutic potential for enhancing corneal stromal repair.