Impaired P4HA1-Driven Hydroxyproline Formation Mediates Scleral Collagen Disruption in Form-Deprivation Myopia

Purpose: Scleral extracellular matrix (ECM) remodeling is a hallmark of myopia progression, yet its metabolic drivers remain poorly defined. This study investigated whether disruption of arginine-proline metabolism-specifically impaired P4HA1-mediated proline hydroxylation-contributes to hydroxyproline deficiency, collagen disorganization, and axial elongation in form-deprivation myopia (FDM). Methods: Monocular FDM was induced in guinea pigs for two weeks. Refractive errors, axial length, scleral morphology, and collagen ultrastructure were assessed. Untargeted and targeted metabolomics were performed to characterize metabolic alterations. AAV8-P4HA1 or control vector was delivered via sub-Tenon's capsule injection to the form-deprived eyes of two animal subgroups to test whether restoration of P4HA1 expression could rescue metabolic and structural abnormalities. Pharmacological inhibition of P4HA (1,4-DPCA) in cultured primary fibroblasts evaluated the cellular consequences of impaired proline hydroxylation. Results: Relative to untreated eyes, FDM resulted in a myopic refractive shift, axial elongation, and scleral thinning, reduced collagen fibril diameter, and ECM disorganization. Metabolomic analyses revealed disruption of arginine-proline metabolism, with elevated arginine and proline but reduced hydroxyproline, accompanied by increased ARG1, OAT, and PYCR1 expression and decreased P4HA1 and P4HA3 expression. P4HA1 overexpression restored hydroxyproline levels, corrected the metabolic imbalance, increased COL1A1, reduced MMP2, reinstated TIMP2, and improved collagen density and alignment, thereby mitigating axial elongation. Pharmacological inhibition of P4HA reduced hydroxyproline and collagen production. Conclusions: Reduced scleral P4HA1 expression in FDM eyes is associated with reduced scleral hydroxyproline content and collagen structural disorganization. That restoration of scleral P4HA1 levels inhibits FD-related excessive axial elongation points to the P4HA1-hydroxyproline axis as a potential target for myopia control.