Dysregulated proline metabolism contributes to retinal fibrosis in neovascular AMD: Therapeutic potential of prolyl-4-hydroxylase inhibition.

Subretinal fibrosis, a major cause of irreversible vision loss in neovascular age-related macular degeneration (nAMD), is driven by excessive deposition of extracellular matrix such as collagens. While proline metabolism is known to play a critical role in collagen biosynthesis and fibrosis, its involvement in subretinal fibrosis remains unclear. Here, we characterized the progression of fibrovascular lesions in JR5558 mice, observing significant molecular alterations as early as 4 weeks of age and phenotypic changes by 8 weeks. Transcriptomic and metabolomic analyses revealed elevated levels of 4-hydroxyproline, an essential component of collagen, alongside significant alterations of other fibrosis-related pathways. P4HA1, a catalytic subunit of prolyl-4-hydroxylase essential for 4-hydroxyproline biosynthesis, was prominently expressed in fibrotic lesions in retinas of JR5558, laser-induced murine models and human eyes with nAMD. Targeting P4HA1 with the small-molecule inhibitor diethyl pythiDC significantly attenuated fibrovascular lesion expansion in the JR5558 mouse model and reduced collagen turnover in human retinal pigment epithelium cells. Treatment responses in JR5558 mice were lesion cluster dependent, with the combination of diethyl pythiDC with aflibercept showing selective antifibrotic benefit in moderate lesion cluster. These findings support a potential role of proline metabolism, particularly proline hydroxylation, in subretinal fibrosis. Inhibiting P4HA1 with diethyl pythiDC inhibited fibrosis in the models we studied, highlighting a potential therapeutic strategy.