Multiomics integration identifies key metabolic modulator Prodh mediated arginine proline pathway in pathological retinal neovascularization

Current anti-VEGF therapies for retinal neovascularization are limited by variable efficacy and transient effects. Retinas from OIR and control mice at P17 underwent untargeted metabolomics, lipidomics and transcriptome sequencing. Multiomics integration was performed using O2PLS. Key findings were validated in vitreous samples of proliferative diabetic retinopathy patients (PDR). Functional relevance was assessed via intravitreal inhibition of proline dehydrogenase ( Prodh ) at P12, followed by vascular phenotyping. Integrated analysis revealed significant dysregulation of the arginine-proline pathway in OIR retinas, characterized by Prodh downregulation and consequent proline accumulation, a signature conserved in human PDR. Lipidomics identified triacylglycerol (TAG) enrichment, likely reflecting a delayed metabolic consequence of prior hypoxia rather than a direct angiogenic driver. Intravitreal Prodh inhibition at P12 enhanced pathological vascular complexity specifically at P17—increasing vessel area and reducing lacunarity in the middle region of the middle capillary plexus (MCP)—accompanied by suppression of interferon-beta (IFN-β) response pathways, a known anti-angiogenic signal; siRNA-mediated Prodh knockdown yielded a complementary pro-angiogenic phenotype, manifested as increased vessel length and endpoint number rather than changes in vessel area or lacunarity. Our study identifies Prodh -mediated proline metabolism as a developmentally timed metabolic modulator of retinal angiogenesis, and dysregulation of the arginine-proline pathway represents a conserved mechanism in pathological neovascularization across species and etiologies, nominating Prodh -proline axis and its downstream effectors as promising molecular determinants that may be exploited to modulate retinal vascular growth in ROP and PDR.