Proteomic Analysis of Aqueous Humor Identified Clinically Relevant Molecular Targets for Neovascular Complications in Diabetic Retinopathy

Diabetic retinopathy (DR) is a leading cause of blindness in adults under 40 in the developed world, with a significant proportion progressing to vision-threatening stages such as proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). This study aims to explore the molecular mechanisms underlying the progression from non-proliferative DR (NPDR) to PDR and NVG, focusing on identifying potential biomarkers and therapeutic targets. Utilizing discovery-based proteomics, specifically label-free quantification (LFQ) and Tandem mass tag (TMT), we analyzed aqueous humor (AH) proteins obtained during cataract surgery or anterior chamber paracentesis from patients with NPDR, PDR, and NVG. Validation of marker candidates for each disease state was conducted using triple quadrupole (QQQ)-MS for targeted protein quantification. Our proteomic analysis identified 2,255 proteins and gene ontology analysis and functional annotation highlighted key biological processes implicated in DR, such as lens development, immune responses and lipid metabolism. Validation of potential biomarkers identified 20 proteins with significant concentration changes, including several candidates with diagnostic utility based on ROC curve analysis. Further investigation into clinical relevance revealed that crystallin gamma-S (CRYGS) is strongly associated with cataract severity, highlighting its role as a potential marker for ocular complications in DR. Importantly, we identified that the pathological factors driving DR progression have a much greater impact than age, a previously known variable, in shaping the proteomic landscape of aqueous humor. Additionally, proteins associated with macular degeneration (CA1, CA2, and HBA1) were uncovered, providing new insights into overlapping mechanisms between DR and other retinal diseases. Finally, proteins linked to panretinal photocoagulation (PRP) treatment, including APOB and CST6, were identified, suggesting their involvement in the therapeutic response and post-treatment adaptation. These findings underscore the potential of AH proteomics in uncovering predictive biomarkers and elucidating the molecular pathogenesis of DR and its complications.