Molecular modulators of anti-VEGF response in neovascular age-related macular degeneration: aqueous humor dynamics of CD93 and endocannabinoids

Background: Neovascular age-related macular degeneration (nvAMD) represents a leading cause of legal blindness in the elderly population of developed countries. Although anti-vascular endothelial growth factor (VEGF) therapy has significantly improved visual outcomes, treatment efficacy is highly variable, with a considerable proportion of patients exhibiting incomplete or transient responses. This variability underscores the need to identify additional molecular players that modulate disease progression and therapeutic response. In this context, CD93 has emerged as a promising candidate. CD93 is a transmembrane glycoprotein involved in endothelial cell adhesion, migration, and angiogenesis. Recent studies have shown that CD93 is upregulated in pathological retinal vasculature and promotes ocular neovascularization in preclinical models. However, its behavior in human aqueous humor during anti-VEGF therapy and its potential role as a therapeutic target remain to be elucidated. Parallel to this, the endocannabinoid system (ECS)—comprising bioactive lipids such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and their receptors (CB1, CB2) has gained attention for its regulatory role in angiogenesis and inflammation. Experimental evidence suggests that endocannabinoids can modulate VEGF signaling and influence endothelial cell behavior. Moreover, the ECS interacts with immune mediators and oxidative stress pathways that are known to be involved in nvAMD pathogenesis. Despite this, the intraocular ECS dynamics in the setting of nvAMD and their interactions with anti-VEGF blockage have not been previously characterized. This study aims to address these gaps by investigating the intraocular concentrations of CD93 and endocannabinoids in nvAMD eyes undergoing intravitreal anti-VEGF treatment. By analyzing their longitudinal changes and associations with therapeutic response, the study seeks to identify novel molecular markers of treatment efficacy and to shed light on alternative pathways contributing to disease persistence or incomplete response. Methods: This prospective study involved aqueous humor sampling from two cohorts: patients affected by nvAMD undergoing anti-VEGF therapy, and healthy controls undergoing cataract surgery. Samples in nvAMD eyes were collected at the time of four consecutive monthly intravitreal bevacizumab injections (baseline, week 4, 8 and 12), and once in control eyes at the beginning of cataract surgery. CD93 concentrations were assessed by enzyme-linked immunosorbent assay (ELISA) in aqueous humor samples from 17 nvAMD eyes and 9 healthy control eyes across all timepoints. Protein normalization was performed to account for intersample variability. Endocannabinoids—anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 10 nvAMD eyes at baseline, week 4, and week 12, and in 10 control eyes at the time of cataract extraction. Statistical analysis included repeated-measures ANOVA and the Friedman test for within-group longitudinal comparisons, with Wilcoxon signed-rank tests for post-hoc analysis. Between-group differences were tested using the Mann–Whitney U test. Bonferroni correction was applied where appropriate, and significance was set at p < 0.05. Results: At baseline, CD93 concentrations in the aqueous humor did not significantly differ between nvAMD and control eyes (0.431 ± 0.35 ng/mL vs 0.359 ± 0.25 ng/mL; p=0.55). Anti-VEGF treatment induced a significant overall reduction of raw CD93 levels over time in nvAMD eyes (repeated-measures ANOVA: F=2.97, df=3,48; p=0.041), with pairwise reductions at week 4 (p=0.015) and week 12 (p=0.003) compared to baseline. Normalized CD93 concentrations showed a similar trend (F=2.46, df=3,48; p=0.074), with a significant decrease at week 4 (p=0.007). Stratification based on response to therapy revealed that CD93 reduction was restricted to good responders (F=3.86, p=0.020), while poor responders showed stable CD93 levels over time (F=1.21, p=0.335). Baseline concentrations of AEA and 2-AG were markedly lower in nvAMD eyes compared to controls (AEA: 0.046 ± 0.040 vs 1.530 ± 0.293 μmol/mL; 2-AG: 0.336 ± 0.293 vs 11.204 ± 6.497 μmol/mL; both p<0.001). Anti-VEGF treatment significantly increased AEA and 2-AG levels over time (Friedman test, p<0.001 for both). Pairwise comparisons showed significant changes between all timepoints (all p=0.006). Notably, AEA levels normalized after a single injection (week 4 vs controls, p=0.97) but decreased again at week 12 (p<0.001). Conversely, 2-AG levels increased progressively, remaining significantly higher than controls at both week 4 (p<0.001) and week 12 (p=0.017).   Conclusions: This study provides novel insights into the molecular modifications induced by anti-VEGF therapy within the aqueous humor of nvAMD eyes. CD93 concentrations significantly decrease during treatment, particularly in patients achieving a favorable anatomical response. In parallel, anti-VEGF administration is associated with a progressive restoration of intraocular endocannabinoid levels, suggesting a potential interaction between VEGF inhibition and ECS homeostasis. These findings expand the understanding of the molecular dynamics in nvAMD and may open new perspectives for biomarker discovery and adjunctive therapeutic strategies.