Serial Aqueous Humor Proteomics in Diabetic Macular Edema: Correlation of Novel Targets with Retinal Fluid Volume

<h4>Objective</h4>Diabetic macular edema (DME) with incomplete response to anti-VEGF therapy is a significant challenge. Here, we integrated advanced proteomics and an artificial intelligence (AI)-driven platform to elucidate molecular mechanisms associated with resistance to anti-VEGF treatment.<h4>Design</h4>Translational longitudinal study.<h4>Subjects</h4>Patients with DME (n = 30) under treatment with anti-VEGF therapy who had reached their plateau phase.<h4>Methods</h4>Aqueous humor samples were obtained at the time of 6 consecutive anti-VEGF injections. OCT scan fluid volumes were assessed by an AI-based platform (Discovery OCT Fluid and Biomarker Detector, RetinAI). Proteomics analysis of the samples collected during the first 3 time points was performed by label-free liquid chromatography-tandem mass spectrometry. Confirmatory enzyme-linked immunosorbent assay (ELISA) was performed for afamin and VEGF for time points 4 to 6.<h4>Main outcome measures</h4>Differentially expressed aqueous humor proteins and their correlation with AI-quantified OCT fluid volumes.<h4>Results</h4>Proteomics identified 202 proteins in ≥70% of the samples. Ten proteins were consistently differentially regulated in patients with substantial edema versus no edema. Upregulated proteins included afamin (highest fold change 1.8 ± 0.4), alpha-1B-glycoprotein, and angiotensinogen, whereas cystatin-C, clusterin, apolipoprotein E, epidermal growth factor-containing fibulin-like extracellular matrix protein, Ig lambda chain V-I, latent transforming growth factor beta-binding protein 2, and reelin were downregulated. Afamin levels correlated with fluid volumes (total, inner retinal, inner macular ring). The upregulation of afamin was confirmed by ELISA.<h4>Conclusions</h4>In this hypothesis-generating study using serial aqueous humor proteomics, multiple novel targets were identified that correlated with retinal fluid volumes. Specifically, afamin, a wingless/integrated (Wnt)-signaling carrier protein and established biomarker of type 2 diabetes, was consistently upregulated in eyes with anti-VEGF-refractory DME. Whether this reflects active Wnt pathway involvement in eyes demonstrating anti-VEGF treatment resistance or vascular dysfunction warrants additional study. Concurrent downregulation of cystatin-C and clusterin suggests impaired endogenous anti-inflammatory pathways in eyes with refractory DME.<h4>Financial disclosures</h4>The authors have no proprietary or commercial interest in any materials discussed in this article.