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

OBJECTIVE

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.

DESIGN

Translational longitudinal study.

SUBJECTS: Patients with DME (n = 30) under treatment with anti-VEGF therapy who had reached their plateau phase.

METHODS

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.

MAIN OUTCOME MEASURES: Differentially expressed aqueous humor proteins and their correlation with AI-quantified OCT fluid volumes.

RESULTS

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.

CONCLUSIONS

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.

FINANCIAL DISCLOSURES: The authors have no proprietary or commercial interest in any materials discussed in this article.