Vascular endothelial growth factor (VEGF) exerts a pathogenic role in proliferative diabetic retinopathy (PDR), a major cause of blindness in the working-age population. Accordingly, anti-VEGF drugs represent a widespread approach in PDR therapy (1). However, even though VEGF is present at picomolar/low nanomolar concentrations in the PDR vitreous as assessed by ELISA (2), high-affinity VEGF-blocking drugs are administered intravitreally at micromolar concentrations and provide a poor response in a significant percentage of patients with PDR (3). Pharmacokinetic considerations, presence of other bioactive molecules, and/or a reduced accessibility of vitreal VEGF may explain, at least in part, the partial efficacy of anti-VEGF drugs and their extremely high drug-to-target stoichiometric ratio. On these bases, we reevaluated the actual concentration of VEGF in PDR vitreous and its accessibility by VEGF traps. With this aim, we established a near-quantitative VEGF Western blot (WB) assay (Fig. 1 A ) using doses of recombinant VEGF (rVEGF) between 5.0 and 17.5 ng per sample that were confirmed by ELISA. Then WB and ELISA were used in parallel to assess VEGF levels in PDR vitreous samples obtained by pars plana vitrectomy from 16 patients with type 2 diabetes (eight males, eight females; mean ± SD age: 66 ± 11 years, diabetes duration: 11 ± 6 years). As shown in Fig. 1 B , ELISA dramatically underestimates the concentration of vitreal VEGF when compared with WB …