3D endothelial cell spheroid/human vitreous humor assay for the characterization of anti-angiogenic inhibitors for the treatment of proliferative diabetic retinopathy

Abstract

Proliferative diabetic retinopathy (PDR) represents a main cause of acquired blindness. Despite the recognition of the key role exerted by vascular endothelial growth factor (VEGF) in the pathogenesis of PDR, limitations to anti-VEGF therapies do exist. Thus, rapid and cost-effective angiogenesis assays are crucial for the screening of anti-angiogenic drug candidates for PDR therapy. In this context, evaluation of the angiogenic potential of PDR vitreous fluid may represent a valuable tool for preclinical assessment of angiostatic molecules. Here, vitreous fluid obtained from PDR patients after pars plana vitrectomy was used as a pro-angiogenic stimulus in a 3D endothelial cell spheroid/human vitreous assay. The results show that PDR vitreous is able to stimulate the sprouting of fibrin-embedded HUVEC spheroids in a time- and dose-dependent manner. A remarkable variability was observed among 40 individual vitreous fluid samples in terms of sprouting-inducing activity that was related, at least in part, to defined clinical features of the PDR patient. This activity was hampered by various extracellular and intracellular signaling pathway inhibitors, including the VEGF antagonist ranibizumab. When tested on 20 individual vitreous fluid samples, the inhibitory activity of ranibizumab ranged between 0 and 100% of the activity measured in the absence of the drug, reflecting a variable contribution of angiogenic mediators distinct from VEGF. In conclusion, the 3D endothelial cell spheroid/human vitreous assay represents a rapid and cost-effective experimental procedure suitable for the evaluation of the anti-angiogenic activity of novel extracellular and intracellular drug candidates, with possible implications for the therapy of PDR.

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