Three-dimensional tubule formation assay as therapeutic screening model for ocular microvascular disorders

Abstract

Purpose: This study is aimed to adapt a three-dimensional (3-D) in vitro angiogenesis model to the ophthalmology field using retinal endothelial cells (REC). This system is applied to assess the angiogenic capacity of aqueous humor (AH) from patients with ocular disorders, and to test the effect of VEGF inhibitor (aflibercept) on induced angiogenesis. Methods: Human REC and umbilical vein endothelial cells (HUVEC) and pericytes were co-cultured in a gel matrix with 25–200 ng/ml pro-angiogenic growth factors (GF). AH from patients with cataract, glaucoma or proliferative diabetic retinopathy (PDR) was tested in the REC-pericyte co-culture. Aflibercept was then introduced to the co-culture containing PDR AH. The surface area and total tubule length were measured using Image J. Results: Optimal GF concentrations at 200 ng/ml induced angiogenesis by REC as well as HUVEC, while vessel formation by both cell types was strongly reduced using 25–50 ng/ml GF. Addition of AH from the PDR patient triggered tubule formation by REC at low GF concentration. Aflibercept, however, significantly inhibited angiogenesis induced by PDR AH, but showed no significant influence on other conditions. Conclusion: REC can be applied efficiently in the 3-D in vitro angiogenesis model as a diagnostic tool to assess the AH angiogenic status and to validate new anti-angiogenic therapeutic compounds prior to clinical trial.