Selective killing of RPE with a vascular endothelial growth factor chimeric toxin

To determine the sensitivity of retinal pigment epithelial (RPE) cells to a vascular endothelial growth factor (VEGF) chimeric toxin.

A targeted toxin was developed using recombinant methods to fuse VEGF165 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165). Human RPE cells, choroidal endothelial cells (CECs), and scleral fibroblasts were isolated, and a dose-response for DT390-VEGF165 was determined by measurement of cell proliferation and cell number. In parallel experiments, cultures were pretreated with transforming growth factor (TGF)-beta2. VEGF-receptor (VEGFR-1 and -2) expression was determined using reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting, and affinity was measured using Scatchard analysis.

RPE cells and CECs were similarly prone to killing by the VEGF-toxin, but scleral fibroblasts were unaffected. Pretreatment with TGF-beta2 selectively increased the sensitivity of RPE cells to the VEGF-toxin. RPE cells expressed both VEGFR-1 and -2 in vitro; however, the expression of VEGFR-1 was very low. Pretreatment with TGF-beta2 (10 ng/ml) was associated with increased expression of the VEGFR-1 in RPE cells and increased receptor affinity for VEGF detected by Scatchard analysis.

Dose-dependent killing of RPE cells by the DT390-VEGF165 conjugate is selectively enhanced by pretreatment with TGF-beta2. This study provides further strong support for the presence of functional VEGFRs on human RPE cells, and demonstrates for the first time the ability to target a normal nonendothelial cell type through VEGFR expression.