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

PURPOSE. To determine the sensitivity of retinal pigment epithelial (RPE) c ells to a vascular endothelial growth factor (VEGF) chimeric toxin. METHODS. A targeted toxin was developed using recombinant methods to fuse V EGF(165) to the diphtheria toxin (DT) translocation and enzymatic domain (D T390-VEGF(165)). Human RPE cells, choroidal endothelial cells (CECs), and s cleral fibroblasts were isolated, and a dose-response for DT390-VEGF(165) w as determined by measurement of cell proliferation and cell number. In para llel experiments, cultures were pretreated with transforming growth factor (TGF)-beta(2). VEGF-receptor (VEGFR-1 and -2) expression was determined usi ng reverse transcription-polymerase chain reaction and fluorescence-activat ed cell sorting, and affinity was measured using Scatchard analysis. RESULTS. RPE cells and CECs were similarly prone to killing by the VEGF-tox in, but scleral fibroblasts were unaffected. Pretreatment with TGF-beta(2) selectively increased the sensitivity of RPE cells to the VEGF-toxin. RPE c ells expressed both VEGFR-1 and -2 in vitro; however, the expression of VEG FR-1 was very low. Pretreatment with TGF-beta(2) (10 ng/ml) was associated with increased expression of the VEGFR-1 in RPE cells and increased recepto r affinity for VEGF detected by Scatchard analysis. CONCLUSIONS. Dose-dependent killing of RPE cells by the DT390-VEGF(165) con jugate is selectively enhanced by pretreatment with TGF-beta(2). This study provides further strong support for the presence of functional VEGFRs on h uman RPE cells, and demonstrates for the first time the ability to target a normal nonendothelial cell type through VEGFR expression.