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Selective Killing of RPE with a Vascular Endothelial Growth Factor Chimeric Toxin

From the Departments of ² Pathology, ⁵ Medicine, and ¹ Ophthalmology, Keck School of Medicine of the University of Southern California; ⁴ Doheny Eye Institute; and ³ Norris Cancer Center, Los Angeles, California.

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

METHODS. 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)-ß₂. 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.

RESULTS. RPE cells and CECs were similarly prone to killing by the VEGF-toxin, but scleral fibroblasts were unaffected. Pretreatment with TGF-ß₂ 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-ß₂ (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.

CONCLUSIONS. Dose-dependent killing of RPE cells by the DT₃₉₀-VEGF₁₆₅ conjugate is selectively enhanced by pretreatment with TGF-ß₂. 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.

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