Selective killing of RPE with a vascular endothelial growth factor chimeric toxin.
OBJECTIVE
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)-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.


RESULTS
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.


CONCLUSIONS
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.
Hoffmann
S
S
Doheny Eye Institute, Los Angeles, California, USA.
Masood
R
R
Zhang
Y
Y
He
S
S
Ryan
S J
SJ
Gill
P
P
Hinton
D R
DR
D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply; D004167:Diphtheria Toxin / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000633:toxicity; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D008222:Lymphokines / Q000633:toxicity; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D017978:Receptors, Growth Factor / Q000096:biosynthesis / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D004167:Diphtheria Toxin; D016228:Endothelial Growth Factors; D000922:Immunotoxins; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D016212:Transforming Growth Factor beta; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1