title: Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.
abstract: BACKGROUND
  The fusion protein VEGF(121)/rGel composed of the growth factor VEGF(121) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF(121)/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.
  
  
  METHODS
  We investigated the binding, cytotoxicity and internalization profile of VEGF(121)/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.
  
  
  RESULTS
  Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with (125)I-VEGF(121)/rGel demonstrated binding specificity that was competed with unlabeled VEGF(121)/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF(121)/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC(50) levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF(121)/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF(121)/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF(121)/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF(121)/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.
  
  
  CONCLUSIONS
  Taken together, these data confirm the selectivity of VEGF(121)/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.
authors.affiliation: Departments of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. kmohamed@mdanderson.org
authors.forename: Khalid A
authors.initials: KA
authors.lastname: Mohamedali
authors.forename: Sophia
authors.initials: S
authors.lastname: Ran
authors.forename: Candelaria
authors.initials: C
authors.lastname: Gomez-Manzano
authors.forename: Latha
authors.initials: L
authors.lastname: Ramdas
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.forename: Sehoon
authors.initials: S
authors.lastname: Kim
authors.forename: Lawrence H
authors.initials: LH
authors.lastname: Cheung
authors.forename: Walter N
authors.initials: WN
authors.lastname: Hittelman
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Johannes
authors.initials: J
authors.lastname: Waltenberger
authors.forename: Philip E
authors.initials: PE
authors.lastname: Thorpe
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rosenblum
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000166:cytology; D002642:Chick Embryo; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D016503:Drug Delivery Systems; D042783:Endothelial Cells / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics / Q000494:pharmacology*; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000494:pharmacology*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D020533:Angiogenesis Inhibitors; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D042461:Vascular Endothelial Growth Factor A; C025674:GEL protein, Gelonium multiflorum; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2