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Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.

ABSTRACT: 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.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.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.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.

SUBMITTER: Mohamedali KA

PROVIDER: S-EPMC3176242 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.

Mohamedali Khalid A KA Ran Sophia S Gomez-Manzano Candelaria C Ramdas Latha L Xu Jing J Kim Sehoon S Cheung Lawrence H LH Hittelman Walter N WN Zhang Wei W Waltenberger Johannes J Thorpe Philip E PE Rosenblum Michael G MG

BMC cancer 20110817

<h4>Background</h4>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.<h4>M ...[more]

PMID: 21849059

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Project description:BackgroundImmune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement.MethodsDAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody.ResultsVEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models.ConclusionsVEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Project description:Recently, vascular endothelial growth factor-C (VEGF-C or VEGF-2) was described as a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3. In vivo data, limited to constitutive overexpression in transgenic mice, have been interpreted as evidence that the growth-promoting effects of VEGF-C are restricted to development of the lymphatic vasculature. The current studies were designed to test the hypothesis that constitutive expression of VEGF-C in adult animals promotes angiogenesis. In vitro, VEGF-C exhibited a dose-dependent mitogenic and chemotactic effect on endothelial cells, particularly for microvascular endothelial cells (72% and 95% potency, respectively, compared with VEGF-A/VEGF-1). VEGF-C stimulated release of nitric oxide from endothelial cells and increased vascular permeability in the Miles assay; the latter effect was attenuated by pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Both VEGFR-2 and VEGFR-3 receptors were shown to be expressed in human saphenous vein and internal mammary artery. The potential for VEGF-C to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model. Ten days after ligation of the external iliac artery, VEGF-C was administered as naked plasmid DNA (pcVEGF-C; 500 microg) from the polymer coating of an angioplasty balloon (n = 8 each) or as recombinant human protein (rhVEGF-C; 500 microg) by direct intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C. Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased to 0.83 +/- 0.03 after pcVEGF-C versus 0.59 +/- 0.04 (P < 0.005) in pGSVLacZ controls and to 0.76 +/- 0.04 after rhVEGF-C versus 0.58 +/- 0.03 (P < 0.01) in control rabbits receiving rabbit serum albumin. Doppler-derived iliac flow reserve was 2.7 +/- 0.1 versus 2.0 +/- 0.2 (P < 0.05) for pcVEGF-C versus LacZ controls and 2.9 +/- 0.3 versus 2.1 +/- 0.2 (P < 0.05) for rhVEGF-C versus albumin controls. Neovascularity was documented by angiography in vivo (angiographic scores: 0.85 +/- 0.05 versus 0.51 +/- 0.02 (P < 0.001) for plasmid DNA and 0.74 +/- 0.08 versus 0.53 +/- 0.03 (P < 0.05) for protein), and capillary density (per mm2) was measured at necropsy (252 +/- 12 versus 183 +/- 10 (P < 0.005) for plasmid DNA and 229 +/- 20 versus 164 +/- 20 (P < 0.05) for protein). In contrast to the results of gene targeting experiments, constitutive expression of VEGF-C in adult animals promotes angiogenesis in the setting of limb ischemia. VEGF-C and its receptors thus constitute an apparently redundant pathway for postnatal angiogenesis and may represent an alternative to VEGF-A for strategies of therapeutic angiogenesis in patients with limb and/or myocardial ischemia.

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Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.

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Cytotoxicity of VEGF(121)/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2.

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