Antiangiogenic gene therapy: Disruption of neovascular networks mediated by inducible caspase-9 delivered with a transcriptionally targeted adenoviral vector

Angiogenesis Research Laboratory, Department of Restorative Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109, USA.
Gene Therapy (Impact Factor: 3.1). 03/2005; 12(4):320-9. DOI: 10.1038/
Source: PubMed


The activation of an inducible caspase (iCaspase-9) mediates apoptosis of neovascular endothelial cells, and overcomes the prosurvival effect of vascular endothelial growth factor or basic fibroblast growth factor. The potential utilization of direct activation of caspases as an antiangiogenic strategy for treatment of angiogenesis-dependent diseases (eg cancer) requires expression of the inducible caspase primarily in the tumor endothelium. The objective of this work was to develop and characterize a transcriptionally targeted adenoviral vector that mediates expression of iCaspase-9 specifically in neovascular endothelial cells. We observed that adenoviral vectors containing the human VEGFR2 promoter induced reporter gene expression primarily in proliferating human dermal microvascular endothelial cells (HDMEC). HDMEC transduced with recombinant adenoviral vectors containing iCaspase-9 under regulation of the VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) and exposed to a cell-permeable dimerizer drug (AP20187), presented higher caspase-3 activity and apoptosis than controls (P < or = 0.05). Using the SCID Mouse Model of Human Angiogenesis, we observed that local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 resulted in endothelial cell apoptosis and local ablation of microvessels. We believe that this constitutes the first report of a transcriptionally targeted antiangiogenic adenoviral vector that mediates neovascular disruption upon activation of a caspase-based artificial death switch.

Download full-text


Available from: Jacques E Nör, Aug 07, 2014
  • Source
    • "Initial evaluation of the Robo4-driven Luc expression in endothelial cells in vitro demonstrated relatively low levels of promoter activity in comparison with AdH5CMVLuc. However, compared with other promoters tested to date (Cefai et al., 2005; Greenberger et al., 2004; Reynolds et al., 2001; Savontaus et al., 2002; Song et al., 2005; Takayama et al., 2007; Wung et al., 2005; Yang et al., 2006), the Robo4 promoter displayed significantly less liver tropism and higher endothelial-specific expression in vivo. Remarkably, the lung endothelial expression was specific and high, while the liver expression was barely detectable by immunohistochemistry analysis . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.
    Virology 12/2013; 447(1-2):312-25. DOI:10.1016/j.virol.2013.09.020 · 3.32 Impact Factor
  • Source
    • "In this model, we seed primary human endothelial cells together with head and neck cancer cells in highly porous biodegradable scaffolds, implant them in the subcutaneous of SCID mice, and observe tumor progression and tumor angiogenesis. Here, we used luciferase-tagged OSCC3 cells to allow us to evaluate tumor progression noninvasively over time, as described [24] [25] [26]. To evaluate the effect of Bcl-2 expression levels in endothelial cells on tumor cell invasion, we removed the primary tumors 35 days after implantation of the cells and evaluated local recurrence of the tumors by in vivo bioluminescence for a period of 3 weeks (Figure 2A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.
    Neoplasia (New York, N.Y.) 03/2008; 10(2):131-9. DOI:10.1593/neo.07815 · 4.25 Impact Factor
  • Source
    • "It is known that scaffolds seeded only with HDMEC-iCaspase-9 or with HDMEC-LXSN presents similar microvascular density before administration of the dimerizer drug AP20187 [32]. Starting eleven days after implantation (time required to form functional human blood vessels in this model), mice received a daily intraperitoneal injection of either vehicle or 2 mg/ kg AP20187, since this concentration was determined previously to be effective in inducing HDMEC-iCaspase-9 apoptosis in vivo [32] [33]. Immunostaining with anti-Factor VIII antibody was used to identify the microvessels in paraffin embedded sections, and demonstrated a direct relationship between increasing proportions of HDMEC-iCaspase-9 cells with decreasing numbers of blood vessels at the end of the experiment (Figs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF(165)) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.
    Experimental Cell Research 11/2007; 313(16):3645-57. DOI:10.1016/j.yexcr.2007.07.023 · 3.25 Impact Factor
Show more