Developing new treatments for brain arteriovenous malformations: molecular responses to radiation in in vitro and in vivo models

Theoretical thesis.Bibliography: pages 162-194.Chapter 1. Introduction -- Chapter 2. General methods -- Chapter 3. Quantitative analysis of gene and protein expression of endothelial cell adhesion molecules after radiation -- Chapter 4.Anatomical localisation and quantification of post-radiosurgery ICAM-1 and VCAM-1 expression in an AVM animal model -- Chapter 5. Phosphatidylserine translocation and anatomical location post-irradiation in an AVM animal model -- Chapter 6. Summary and conclusions.Brain arteriovenous malformations are a leading cause of stroke in children and young adults. They account for 4% of haemorrhagic strokes. Arteriovenous malformations (AVMs) are complex vascular lesions, characterised by abnormal connections between arteries and veins that lack a capillary network. The treatment options for AVMs include surgery, radiosurgery and embolisation. However, over one third of AVM patients with large and deep AVMs cannot be safely and effectively treated with current methods. Therefore, a new treatment method is required for these patients with life-threatening AVMs. A biological technique that can be harnessed to enhance the rate of occlusion or thrombosis inside the AVM blood vessels is highly attractive. A proposed method to achieve this goal is vascular targeting. This approach has been applied in cancer therapy where unique molecular markers expressed on the surface of tumour vessels are targeted by conjugated antibodies and pro-thrombotic factors to induce thrombosis inside the tumour vessels. If such a technique can be used for AVM treatment, it will open a new window toward treatment of life-threatening AVMs. In order to follow this path, specific markers on the surface of the AVM endothelium need to be identified for selective targeting. However, previous studies have shown that AVM endothelial cells are not dramatically different to normal endothelial cells. Therefore, a priming mechanism is required. It is hypothesised that radiosurgery induces molecular changes on the surface of endothelial cells that can be used to discriminate irradiated vessels from normal vessels. Previous studies have shown radiation can induce endothelial membrane changes, such as phosphatidylserine (PS) translocation and up-regulation of various cell adhesion molecules (CAMs), including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), P-selectin and E-selectin, both in vitro and in vivo. Therefore, the aims of this study were as follows: 1) to examine which of the CAMs elicits the greatest response to radiation in endothelial cells in vitro and may provide the best candidate for a vascular targeting approach; 2) to determine the lowest radiation dose (5, 15 or 25 Gy) able to elicit a significant response in these molecules in vitro, as lower doses reduce the risk of off-target radiation damage to normal cells; 3) to determine the most discriminating CAMs in an AVM animal model; and 4) to examine the in vivo externalisation of PS in response to radiosurgery in the AVM animal model. While all four CAMs were up-regulated by irradiation in vitro, among the candidate molecules, ICAM-1 and VCAM-1 demonstrated the highest level of expression, followed by P-selectin. A dose of 15 Gy was as effective as 25 Gy at inducing expression while minimal response was evident at a dose of 5 Gy. The results of these studies led to the selection of candidate molecules for in vivo imaging, (ICAM-1 and VCAM-1), with 15 Gy as the treatment dose. In the AVM animal model, ICAM-1 and VCAM-1 were expressed at the luminal endothelial surface in the AVM region only. Expression of the two molecules was high in the AVM prior to radiosurgery. No significant increases in ICAM-1 and VCAM-1 were found in response to the 15 Gy radiation dose. The inability to detect differences in vivo suggested that the dose was not sufficient to further induce surface expression above the high background level, at least in this model. However, in vivo imaging of phosphatidylserine externalisation in the rat AVM model demonstrated that radiation could significantly increase PS exposure at the luminal surface. This was despite rat AVMs also displaying significantly elevated PS externalisation relative to the normal vasculature. This molecule may provide the most promising candidate to move toward vascular targeting of AVMs.Mode of access: World wide web1 online resource (198 pages) illustrations (some colour

Angiographic, hemodynamic, and histological changes in an animal model of brain arteriovenous malformations treated with Gamma Knife radiosurgery

Object: Brain arteriovenous malformations (AVMs) are a major cause of stroke. Many AVMs are effectively obliterated by stereotactic radiosurgery, but such treatment for lesions larger than 3 cm is not as effective. Understanding the responses to radiosurgery may lead to new biological enhancements to this treatment modality. The aim of the present study was to investigate the hemodynamic, morphological, and histological effects of Gamma Knife surgery (GKS) in an animal model of brain AVM. Methods: An arteriovenous fistula was created by anastomosing the left external jugular vein to the side of the common carotid artery in 64 male Sprague-Dawley rats (weight 345 ± 8.8 g). Six weeks after AVM creation, 32 rats were treated with a single dose of GKS (20 Gy); 32 animals received sham radiation. Eight irradiated and 8 control animals were studied at each specified time point (1, 3, 6, and 12 weeks) for hemodynamic, morphological, and histological characterization. Results: Two AVMs showed partial angiographic obliteration at 6 weeks. Angiography revealed complete obliteration in 3 irradiated rats at 12 weeks. Blood flow in the ipsilateral proximal carotid artery (p < 0.001) and arterialized jugular vein (p < 0.05) was significantly lower in the irradiated group than in the control group. The arterialized vein's external diameter was significantly smaller in GKS-treated animals at 6 (p < 0.05) and 12 (p < 0.001) weeks. Histological changes included subendothelial cellular proliferation and luminal narrowing in GKS-treated animals. Neither luminal obliteration nor thrombus formation was identified at any of the time points in either irradiated or nonirradiated animals. Conclusions: GKS produced morphological, angiographic, and histological changes in the model of AVM as early as 6 weeks after treatment. These results support the use of this model for studying methods to enhance radiation response in AVMs.7 page(s

In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation.

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs

Time course of ICAM-1 and VCAM-1 expression in the rat AVM model.

<p>Xenolight-750 probe binding was examined over a period of 84 days for ICAM-1 (A) and VCAM-1 (B) in control and irradiated (15 Gy) animals (n = 7 per group). Raw MFI (mean fluorescence intensity) was normalized to day 1 in matched animals to reduce inter-animal variation. No significant differences were detected at any time between irradiated and control AVMs. <i>Ex vivo</i> image analysis of Xenolight 750-ICAM-1 (C) and Xenolight 750-VCAM-1 (D) probe binding in excised tissue: common carotid artery (CCA), external jugular vein (EJV) (n = 7).</p

Quantitative real-time PCR and western analysis of ICAM-1 and VCAM-1 expression in irradiated bEnd.3 cells.

<p>qRT-PCR analysis of ICAM-1 (A) and VCAM-1 (E) gene expression, n = 4 independent experiments. Representative western blots (time course) of ICAM-1 (B) and VCAM-1 (F) protein at 25 Gy. Representative western blots (dose response) of ICAM-1 expression (120 h) (C) and VCAM-1 expression (72 h) (G) post-irradiation. ICAM-1 (D) and VCAM-1 (H) protein expression quantitated using Image J, n = 4. Values are mean ± SEM. Data were normalized to GAPDH (westerns) or HPRT (qRT-PCR).</p

Effect of radiation on bEnd.3 cell morphology and viability.

<p>(A) Representative images of bEnd.3 cells after radiation at doses of 15 and 25 Gy. Scale bar = 20 μm. All images at 200× magnification. (B) Cell viability was determined by trypan blue assay. Values are mean ± SEM, n = 3 for each group.</p

ELISA and immunocytochemical analysis of ICAM-1 and VCAM-1 expression in irradiated bEnd.3 cells.

<p>ELISA analysis of surface ICAM-1 (A) and VCAM-1 (D) expression in irradiated bEnd.3 cells normalized to Janus green absorbance to account for changes in cell number. Immunocytochemistry was performed on bEnd.3 cells using CF555-conjugated ICAM-1 or CF640-conjugated VCAM-1 antibodies (red). Staining was quantitated as integrated density using Image J (arbitrary units) for ICAM-1 (B) and VCAM-1 (E). Representative images are shown at 120 h (ICAM-1) (C) or 72 h (VCAM-1) (F) post-radiation at doses of 0–25 Gy. Isotype controls for ICAM-1 (IgG1-CF555) and VCAM-1 (IgG2b-CF640) showed no staining (representative images shown at 25 Gy, 72h). Cells were counterstained with DAPI to visualize nuclei (blue). All images were acquired at a magnification of 200× (scale bar = 100 μm). Values are mean ± SEM, n = 3 for each group.</p

<i>In vivo</i> near-infrared fluorescence imaging of Xenolight 750 probes in the rat AVM model.

<p>Rats with an AVM creation were sham treated or irradiated with a 15 Gy marginal dose to the AVM region by Gamma Knife and imaging performed 12 h after conjugate dye injection (25 μg/kg). Representative montages of x-ray (left), fluorescent (centre) and merged (right) images after injection of Xenolight 750 probes: (A) Xenolight-750 isotype control in irradiated animal; (B) Xenolight 750-ICAM-1 probe and; (C) Xenolight 750-VCAM-1 probe, at day 21 after sham (top panels) or radiation (bottom panels). Image J quantitation of fluorescence at day 21 post-irradiation or sham with Xenolight 750-ICAM-1 (D) or Xenolight 750-VCAM-1 (E) probes and Xenolight-750 isotype control probe.</p