01whole.pdf (6.04 MB)
Developing new treatments for brain arteriovenous malformations: molecular responses to radiation in in vitro and in vivo models
thesisposted on 2022-03-29, 02:02 authored by Newsha Raoufi-Rad
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.