Radiation-induced markers for vascular targeting

<p><strong>Background: </strong>Brain arteriovenous malformations (bAVMs) are vascular disorders associated with a high lifetime risk of haemorrhagic stroke as they occur in children and young adults. Despite traditional therapies, about one-third of bAVMs remain incurable. It is critical, therefore, to develop innovative new treatments. Vascular targeting is a therapy that has shown some promise in tumour targeting. This technique may offer an alternate way of treating bAVMs if combined with the use of focussed radiosurgery as a priming tool to induce molecular markers selectively on bAVM vessels. These markers can then be used for targeted drug delivery. Intracellular proteins translocated atypically to the cell surface of irradiated vessels have recently been identified as putative radiation-induced endothelial biomarkers. In this thesis, it was hypothesised that one mitochondrial protein, PDCE2 (the mitochondrial E2 subunit of pyruvate dehydrogenase complex), could be targeted after AVM radiosurgery with a ligand-directed coagulant (coaguligand) to simulate local thrombosis and bAVMs closure and cure.</p> <p><strong>Aims: </strong>The overall purpose of this thesis was to study radiation-stimulated PDCE2 ectopic cell surface PDCE2, its mechanism of atypical translocation, and its potential as a novel target for ligand-directed vascular targeting and thrombosis induction using <em>in vitro </em>and <em>in vivo </em>bAVMs models.</p> <p><strong>Methods: </strong>Human cerebral microvascular endothelial cells were used as an <em>in vitro </em>model of human brain endothelial cells. Delivery of ionising radiation to monolayer cultures was by a linear accelerator. Immunocytochemistry, western analysis and co-localisation assays were used to analyse PDCE2 expression and the underlying mechanism of its ectopic localisation. PDCE2-targeting coaguligand was prepared via crosslinking of thrombin and a monoclonal antibody. Specificity and efficacy of thrombosis of this agent were tested in a dynamic closed-loop parallel-plate flow chamber containing whole human blood and human endothelial cells. Static tests were also developed for rapid thrombosis analysis. Blood clot formation was measured via confocal imaging. The binding of monoclonal and polyclonal antibodies to externalised PDCE2 was preliminarily assessed in a bAVMs rat model using <em>in vivo </em>imaging.</p> <p><strong>Results and conclusion: </strong>Radiation induces PDCE2 ectopic cell surface expression in human endothelial cells via blockade of the degradative autophagy pathway and up-regulation of the secretory autophagy pathway. Anti-PDCE2-thrombin coaguligands assessed in static and dynamic thrombosis assays can induce rapid and significant thrombosis on irradiated endothelial cells. Efficacy is dependent on a combination of both radiation and coaguligand dose. Preliminary data demonstrate that externalised PDCE2 can be targeted on the luminal surface of irradiated bAVMs in a rat model. The findings support continued investigation of radiation-induced PDCE2 as a valid, discriminatory marker for radiation-guided vascular targeting in the rat model.</p>