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Proteomics analysis of brain AVM endothelium post irradiation in pursuit of targets for AVM molecular therapy
thesisposted on 2022-03-28, 02:07 authored by Margaret Simonian
Brain arteriovenous malformations (AVMs) are congenital abnormalities that consist of direct connections between arteries and veins. Ruptured AVMs are the major cause of haemorrhagic stroke in children and young adults. Treatment of AVM depends on their size and location. Radiosurgery is the treatment recommended for lesions < 3 cm; however vascular occlusion after radiosurgery can take up to 3 years to complete, while patients remain at risk of haemorrhage. Approximatly one third of AVMs are unsuitable for current treatment methods of surgery and radiosurgery, such as large and deep AVMs, therefore there is a need for a new treatment that is safer and more effective than current treatment methods. This thesis research will be focused on identifying proteins on the surface of AVM vessels in response to radiosurgery that can be used as targets for AVM molecular therapies, as a new treatment method. Project aims - Specifically, this project aimed to identify proteins in irradiated AVM endothelium that are different from those expressed in normal vessels. Protein candidates could then be investigated for a ligand-directed treatment to promote rapid thrombosis in AVM vessels post radiosurgery. In vitro and in vivo biotinylation methods to label membrane proteins were first optimized then employed in murine cerebral endothelial cell cultures (bEnd.3) and the rat model of AVM. Membrane protein changes in response to irradiation were assessed using proteomics analysis. This is the first time that proteomics has been employed in the study of AVM endothelium. Hypothesis - The central hypothesis is that radiosurgery induces changes in AVM endothelial membrane proteins that allow discrimination from normal endothelial cells, providing protein targets that can be used for a ligand-based vascular targeting therapy. Results - Cell surface protein biotinylation and quantitative proteomics analyses successfully identified membrane proteins from endothelial cell cultures and the animal model of AVM in response to irradiation. The most significant in the cell cultures were, PECAM, cadherin 5, PDI, integrin alpha5, integrin alpha6, integrin beta1, CD109, EPCR and multimerin2, and in the rat model were, profilin1, potassium voltage gated channel protein, chloride intracellular channel protein 2 and ESAM-1. Most up-regulations were observed at 24h post irradiation. The upregulated membrane proteins identified from this thesis novel research are currently being investigated as potential targets for the ligand-directed molecular targeting trials in the rat model of AVM.