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Proteomics analysis of brain AVM endothelium post irradiation in pursuit of targets for AVM molecular therapy

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thesis
posted on 28.03.2022, 02:07 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.

History

Table of Contents

Chapter 1. General introduction -- Chapter 2. In vitro biotinylation protocol optimizations of murine endothelial cell cultures (bEnd3) -- Chapter 3. In vitro biotinylation and iTRAQ-mass spectrometry of bEnd.3 cells -- Chapter 4. In vitro biotinylation and mass spectrometry expression (MSE) of bEnd.3 cell cultures post irradiation -- Chapter 5. In vivo biotinylation optimization of the rat model of AVM -- Chapter 6. In vivo biotinylation and response of endothelial cells to irradiation in the rat model of AVM -- Chapter 7. General discussion.

Notes

Bibliography: pages 168-179 Theoretical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Medicine and Health Sciences, Australian School of Advanced Medicine

Department, Centre or School

Australian School of Advanced Medicine

Year of Award

2016

Principal Supervisor

Marcus Stoodley

Additional Supervisor 1

Mark Molloy

Rights

Copyright Margaret Simonian 2016. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (192 pages) illustrations (some colour)

Former Identifiers

mq:57458 http://hdl.handle.net/1959.14/1164012