title: Radiosurgery Alters the Endothelial Surface Proteome: Externalized Intracellular Molecules as Potential Vascular Targets in Irradiated Brain Arteriovenous Malformations.
abstract: Stereotactic radiosurgery (SRS) is an established treatment for brain arteriovenous malformations (AVMs) that drives blood vessel closure through cellular proliferation, thrombosis and fibrosis, but is limited by a delay to occlusion of 2-3 years and a maximum treatable size of 3 cm. In this current study we used SRS as a priming tool to elicit novel protein expression on the endothelium of irradiated AVM vessels, and these proteins were then targeted with prothrombotic conjugates to induce rapid thrombosis and vessel closure. SRS-induced protein changes on the endothelium in an animal model of AVM were examined using in vivo biotin labeling of surface-accessible proteins and comparative proteomics. LC-MS/MS using SWATH acquisition label-free mass spectrometry identified 280 proteins in biotin-enriched fractions. The abundance of 56 proteins increased after irradiation of the rat arteriovenous fistula (20 Gy, ≥1.5-fold). A large proportion of intracellular proteins were present in this subset: 29 mitochondrial and 9 cytoskeletal. Three of these proteins were chosen for further validation based on previously published evidence for surface localization and a role in autoimmune stimulation: cardiac troponin I (TNNI3); manganese superoxide dismutase (SOD2); and the E2 subunit of the pyruvate dehydrogenase complex (PDCE2). Immunostaining of AVM vessels confirmed an increase in abundance of PDCE2 across the vessel wall, but not a measurable increase in TNNI3 or SOD2. All three proteins co-localized with the endothelium after irradiation, however, more detailed subcellular distribution could not be accurately established. In vitro, radiation-stimulated surface translocation of all three proteins was confirmed in nonpermeabilized brain endothelial cells using immunocytochemistry. Total protein abundance increased modestly after irradiation for PDCE2 and SOD2 but decreased for TNNI3, suggesting that radiation primarily affects subcellular distribution rather than protein levels. The novel identification of these proteins as surface exposed in response to radiation raises important questions about their potential role in radiation-induced inflammation, fibrosis and autoimmunity, but may also provide unique candidates for vascular targeting in brain AVMs and other vascular tissues.
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Lucinda S
authors.initials: LS
authors.lastname: McRobb
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Vivienne S
authors.initials: VS
authors.lastname: Lee
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.|c   Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, California.
authors.forename: Margaret
authors.initials: M
authors.lastname: Simonian
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Zhenjun
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Santhosh George
authors.initials: SG
authors.lastname: Thomas
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Markus
authors.initials: M
authors.lastname: Wiedmann
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Jude V Amal
authors.initials: JV
authors.lastname: Raj
authors.affiliation: d   Genesis Cancer Care, Macquarie University Hospital, Sydney, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Grace
authors.affiliation: d   Genesis Cancer Care, Macquarie University Hospital, Sydney, New South Wales, Australia.
authors.forename: Vaughan
authors.initials: V
authors.lastname: Moutrie
authors.affiliation: b   Australian Proteome Analysis Facility, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: McKay
authors.affiliation: b   Australian Proteome Analysis Facility, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
authors.affiliation: a   Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Stoodley
mesh_terms: D000818:Animals; D001165:Arteriovenous Malformations / Q000378:metabolism* / Q000473:pathology / Q000532:radiotherapy*; D001921:Brain / Q000473:pathology* / Q000528:radiation effects; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism / Q000528:radiation effects*; D042541:Intracellular Space / Q000378:metabolism / Q000528:radiation effects*; D008297:Male; D021381:Protein Transport / Q000528:radiation effects; D020543:Proteome / Q000378:metabolism*; D016634:Radiosurgery; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020543:Proteome