title: Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.
abstract: OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation causes remarkable cellular changes in endothelial cells. Significant PS externalization is induced by radiation at doses of 15 Gy or higher, concomitant with a block in the cell cycle. Radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for AVM treatment.
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Zhenjun
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Faculty of Science, University of Technology; and.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Biyi
authors.initials: B
authors.lastname: Chen
authors.affiliation: Genesis Cancer Care, Macquarie University Hospital, Sydney, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Grace
authors.affiliation: Genesis Cancer Care, Macquarie University Hospital, Sydney, New South Wales, Australia.
authors.forename: Jaysree
authors.initials: J
authors.lastname: Ukath
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Vivienne S
authors.initials: VS
authors.lastname: Lee
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Lucinda S
authors.initials: LS
authors.lastname: McRobb
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Sedger
authors.affiliation: Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Stoodley
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects*; D016923:Cell Death / Q000502:physiology / Q000528:radiation effects; D049108:Cell Enlargement / Q000528:radiation effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects*; D005434:Flow Cytometry / Q000379:methods; D002538:Intracranial Arteriovenous Malformations / Q000378:metabolism / Q000532:radiotherapy; D051379:Mice; D010315:Particle Accelerators; D010718:Phosphatidylserines / Q000378:metabolism*; D011832:Radiation Injuries / Q000378:metabolism / Q000473:pathology; D011839:Radiation, Ionizing; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D010718:Phosphatidylserines
keywords: AVM = arteriovenous malformation; FWD = forward; PI = propidium iodide; PS = phosphatidylserine; SRS = stereotactic radiosurgery; brain arteriovenous malformations; cell cycle; endothelial cells; ionizing radiation; live-cell imaging; pSIVA = polarity-sensitive indicator of viability and apoptosis; phosphatidylserine externalization; stereotactic radiosurgery; vascular disorders