Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations

Abstract

Brain arteriovenous malformations (AVMs) are congenital abnormalities that consist of direct connections between arteries and veins, allowing highpressure arterial blood to flow into fragile cerebral veins, resulting in a high risk of hemorrhagic stroke. 16,37 The aim of AVM treatment is to prevent hemorrhage. 29 Treatment is effective only with complete AVM removal or obliteration. 14 Current treatments-namely surgery, endovascular occlusion, or stereotactic radiosurgery-are all associated with significant limitations.1,42 The surgical risk is generally unacceptable for lesions that are large, located in critical brain structures, or involve deep perforating arterabbreviatioNs AVM = arteriovenous malformation; FWD = forward; PI = propidium iodide; PS = phosphatidylserine; pSIVA = polarity-sensitive indicator of viability and apoptosis; SRS = stereotactic radiosurgery. obJective 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. Ra...

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations

“…In addition, radiation therapy has been reported to enhance PS exposure on tumor endothelium, serving as a potential radiation-stimulated target in future clinical applications [25,26]. Our recent studies also demonstrated significant externalization of endothelial PS upon radiation both in vitro, in brain microvascular endothelial cells, and in vivo, using a rat model of AVM [11,12]. This suggests PS could be used as a vascular target in irradiated AVMs.…”

“…Hence, we aim to use radiosurgery to prime the AVM endothelium to induce potential targets. Our previous studies demonstrated that radiation can induce upregulation of surface biomarkers on the AVM endothelium thus discriminating it from normal vessels [10][11][12][13][14]. These proteins may serve as potential targets for ligand-directed pro-thrombotic conjugates, enhancing the thrombotic response when induced with radiation.…”

Ionizing radiation reduces ADAM10 expression in brain microvascular endothelial cells undergoing stress-induced senescence

“…Cells were used between passages 18-24 and passaged with 0.1% Trypsin/EDTA. Cells were seeded in 6-well plates for protein extraction, 8-well chamber slides (Thermoscientific) for immunocytochemistry or 75 cm 2 flasks for biotinlabelling at 30% confluence and irradiated with X-rays (20 Gy) generated by a 6 MV linear accelerator (LINAC, Elekta Synergy, Crawley, UK) at Macquarie University Hospital (Sydney, Australia) as previously described [67]. Control cells were treated identically but received no radiation.…”

Ionizing Radiation-Induced Immune and Inflammatory Reactions in the Brain

“…Several in vitro and in vivo studies demonstrated endothelial cell apoptosis as an early event after irradiation. However, it was induced only by high doses of irradiation and was accompanied by strong inhibition of endothelial cell proliferation capacity (110, 111). The rate of apoptotic endothelial cells was estimated to be around 15% within 24 h after irradiation with high doses (112, 113).…”