Zhao Zhenjun, Johnson Michael S, Chen Biyi, Grace Michael, Ukath Jaysree, Lee Vivienne S, McRobb Lucinda S, Sedger Lisa M, Stoodley Marcus A
Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University;
Faculty of Science, University of Technology; and.
J Neurosurg. 2016 Jun;124(6):1780-7. doi: 10.3171/2015.4.JNS142129. Epub 2015 Oct 2.
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.
目的 立体定向放射外科手术（SRS）是治疗脑动静脉畸形（AVM）的一种既定干预措施。SRS后AVM血管闭塞的过程尚不清楚。为提高SRS疗效，了解血管对辐射的细胞反应很重要。照射后AVM内皮细胞表面的分子变化也可用于血管靶向。本研究使用活细胞成像研究辐射诱导的内皮细胞磷脂酰丝氨酸（PS）外化。方法 培养从小鼠脑内皮生成的永生化细胞系bEnd.3细胞，并用直线加速器以不同辐射剂量进行照射。随后使用极性敏感的活力和凋亡指示剂（pSIVA）-IANBD（一种极性敏感探针）可视化细胞中的PS外化。使用活细胞成像实时监测PS外化。还通过流式细胞术检查辐射对bEnd.3细胞周期的影响。结果 电离辐射效应具有剂量依赖性。暴露于5 Gy辐射后观察到细胞增殖率降低，而更高的辐射剂量（15 Gy和25 Gy）完全抑制增殖。与假辐射处理的细胞相比，照射后的细胞显示出明显的伪足伸长，而细胞体很少或没有伸展。在接受25 Gy和15 Gy辐射剂量的培养物中，处理后24小时pSIVA阳性细胞的百分比显著更高（p = 0.04）。这种效应一直持续到实验结束（3天）。与假辐射对照组相比，5 Gy辐射在任何时间点均未诱导明显的PS外化（p > 0.15）。流式细胞术分析数据表明，照射诱导bEnd.3细胞生长停滞，细胞积聚在细胞周期的G2期。结论 电离辐射导致内皮细胞发生显著的细胞变化。15 Gy或更高剂量的辐射诱导显著的PS外化，同时伴有细胞周期阻滞。辐射诱导的标志物/靶点在AVM治疗的血管靶向中可能具有很高的鉴别能力。
