Internalization of annexin A 5-USPIOs by apoptotic cells
Internalization of annexin A 5-USPIOs by apoptotic cells
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凋亡细胞对膜联蛋白 A 5-USPIO 的内化
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发表时间：
2005
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通讯作者：
K. Nicolay
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作者：
G. V. Tilborg;G. Strijkers;H. Duimel;P. Bomans;P. Frederik;C. Reutelingsperger;K. Nicolay
G. A. van Tilborg、G. J. Strijkers、H. Duimel、P. Bomans、P. M. Frederik、C. P. Reutelingsperger、K. Nicolay 生物医学 NMR，埃因霍温理工大学生物医学工程系，荷兰埃因霍温，EM 单位，马斯特里赫特大学病理学系，马斯特里赫特，荷兰，马斯特里赫特心血管研究所，荷兰马斯特里赫特大学生物化学系介绍细胞凋亡在多种心血管疾病的病因学中发挥着重要作用，包括心肌梗死、心力衰竭和动脉粥样硬化。体内细胞凋亡的早期检测将有助于在早期阶段评估疾病治疗。膜联蛋白 A5 是一种在 Ca 存在下与磷脂酰丝氨酸 (PS) 特异性结合的蛋白质。磷脂 PS 暴露在凋亡细胞膜的外层，因此易于与细胞外膜联蛋白 A5 结合。因此，膜联蛋白 A5 结合的氧化铁颗粒能够通过 MRI 检测细胞凋亡。氧化铁造影剂的T1、T2和T2降低能力取决于多个参数，包括含铁细胞的细胞密度和细胞内氧化铁颗粒的区室化程度。对于给定的铁浓度，与聚集在核内体中的氧化铁颗粒相比，均匀分布的颗粒可诱导更短的 T1 和 T2 弛豫时间。相比之下，与更均匀分散的氧化铁颗粒相比，细胞区室中的颗粒积累可诱导更短的 T2 * 弛豫时间。最近显示，膜联蛋白 A5 蛋白可被同时暴露于凋亡刺激（包括抗 Fas）的细胞内化到内吞囊泡中。因此，膜联蛋白 A5-USPIO 也可能在体内内化。因此，可以通过选择正确的成像序列来优化 A5-USPIO 体内凋亡细胞的检测限。在这项研究中，我们通过 MRI 和透射电子显微镜 (TEM) 研究了可能的膜联蛋白 A5-USPIO 内化。为此目的，进行了一些实验，其中将在野生型膜联蛋白 A5 仅与细胞膜外层结合的条件下暴露于造影剂的细胞与观察到磷脂酰丝氨酸-膜联蛋白 A5 介导的胞饮作用的实验进行比较。材料和方法 T 淋巴瘤细胞系 Jurkat (ATCC) 在 RPMI1640 培养基中于 37°C、腐殖化气氛和 5% CO2 下生长。膜联蛋白 A5 功能化和非功能化 MACS 微珠均用作 T2 还原造影剂（Miltenyi Biotec，Auburn，CA）。为了诱导细胞凋亡，收获细胞并重悬于10个细胞/ml培养基中。 RPMI1640培养基用于结合实验，而含Ca培养基M199用于内化实验。 • 结合：用200 ng ml 抗Fas 处理细胞3 小时以诱导细胞凋亡，收获细胞并重悬于2.5 mM Ca 结合缓冲液中至终浓度为2.5·10 cells ml。随后细胞未经处理或与 USPIO 或膜联蛋白 A5-USPIO (0.5 μg ml Fe) 在室温下孵育 15 分钟。 • 内化：将USPIO 或膜联蛋白A5-USPIO (0.5 μg ml Fe) 添加到培养基M199 中。另外添加200 ng ml抗Fas，并将细胞在37°C、腐殖化气氛和5% CO2中孵育3小时。两个实验的孵育期结束后，在含 Ca2+ 的结合缓冲液或含有 Ca2+ 螯合剂 EDTA (5 mM) 的缓冲液中广泛洗涤细胞，已知 ETA 可以从细胞膜上去除结合的膜联蛋白 A5-USPIO。最后将细胞固定在分别含有 2.5 mM Ca 或 5 mM EDTA 的 2.5% 戊二醛溶液中，并在室温下在 250 μl 管中沉降成松散堆积的细胞沉淀过夜。细胞沉淀的 T2 弛豫时间在 6.3 T 下测量。用透射电子显微镜 (TEM) 确定为凋亡细胞沉淀提供降低的 T2 值的膜联蛋白 A5-USPIO 的空间位置。对于 TEM 电池，将其后固定在 1% 四氧化锇溶液中，脱水并嵌入环氧树脂中。进行额外的 EDX 光谱测定以确认 TEM 图像中膜联蛋白 A5-USPIO 被目视识别的区域中是否存在氧化铁。结果 在结合和内化实验中，膜联蛋白 A5-USPIO 在 Ca 存在下均能增加凋亡细胞沉淀的弛豫率 R2 (T2)，而 R2 值不受与非功能化 USPIO 一起孵育的影响（图 1）。通过在含有 EDTA 的缓冲液中进行大量洗涤来去除 Ca 2+ 可以降低用抗 Fas 刺激后与膜联蛋白 A5-USPIO 一起孵育的细胞的 R2 值至对照水平（图 1. 结合）。与膜联蛋白 A5-USPIO 和抗 Fas 共孵育的细胞的 R2 值也因 Ca 的去除而降低，但 R2 值并未恢复到对照水平（图 1 内化）。用抗-Fas 刺激后与膜联蛋白 A5-USPIO 一起孵育并随后在结合缓冲液中洗涤的细胞的 TEM 图像显示膜联蛋白 A5-USPIO 与细胞膜的结合（图 2 左）。另一方面，在与膜联蛋白 A5-USPIO 与抗 Fas 组合一起孵育的细胞中，发现了内化的膜联蛋白 A5-USPIO（图 2 右）。 EDX 光谱在 TEM 图像上识别出膜联蛋白 A5-USPIO 的区域显示出大的铁峰，而在周围区域没有发现铁峰（图 3）。讨论和结论 如果细胞同时暴露于膜联蛋白 A5-USPIO 和凋亡刺激，则膜联蛋白 A5-USPIO 会被内化到囊泡中，这表明磷脂酰丝氨酸 - 膜联蛋白 A5 介导的胞饮作用不受与高分子量造影剂缀合的限制。这里提出的体外结果表明这些造影剂也有望在体内内化。因此，当大量氧化铁颗粒内化时，与 T2 加权图像相比，在 T2* 加权图像中可以更早地检测到细胞凋亡。需要进一步研究来量化膜联蛋白 A5-USPIO 的空间位置对弛豫率 r2 * 与 r2 之比的影响。参考文献 1. Fadok V.A. 等人，《自然》，4:405(6782):85-90 (2000)。 2. Billotey C. 等人，医学磁共振，49：646-654 (2003)。 3. 鲍文 C.V.等人，《医学磁共振》，48：52-61 (2002)。 4. Kenis H. 等人，《生物化学杂志》，10；279(50)：52623-9 (2004)。
G. A. van Tilborg, G. J. Strijkers, H. Duimel, P. Bomans, P. M. Frederik, C. P. Reutelingsperger, K. Nicolay Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands, EM unit, Department of Pathology, University of Maastricht, Maastricht, Netherlands, Cardiovascular Research Institute of Maastricht, Department of Biochemistry, Maastricht University, Maastricht, Netherlands Introduction Apoptosis plays an important role in the etiology of a variety of cardiovascular diseases, including myocardial infarction, heart failure and atherosclerosis. Early detection of apoptosis in-vivo would allow for evaluation of disease treatment at an early stage. Annexin A5 is a protein that binds specifically to phosphatidylserine (PS) in the presence of Ca. The phospholipid PS is exposed on the outer layer of the apoptotic cell membrane, and hence becomes accessible for binding to extracellular annexin A5. Therefore annexin A5 conjugated iron oxide particles enable the detection of apoptosis with MRI. The T1, T2 and T2 -reducing ability of iron oxide contrast agent depends on multiple parameters, including the cellular density of iron containing cells and the degree of iron oxide particle compartmentalization within the cell. For a given iron concentration homogeneously distributed particles were shown to induce shorter T1 and T2 relaxation times compared to iron oxide particles that are clustered for example in endosomes. In contrast, particle accumulation in cellular compartments was shown to induce shorter T2 * relaxation times compared to more homogeneously dispersed iron oxide particles. The protein annexin A5 was recently shown to be internalized into endocytic vesicles by cells that are co-exposed to apoptotic stimuli, including anti-Fas. Consequently annexin A5-USPIOs may be internalized in-vivo as well. Therefore the detection limit of apoptotic cells in-vivo with A5-USPIOs can be optimized by choosing the correct imaging sequence. In this study we investigated possible annexin A5-USPIO internalization both with MRI and transmission electron microscopy (TEM). For this purpose experiments were performed in which cells that were exposed to the contrast agents under conditions for which wild type annexin A5 would only bind to the outer leaflet of the cell membrane were compared to experiments in which phosphatidylserine-annexin A5-mediated pinocytosis would be observed. Materials and methods The T-lymphoma cell line Jurkat (ATCC) were grown in RPMI1640 medium at 37 C in a humified atmosphere and 5% CO2. Both annexin A5-functionalized and nonfunctionalized MACS microbeads were used as T2 reducing contrast agents (Miltenyi Biotec, Auburn, CA). For the induction of apoptosis cells were harvested and resuspended to 10 cells ml medium. RPMI1640 medium was used for binding experiments whereas Ca-containing medium M199 was used for internalization experiments. • Binding: Cells were treated for 3 hours with 200 ng ml anti-Fas in order to induce apoptosis, harvested and respuspended in 2.5 mM Ca-containing binding buffer to a final concentration of 2.5·10 cells ml. Subsequently cells were left untreated or incubated with USPIOs or annexin A5-USPIOs (0.5 μg ml Fe) for 15 minutes at room temperature. • Internalization: USPIOs or annexin A5-USPIOs (0.5 μg ml Fe) were added to medium M199. Additionally 200 ng ml anti-Fas was added, and cells were incubated for 3 hours at 37C in a humified atmosphere and 5% CO2. Following the incubation periods of both experiments the cells were washed extensively either in Ca-containing binding buffer or in a buffer that contained the Ca chelator EDTA (5 mM), which is known to remove bound annexin A5-USPIOs from the cell membrane. Finally cells were fixed in a 2.5% gluteraldehyde solution, containing 2.5 mM Ca or 5 mM EDTA respectively, and allowed to settle to a loosely packed cell pellet in a 250 μl tube overnight at room temperature. T2 relaxation times of the cell pellets were measured at 6.3 T. The spatial location of the annexin A5-USPIOs that provided the apoptotic cell pellets with reduced T2 values was determined with transmission electron microscopy (TEM). For TEM cells were postfixed in 1% osmium tetroxide solution, dehydrated and embedded in epoxy resin. Additional EDX spectrometry was performed to confirm the presence of iron oxide in areas of the TEM images where annexin A5-USPIOs were visually identified. Results Annexin A5-USPIOs were shown to increase the relaxation rate R2 (T2 ) of apoptotic cell pellets in the presence of Ca for both the binding and the internalization experiments, whereas R2 values remained unaffected by incubation with non-functionalized USPIOs (Fig1). Removal of Ca 2+ by extensive washing in an EDTAcontaining buffer reduced the R2 values of cells that were incubated with the annexin A5-USPIOs after stimulation with anti-Fas to control levels (Fig1. Binding). R2 values of cells that were co-incubated with annexin A5-USPIOs and anti-Fas were also decreased by removal of Ca, however R2 values did not return to control levels (Fig1 Internalization). TEM images of cells that were incubated with annexin A5-USPIOs following stimulation with anti-Fas and subsequently washed in binding buffer showed binding of the annexin A5-USPIOs to the cell membrane (Fig2 left). On the other hand internalized annexin A5-USPIOs were found for cells that were incubated with annexin A5-USPIOs in combination with anti-Fas (Fig2 right). EDX spectra displayed large iron peaks in the areas where annexin A5-USPIOs were identified on the TEM images, whereas no iron peaks were found in the surrounding areas (Fig3). Discussion and conclusion Annexin A5-USPIOs were shown to be internalized into vesicles if cells are co-exposed to annexin A5-USPIOs and apoptotic stimuli, indicating that phosphatidylserine-annexin A5-mediated pinocytosis is not restricted by conjugation to high molecular weight contrast agents. In vitro results presented here suggest that these contrast agents can be expected to be internalized in-vivo as well. Consequently, when large number of iron oxide particles are internalized apoptosis may be detected at an earlier stage in T2*-weighted images compared to T2-weighted images. Further research is required to quantify the effect of the spatial location of the annexin A5-USPIOs on the ratio of relaxivity r2 * to r2. References 1. Fadok V.A.et al., Nature, 4:405(6782):85-90 (2000). 2. Billotey C. et al., Magnetic Resonance in Medicine, 49:646-654 (2003). 3. Bowen C.V. et al., Magnetic Resonance in Medicine, 48:52-61 (2002). 4. Kenis H. et al., The Journal of Biological Chemistry, 10;279(50):52623-9 (2004).