In vivo evidence of angiogenesis inhibition by β2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.血管网络的扩张与功能是影响宫内胎儿正常发育的关键因素。本研究针对此前报道的β2-糖蛋白I（beta-2-glycoprotein I，β2GPI）在鸡胚血管生成体内模型中的抗血管生成潜能展开探究。本研究考察了两种天然存在的β2GPI形式对尿囊绒膜（chorioallantoic membrane，CAM）血管及鸡胚发育的影响。研究人员通过分级纯化获得β2GPI单体与二聚体，并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）、免疫印迹（immunoblot）及酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）对其进行表征。通过向鸡胚卵内注射小体积的2.5 μg/mL β2GPI亚组分溶液完成给药操作。研究人员采用纹理对比度计算分析与计算机视觉技术，对采集得到的CAM图像中的血管架构参数进行定量测量，以此评估血管生成情况，并将定量数据与CAM血管系统的形态变化相关联。体内实验结果显示，β2GPI二聚体可完全阻断CAM血管的形成，并在给药暴露48小时后导致鸡胚死亡。尽管CAM血管出现早期形态改变，β2GPI单体仍可支持鸡胚发育至第10天；正常血管生长的受损过程呈现出自限性特征。经β2GPI单体处理的鸡胚卵，在孵化第6天时血管化程度降低，但至孵化第10天时鸡胚仍存活，此时CAM血管出现充盈，提示血管生成抑制存在后续效应。两种β2GPI亚组分均会损害CAM血管系统的发育。实验证实，β2GPI二聚体的危害性远高于β2GPI单体。通过裂解或二聚化修饰β2GPI，可能在体内血管生成调控中发挥作用。