Vascular Biology Lab, Department of Biotechnology and AU-KBC Research Centre, MIT Campus, Anna University, Chennai, India.
Vascular Biology Lab, Department of Biotechnology and AU-KBC Research Centre, MIT Campus, Anna University, Chennai, India.
Microvasc Res. 2018 Sep;119:47-59. doi: 10.1016/j.mvr.2018.04.001. Epub 2018 Apr 9.
Existing animal models for screening tumor angiogenic process have various setbacks that necessitate further investigations. In this study, we developed an ex-ovo egg yolk angiogenesis model to screen the angiogenic potency of tumor cells (HeLa and SiHa cell lines). The egg yolk angiogenesis assay was applied to study the nitric oxide (NO) influence on switching from sprouting angiogenesis (SA) to intussusceptive angiogenesis (IA) under tumor microenvironment. Morphological analysis and SA-like or IA-like markers expression were determined during the development of chicken chorioallantoic membrane (CAM) from day 5 to 13. Expression of Notch1, Notch2, EphrinB2, and Tie2 were considered as SA-like while TEM8, CALD1, CXCR4 and HOMX1 were followed as IA-like markers. The HeLa and SiHa cell lines embedded CAM showed an increase in micro and macro blood vessels and vascular size, junction and length which are the pivotal morphological parameters of angiogenesis. Further, the study revealed that HeLa is more aggressive than SiHa in inducing tumor angiogenesis. To determine the NO signaling implication in tumor milieu, NO donor (Spermine NONOate (SPNO)), NOS inhibitor (L-nitro-L-arginine-methyl ester (L-NAME) and VEGF inhibitor (Avastin) were administrated to chick embryo vascular bed with and without HeLa cells. The results demonstrated that HeLa cells promote IA through NO signaling, VEGF and eNOS and it was documented by angiogenic morphological parameters and SA-like or IA-like markers expression. Therefore, our study claims that ex-ovo egg yolk angiogenesis model could be used to study tumor angiogenesis and NO plays a key role in switching of IA under tumor microenvironment.
现有的肿瘤血管生成过程筛选动物模型存在各种缺陷，需要进一步研究。在这项研究中，我们开发了一种卵黄外鸡蛋血管生成模型，以筛选肿瘤细胞（HeLa 和 SiHa 细胞系）的血管生成能力。应用蛋黄血管生成试验研究了肿瘤微环境下一氧化氮（NO）对从发芽血管生成（SA）向体腔血管生成（IA）转变的影响。在鸡胚绒毛尿囊膜（CAM）从第 5 天到第 13 天的发育过程中，对形态学分析和 SA 样或 IA 样标志物的表达进行了测定。Notch1、Notch2、EphrinB2 和 Tie2 的表达被认为是 SA 样标志物，而 TEM8、CALD1、CXCR4 和 HOMX1 则被认为是 IA 样标志物。嵌入 CAM 的 HeLa 和 SiHa 细胞系显示出微血管和大血管以及血管大小、连接和长度的增加，这些都是血管生成的关键形态学参数。此外，研究表明 HeLa 比 SiHa 更具侵袭性，能诱导肿瘤血管生成。为了确定 NO 信号在肿瘤微环境中的意义，向鸡胚血管床中给予了 NO 供体（精脒亚硝脲（SPNO））、NOS 抑制剂（L-硝基-L-精氨酸甲酯（L-NAME）和 VEGF 抑制剂（Avastin），并与 HeLa 细胞一起给予。结果表明，HeLa 细胞通过 NO 信号、VEGF 和 eNOS 促进了 IA，这可以通过血管生成的形态学参数和 SA 样或 IA 样标志物的表达来证明。因此，我们的研究表明，卵黄外鸡蛋血管生成模型可用于研究肿瘤血管生成，NO 在肿瘤微环境下 IA 转变中发挥关键作用。
