Supplementary Material for: The Endothelin-1 Pathway and the Development of Cardiovascular Defects in the Haemodynamically Challenged Chicken Embryo

<i>Background/Aims:</i> Ligating the right lateral vitelline vein of chicken embryos (venous clip) results in cardiovascular malformations. These abnormalities are similar to malformations observed in knockout mice studies of components of the endothelin-1 (ET-1)/endothelin-converting enzyme-1/endothelin-A receptor pathway. In previous studies we demonstrated that cardiac <i>ET-1</i> expression is decreased 3 h after clipping, and ventricular diastolic filling is disturbed after 2 days. Therefore, we hypothesise that ET-1-related processes are involved in the development of functional and morphological cardiovascular defects after venous clip. <i>Methods:</i> In this study, ET-1 and endothelin receptor antagonists (BQ-123, BQ-788 and PD145065) were infused into the HH18 embryonic circulation. Immediate haemodynamic effects on the embryonic heart and extra-embryonic vitelline veins were examined by Doppler and micro-particle image velocimetry. Ventricular diastolic filling characteristics were studied at HH24, followed by cardiovascular morphologic investigation (HH35). <i>Results:</i> ET-1 and its receptor antagonists induced haemodynamic effects at HH18. At HH24, a reduced diastolic ventricular passive filling component was demonstrated, which was compensated by an increased active filling component. Thinner ventricular myocardium was shown in 42% of experimental embryos. <i>Conclusion:</i> We conclude that cardiovascular malformations after venous clipping arise from a combination of haemodynamic changes and altered gene expression patterns and levels, including those of the endothelin pathway.

背景与目的：结扎鸡胚右侧卵黄静脉（静脉夹法）可诱导心血管畸形。此类畸形与内皮素-1（endothelin-1, ET-1）/内皮素转换酶-1/内皮素A受体通路相关组分的基因敲除小鼠研究中观察到的畸形高度相似。既往研究证实，静脉夹闭术后3小时，胚胎心脏的ET-1表达水平显著下降，而术后2天则出现心室舒张充盈功能紊乱。据此，本研究提出假说：内皮素-1相关通路参与了静脉夹法所致功能性与形态学心血管缺陷的发生发展过程。

方法：本研究向HH18期鸡胚的循环系统中输注ET-1及内皮素受体拮抗剂（BQ-123、BQ-788与PD145065）。采用多普勒技术与微粒子图像测速技术，检测其对胚胎心脏及卵黄静脉的即时血流动力学效应。于HH24期分析心室舒张充盈特征，并在HH35期开展心血管形态学检测。

结果：ET-1及其受体拮抗剂在HH18期即可引发血流动力学改变。在HH24期，实验组胚胎的心室舒张被动充盈成分降低，该缺陷可通过主动充盈成分的增加得到代偿。42%的实验组胚胎出现心室肌层变薄的表型。

结论：本研究认为，静脉夹闭术后的心血管畸形，是血流动力学改变与包括内皮素通路在内的基因表达模式及表达水平异常共同作用的结果。