Endothelin-1, a Regulator of Angiogenesis in the Chick Chorioallantoic Membrane

Abstract

We investigated the angiogenic properties of endothelin-1 (ET-1) using a novel experimental approach involving the constant production and release of ET-1, which was achieved by grafting stably transfected Chinese hamster ovary (CHO) (CHO-ET-1) cell aggregates onto the chorioallantoic membrane (CAM) ectoderm. Macroscopic observation showed that CHO-ET-1 cell aggregates formed highly vascularized nodules surrounded by radially rearranged vessels, with a strong angiogenic response. 5-Bromo-2′-deoxy-uridine (BrdU) studies showed an increase in endothelial cell proliferation in the CAM vasculature around CHO-ET-1 nodules. An angiogenic response was also observed with gelatin sponges containing conditioned medium from CHO-ET-1 cells. The specific involvement of ET-1 in the angiogenic effect mediated by CHO-ET-1 was demonstrated by the reduction or abolition of neovascularized CHO-ET-1 nodules by (1) bosentan, a mixed antagonist of ET_A/ET_B receptors, (2) an ET_A receptor antagonist (Ru69986) and (3) phosporamidon, an inhibitor of endothelin-converting enzyme-1 (ECE-1). We also demonstrated that VEGF was involved in CHO-ET-1-mediated angiogenesis, by using a specific inhibitor of VEGF tyrosine kinase receptor activity (PTK787/ZK 222584), which abolished CHO-ET-1 nodule formation and CAM neovascularization. Thus, our results show that exogenous ET-1 mediates angiogenesis in vivo.

Bone-specific growth inhibition of prostate cancer metastasis by Atrasentan

“…23,24 In vivo studies have yielded conflicting results, with ET-1 failing to induce angiogenesis in chick embryo chorioallantoic membrane and rat sponge models and inducing angiogenesis, via ETA, in a rat cornea model and, when combined with VEGF, in the matrigel plug and chick embryo chorioallantoic membrane assays. [24][25][26][27][28] ET-1, via ETA, has been shown to upregulate VEGF in normoxic ovarian carcinoma cells by stabilization of the VEGF regulatory protein HIF-1α and has been correlated with VEGF expression and microvessel density (MVD) in breast and ovarian carcinomas. 25,29,30 However, whether ET-1 plays either a direct or indirect role in angiogenesis of prostate cancer has not been fully investigated.…”

Hyperglycemia-Induced Defects in Angiogenesis in the Chicken Chorioallantoic Membrane Model

“…Proliferation. BrdU (10 mol/l; Roche) was laid onto the CAM in two siliconized rings, 25 l each, overnight, as previously described (9). Detection was performed using the BrdU detection kit (Roche) according to the manufacturer's instructions, with the anti-BrdU antibody at a 1:50 dilution.…”

A Phase II study of bosentan, a dual endothelin receptor antagonist, as monotherapy in patients with stage IV metastatic melanoma

“…Bosentan reversed ET-1-induced inhibition of apoptosis [19] and potentiated Fas ligand-mediated apoptosis in melanoma, as well as in colon carcinoma, and glioblastoma cells [19][20][21]. Furthermore, bosentan inhibited ET-1-mediated angiogenesis [22]. The role of the ET system in the pathophysiology of melanoma and the experimental data with endothelin receptor antagonists provide the rationale to assess bosentan in a proof-of-concept study in patients with metastatic melanoma.…”