Endothelin-1 Promotes Proliferation and Angiogenesis in Cultured Endothelial Cells via The ETB Receptor

Background: Endothelin-1 (ET-1) is a 21 amino acid polypeptide with potent vasoconstrictor and mitogenic properties. We have previously demonstrated ET-1 expression around areas of neovascularization in critically ischaemic skeletal muscle in human. Here, we aim to investigate whether ET-1 and the ET-1 receptor subtypes provide an angiogenic stimulus in human microvascular endothelial cells. Methods: Cell proliferation - Human microvascular endothelial cells (HMEC-1) were incubated in low-serum (0.5%) conditions with different concentrations of ET-1. After 24 hours the cells were harvested and counted in a haemocytometer. We assessed the impact of selective blockade of the endothelin receptors with either Bosentan (a non-peptide competitive dual antagonist of both ET_A and ET_B receptor subtypes), BQ788 (ET_B receptor antagonist), BQ123 (ET_A receptor antagonist) and IRL-1620 (ET_B receptor agonist). In-vitro angiogenesis assays - Morphometric capillary tube formation was measured by culturing HMEC-1 on growth factor reduced matrigel. In-vitro angiogenesis was investigated using the rat aortic ring assay. Angiogenesis was measured after 24 hours treatment by digital image analysis of the angiogenic field. Results: ET-1 enhances the in-vitro proliferation of HMEC-1 in a dose-dependent manner (p<0.001). Bosentan alone attenuates HMEC-1 proliferation to below control levels (p<0.015) and had a dose-dependent inhibitory effect on ET-1 proliferation (p<0.03). The attenuation of proliferation appears to be a property of the ET_B receptor (p<0.001). ET-l significantly enhanced angiogenesis (p<0.02) whilst BQ788 suppressed endogenous angiogenesis (p<0.04). BQ123 had no effect. BQ788 and IRL-1620 attenuated and enhanced ET-1-mediated angiogenesis respectively (both p<0.05). Conclusions: The data suggests that ET-1 may be an important autocrine growth factor for human microvascular endothelial cells stimulating angiogenesis. Our observations support the hypothesis that the angiogenic properties of ET-1 are ET_B receptor mediated, unlike ET-l’s effect on vasomotor tone, which is predominantly ET_A receptor dependent. The separation of the distinct actions provides important information regarding the mechanism of ET-1 action and might benefit patients with peripheral vascular disease, where selective ET_A antagonism might promote vasodilatation while preserving angiogenesis.