Abstract

Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.

Highlights

  • The Notch pathway is highly conserved from invertebrates to mammals [1] since it plays a crucial role in determining cell fate and differentiation during development and postnatal life

  • We evaluated by Western blot analysis the expression of ERa and ERb in 6 different pools of commercially available HUVECs and in cells derived from the umbilical vein of a single donor

  • While the effects of various mediators of cell growth or cell activation on Notch pathway in endothelial cells have been well characterized [36] [37], less is known on how estrogens affect Notch in the endothelium

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Summary

Introduction

The Notch pathway is highly conserved from invertebrates to mammals [1] since it plays a crucial role in determining cell fate and differentiation during development and postnatal life. This allows the second proteolytic cut of NotchTM by a surface protease, generally ADAM10 (A Disintegrin And Metalloprotease 10), which creates a membrane-tethered intermediate NotchIC binding displaces a co-repressor complex, promotes the recruitment of co-activator molecules and activates transcription of Notch target genes such as Hes (hairy/enhancer of split), Hey (Hes-related proteins) and Nrarp (Notch-regulated ankyrin repeat protein). These factors, in turn, regulate downstream genes, some of which can either maintain cells in an uncommitted state or induce differentiation. Genes that control cell proliferation and apoptosis are regulated by Notch activity [2]

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