April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Tip cell signalling in Endothelial Cells
Author Affiliations & Notes
  • Elmira Jalilian
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • Michael B Powner
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • Marcus Fruttiger
    Cell Biology, UCL Institute of Ophthalmology, London, United Kingdom
  • Footnotes
    Commercial Relationships Elmira Jalilian, None; Michael Powner, None; Marcus Fruttiger, AstraZeneca (F), Novartis (C), Novartis (F)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5404. doi:
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      Elmira Jalilian, Michael B Powner, Marcus Fruttiger, UCL institute of Ophthalmology; Tip cell signalling in Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5404.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Angiogenesis plays an important role in pathogenesis of many ischemic eye diseases, such as diabetic retinopathy and retinopathy of prematurity. Sprouting of new blood vessels requires endothelial cells to take on specialised phenotypes, such as the ‘tip cell’ phenotype at the tip of angiogenic sprouts and the ‘stalk cell’ phenotype in a more proximal positions. This differential behaviour of endothelial cells is regulated by signalling pathways acting through Notch and vascular endothelial growth factor (VEGF) receptors. In the past, numerous studies have used human umbilical vein endothelial cells (HUVECs) to investigate endothelial cell signalling in vitro. However, tip and stalk cell behaviours are more difficult to address in HUVECs because they do not typically form angiogenic sprouts under normal culture conditions. Nevertheless, a recent study has demonstrated a subset of CD34+ migratory cells in HUVEC monolayers, showing a similar gene expression profile to tip cells. However, the mechanisms that regulate the differentiation of these CD34+ cells in HUVEC cultures are not known. Since VEGF and Notch signalling are known to regulate tip cells we hypothesised that these signalling pathways may also regulate the differentiation of CD34+ HUVECs.

Methods: HUVECs were plated in 24 well plates and grown to either confluency or sub-confluency. Cell were treated with various concentrations of VEGF and a Notch inhibitor (DAPT) for 24 hours and then frequency of CD34+ was assessed by immunohistochemistry and Fluorescent Microscopy. Results were analysed using ImageJ.

Results: We found that in the presence of DAPT alone the percentage of CD34+ cells did not increase. However, a significant increase of CD34+ cells was observed when cells were exposed to DAPT and high levels of VEGF in combination. This mirrors the behaviour of tip cells, which are in-vivo exposed to high levels of VEGF and are supressed by Notch signalling.

Conclusions: This suggests that HUVECs cultured in monolayers display elementary forms of tip and stalk cell phenotypes with functional consequences. It may therefore be possible to use HUVEC monolayer cultures as an in-vitro model to study the molecular mechanisms of tip cell biology.

Keywords: 447 cell-cell communication • 748 vascular endothelial growth factor  
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