Abstract
Purpose :
The Notch-DLL 4 signaling pathway regulates the proliferation and specification of endothelial cells (EC) into either tip or stalk cells. Its precise spatial and temporal regulation is critical to establishing a functional vascular network. We have previously shown that eNOS (endothelial nitric oxide synthase) augmentation increases EC proliferation as well as branch point formation in the retinal vasculature suggesting an interaction between eNOS and Notch. Our aim here is to investigate this potential cross-talk.
Methods :
Methods: HEK 293 cells were stably transfected with a plasmid encoding an eNOS-GFP fusion construct (eNOS-GFP HEKs) and functionality of eNOS confirmed by nitrite assay and normal trafficking of eNOS-GFP to the plasma membrane and Golgi compartments. HEK 293 and eNOS-GFP HEK were transfected with DLL 4 and basal and DLL 4 mediated cleavage of Notch 1 intracellular domain (NICD) quantified by western blotting in the presence or absence of the Notch inhibitor DAPT. The phenotype of the cells was analysed by microscopy and their response to activation analysed by comparative expression of the Notch target genes in transfected and non-transfected cell lines by qPCR.
Results :
Results: Basally eNOS-GFP expressing cells demonstrated elevated Notch 1 and DLL 1 expression compared to the untransfected control HEK cells and displayed a more adhesive phenotype suggestive of a transition to an epithelial cell-type morphology. This correlated with an increase in expression of N-Cadherin and elevated sub-cellular localisation to cell-cell junctions. Despite increased Notch 1 expression and NICD levels there was only a marginal increase in expression of the Notch target gene Hes1. By comparison, in the presence of high levels of DLL 4 achieved by DLL 4 overexpression, the presence of eNOS amplified the Notch signalling output evident as enhanced Hes 1 expression and a corresponding decrease in cell number in eNOS-GFP expressing cells.
Conclusions :
Conclusion: Taken together, using a cell culture model to manipulate eNOS and DLL 4 levels, we show that in the presence of low DLL 4 eNOS moderates the DLL 4-Notch 1 signaling axis. However, in the presence of high DLL 4 eNOS amplifies Notch responsiveness, suggesting a context specific role for the modulation of eNOS on Notch signalling.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.