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Molecular control of vascular remodelling: A role for Wnt signalling

Final Report Summary - WNT VASCULAR REMODEL (Molecular control of vascular remodelling: A role for Wnt signalling)

The cardiovascular system is the first organ to develop during embryogenesis and the correct function of blood vessels is essential for the viability of the embryo and for adult life. Various diseases related to vascular defects are associated to dysfunctional vascular remodelling, such as cancer, diabetic retinopathy and familial exudative vitreoretinopathy. During embryonic development, together vasculogenesis and angiogenic sprouting establish the major axial vessels and a rough draft of a network. However, sprouting angiogenesis creates a highly dense and poorly perfused vascular network, which needs to undergo extensive remodelling to become functional. During vascular remodelling, superfluous blood vessels regress possibly by endothelial cell retraction and apoptosis, in a process named vascular pruning. In contrast to the molecular mechanisms controlling vascular sprouting, vascular pruning has been less studied and very little is known about the cellular and molecular mechanisms controlling this process. The original aim of the project was to elucidate the involvement of the Wnt signalling pathway in vascular pruning and remodelling. The research plan used combinatorial approaches in mouse, zebrafish and cell culture systems to describe precisely and accurately the process of vascular remodelling during angiogenesis.

Using new innovative methodologies, we have characterised with unprecedented detail the endothelial cell cytoarchitecture in a remodelling vascular plexus. Our analysis has for the first time clearly demonstrated that vessel regression and remodelling are mostly independent of endothelial cell death. We propose that regression involves four discrete steps:

1) selection of the regressing branch;
2) physical lumen constriction;
3) endothelial cell migration and rearrangement; and
4) resolution of the vessel segment regression.

Our previous work illustrated that an imbalance in Notch and Wnt/b-catenin signalling due to loss of the Notch regulated ankyrin repeat protein (Nrarp) leads to premature vessel regression. To investigate which tissues are expressing the relevant Wnt ligands, we employed conditional inactivation of Evi/Gpr177/Wls to block Wnt ligand secretion from different cell types. Surprisingly, we observed that the relevant cell type secreting Wnt ligands important for regulating vessel regression are the endothelial cells, indicating that endothelial cells regulate vessel cell stability via an autocrine activation of Wnt signalling. Deletion of Evi/Gpr177/Wls from endothelial cells lead to a vascular retinal phenotype characterised by decreased vascular density and increased vessel regression. The vascular defects were not associated with increased apoptosis or impaired proliferation of endothelial cell. Mechanistically, vessel regression in the absence of endothelial Wnt-ligands followed the same sequence of discrete steps, suggesting that Wnt-signalling protects vessels from premature regression during the early phase of plexus formation and stabilisation. We used a chimeric approach to analyse functional endothelial cell behaviour and interaction dependent on Wnt ligand secretion. We concluded that Wnt signalling is not required to stabilise individual cells in a cell-autonomous manner, but acts as a communication mechanism between endothelial cells during angiogenesis.

To further clarify which branch of Wnt signalling could be important in regulating vessel regression, we used the Wnt11, Wnt5a, Lrp5 and Lrp6 mutant mouse models to discriminate between canonical and non-canonical Wnt signalling. We observed a similar regression phenotype in the double Wnt5a/Wnt11 mutant mice. Computational analysis of endothelial golgi polarity in vivo and in vitro showed that non-canonical Wnt ligands are important for coordination of endothelial cell polarity and migration, especially during vessel regression.

We propose that non-canonical Wnt-signalling is involved in the initial selection step of the remodelling branch and affects coordinated and polarised endothelial cell migration.

In the near future, we are going to explore which are the molecular mediators in vessel regression acting downstream of Wnt5a/Wnt11 in non-canonical activation.

Our mechanistic conceptualisation of vessel regression uncovered an extraordinary level of endothelial cell plasticity during vascular remodelling and opens new therapeutic perspectives on vascular diseases, which could be particularly important for ischemia-associated diseases such as stroke.