Regulation of endothelial cell quiescence by Zeb1 as a regulator of angiogenesis

The growth of new blood vessels from the pre-existing vasculature (angiogenesis) is an important physiological process driven by hypoxia. All blood vessels are lined by endothelial cells (EC) which under normal conditions form a smooth monolayer, which is non-proliferative, non-motile, tightly controlled junctions, non-adherent surface with no inflammation or leukocyte binding. Under these conditions the endothelium is in a state of 'quiescence' allowing efficient oxygen and other solute exchange into the interstitium and respiring tissues. The EC maintains this quiescent phenotype through the activity of a combination of epigenetic, surface receptor and junctional receptor activity, metabolic and of course transcriptional control of key genes. As tissues change their metabolic needs, the endothelium responds. A responding EC can be considered activated and depending on the specifics of the activation, can result in a phenotypic remodelling causing increased leukocyte adherence, increased inflammatory marker expression, proliferation, motility and significant changes in signalling pathways 1. Endothelial cell dysfunction occurs across a wide variety of diseases. During diabetes the altered microenvironment leads to EC dysfunction resulting in hypoxia and inflammation which results in excessive vascular leakage and growth. Diabetic retinopathy is a frequent complication of diabetes in which vessel growth in the retina results in loss of vision. In cardiovascular diseases (including coronary and peripheral vascular disease, stroke and diabetes) and vascular related diseases (neurodegeneration cancer, arthritis and sepsis) the EC is activated, and disease progression results in vascular remodelling, inducing the growth of new vessels (angiogenesis) or inflammatory conditions. Understanding how the quiescence-activation switch occurs will enable modification of this process for therapeutic benefit: if we can understand how to restore (or generate methods to maintain) EC quiescence we will be able to inhibit undesired EC activation.

We have recently identified Zeb1 (a transcription factor with known roles in tumour progression and epithelial to mesenchymal transition) as being expressed in quiescent ECs. This was originally identified in vitro, and subsequently in vivo using the angiogenic mouse retina as an experimental model. Using inducible and endothelial specific Zeb1 knockout mice we have demonstrated preliminary evidence to show mice spontaneously undergo aberrant endothelial growth in adult retinae and increase solute leakage during choroidal neovascularisation. These preliminary data suggest altered Zeb1 signalling contributes to retinal angiogenesis and vessel leakage in adult mice, which could be important in the progression of DR. Therefore, this project will aim to investigate angiogenic remodelling in a series of developmental and pathological angiogenesis models.
Aim 1: Does Zeb1 alter angiogenic phenotype in development? Zeb1 will be knocked out using a EC specific, conditional transgene. Retinal angiogenesis will be quantified in neonatal mice, and adult mice. Pathological angiogenesis will be induced using hindlimb ischaemia and choroidal neovascularisation models. Characterisation of EC phenotypes and interactions with angiogenesis modulating leukocyte populations
Aim 2: Contribution of EC Zeb1 to the progression of diabetic retinopathy. Diabetes will be induced within the inducible EC specific KO mice and retinal permeability will be measured in vivo. Characterization of EC phenotype will be made by immunohistochemistry and molecular characterisation
Aim 3: Is Zeb1 regulated angiogenesis VEGF dependent? Signalling pathways linking VEGFR2, hypoxia and glycolysis will be explored in vitro. This will also be explored in vivo with the use of VEGF antibodies to prevent VEGF stimulation within the EC Zeb1 KO diabetic model.