The role of endothelial cell metabolism in driving dynamic cell rearrangement during vessel sprouting

Angiogenesis, the formation of blood vessels from pre-existing ones, contributes to numerous malignant and other diseases of which cancer is the best known. Since five decades researchers have been striving to find drug targets that could block this process. Unraveling the molecular players involved in vessel sprouting has led to the approval of ten anti-angiogenic drugs since 2004 and many more are in clinical trials. These approved drugs all target the vascular endothelial growth factor (VEGF) or its receptors, which together constitute the main signaling axis in angiogenesis. However, due to problems such as intrinsic refractoriness, acquired resistance or toxicity, the clinical benefit of these anti-angiogenic drugs has unfortunately been limited. Hence, there is a clinical need to discover conceptually distinct anti- angiogenic strategies that bypass the resistance mechanisms and / or can be used in combination with the existing anti-VEGF therapies. Endothelial cells (ECs), which line the luminal side of blood vessels, attain specialized phenotypes, i.e. tip and stalk cells, during angiogenesis to form a vessel sprout. The tip cell spearheads and directs the vessel sprout towards the chemoattractant(s), e.g. VEGF, while the stalk cells trail behind the tip cell and proliferate to elongate the sprout. Classicaly, it was believed that ECs acquire their phenotype at the onset of vessel sprouting and that these phenotypes persist until new blood vessel loops are established. However, in contrast to this classical dogma, the phenotypes were found to be reversible and ECs have been shown to frequently interchange position in the sprout (EC rearrangement). These processes are essential for proper vessel sprouting and rely on EC heterogeneity within a sprout, which is acquired through lateral inhibition via the Delta-like 4 (Dll4)/Notch signaling pathway. Elevated VEGF levels are characteristic for pathological angiogenesis and give rise to synchronous Dll4/Notch signaling oscillations, which abrogate lateral inhibition and remove all EC heterogeneity within the sprout. Hence, EC specialization and rearrangement are severely impaired and proper vessel sprouting is impeded. Consequently, the pathological tissue is poorly perfused and secretes additional VEGF, which renders vessel sprouting and the formed pathological blood vessels even more abnormal. This vessel disorganization does not only affect the delivery of oxygen and nutrients to the tissue but also the delivery (and effectiveness) of drugs targeting the pathology. In addition, anti-angiogenic therapies that aim to prune vessels (further) decrease tissue perfusion and have been linked with increased malignancy and metastasis in case of cancers. This knowledge caused a paradigm shift in the field, where rather than destroying the abnormal vessels characteristic for pathology, the current objective is to normalize these vessels, resulting in a less hostile microenvironment and increased delivery of therapeutics. We hypothesize that counteracting the Dll4/Notch signaling oscillations and restoring EC rearrangement is a potential strategy to normalize pathological vessel sprouting and the pathological vasculature in general. Recently, EC metabolism has emerged as a novel determinant of vessel sprouting. Indeed, inhibiting EC migration and proliferation by blocking the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) or specifically lowering EC proliferation by impeding fatty acid oxidation (FAO) reduces pathological angiogenesis. Interestingly, since signaling pathways converge on metabolism, this novel anti-angiogenic approach might circumvent the resistance problems that the current therapies are facing. In this thesis, I developed a computational model that allowed us to study how PFKFB3-driven glycolysis steers EC rearrangement. Using this model I investigated whether targeting EC metabolism can not only be used as an anti-angiogenic therapy but also has the potential to normalize disorganized vessels in pathology. In addition, I assessed whether targeting EC metabolism is compatible with current anti-VEGF therapies. The in silico simulations predicted that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular vascular endothelial cadherin (VE-cadherin)-dependent adhesion, which I validated experimentally. Moreover, I found that blocking PFKFB3 normalized the disturbed EC signaling and rearrangement in high VEGF conditions. In addition, PFKFB3 blockade and different anti-VEGF therapies were predicted to synergize in normalizing pathological EC dynamics. Hence, by applying a multi-disciplinary approach integrating computation and experimentation, I identified a novel therapeutic strategy, namely normalizing pathological (tumor) vessels by targeting EC metabolism (glycolysis).nrpages: 162status: publishe