Study on the role of mitochandria during angiogenesis: can they provide a novel angiogenic target?

Angiogenesis, the growth of new blood vessels, is critical during development. Indeed blockade of this process results in embryonic lethality. Furthermore angiogenesis also plays a major role during several physiological but also pathological conditions. Aberrant blood vessel growth contributes to several pathologies such as ocular disease and cancer, thus blocking angiogenesis has become an attractive therapeutic strategy. Current anti-angiogenic therapies focus on blockade of the growth factors that initiate angiogenesis. While this strategy is successful for treatment of ocular disease, its success for cancer treatment is limited due to toxicity and resistance. There is thus an urgent need for novel anti-angiogenic strategies.

      Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. We hypothesized that pro-angiogenic growth factors converge onto metabolism to initiate a metabolic reprogramming that allows sufficient biomass and energy production to fuel proliferation and migration of ECs during angiogenesis. In this PhD thesis we therefore investigated the role of metabolism during vessel sprouting, thereby focusing on glycolysis and fatty acid oxidation.

      In a first study, we showed that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator phosphofructokinase-fructose-2,6-bisphophatase 3 (PFKFB3) in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalization with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the prostalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

      Having identified PFKFB3 as a potential anti-angiogenic target, we next explored its therapeutic potential in several in vivo models of physiological and pathological angiogenesis. We demonstrate that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in zebrafish embryos and in the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) signaling and amplified the anti-angiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models, with minimal systemic effects. These insights may offer therapeutic anti-angiogenic opportunities.

            Besides glycolysis, the metabolism of endothelial cells during vessel sprouting remains poorly studied. More specifically, the role of oxidative mitochondrial metabolism during angiogenesis is unknown. Even more, current literature does not ascribe a crucial role for oxidative metabolism during vessel sprouting. We therefore evaluated the role of fatty acid oxidation (FAO) during vessel sprouting and demonstrated that endothelial loss of carnitine palmitoyl transferase 1a (CPT1a), a rate-limiting enzyme of FAO, causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.

            Overall, our research has uncovered two potential metabolic targets for anti-angiogenic therapy. We have provided proof of principle for each of these targets in pre-clinical models of pathological angiogenesis. Future research will allow further drug development for these targets towards clinical use.