Blood vessels are remarkably specialized structures that present morphological, functional, and transcriptional heterogeneity. Hence, proper specification into arteries, veins, capillaries and lymphatic vasculature is crucial and under intense investigation. Endothelial cell (EC) metabolism is an important regulator of vessel formation in physiological and pathological conditions. ECs are highly glycolytic and metabolism crucially controls the angiogenic potential of ECs. However, recent single cell studies have also suggested that arterial and venous ECs have different metabolic properties. Even though it has already been suggested that a metabolic arrest precedes arterialization, functional evidence that endothelial metabolism can (co)define arteriovenous specification is so far missing.

We use the postnatal mouse retina as a model system of angiogenesis. Previous studies have shown that the pool of proliferating ECs that expands the growing vascular network derives from venous endothelium. ECs that get exposed to the highest levels of VEGF are subsequently selected to become tip cells, which are specialized, migratory ECs at the growing vessel front. Arterial ECs are specified at the tip cell position from where they migrate into the artery or capillary system, but rarely back into the vein. Using a tip-cell specific Cre line (Esm1-CreERT2) combined with different fate tracing strategies, we are studying the sorting of tip ECs into arteries after PFKFB3 deletion (PFKFB3ΔtipEC) or overexpression (PFKFB3-tip-OE).

PFKFB3 deletion promotes the migration of tip ECs into the arteries in PFKFB3ΔtipEC mice. However, arteries were overall less complex than in WT littermates. Interestingly, when tracing PFKFB3-tip-OE ECs in which one extra copy of HA-tagged PFKFB3 is expressed under the Rosa26 promoter, we found a very low number of HA-tagged ECs, suggesting that PFKFB3 is also downregulated on post-transcriptional level. Indeed, when PFKFB3-tip-OE pups were injected with a proteasome inhibitor (MG132), a significant number of HA-tagged ECs was found in arteries, capillaries and, surprisingly, veins.

Our data suggests that PFKFB3 expression is tightly regulated both on a transcriptional and translational level. This is required for the proper formation of arteries, and sorting of tip-cell derived ECs specifically into the arterial and not venous compartment.