Objective: Cholesterol is an important membrane constituent and is involved in cell signaling. Cellular cholesterol is determined by uptake and de novo synthesis. Endothelial cells are capable of cholesterol metabolism through a pathway that requires the cytochrome P450 reductase (POR) as well as cyp51 monooxygenase. High circulating cholesterol is linked to cardiovascular diseases. The role of endogenous cholesterol synthesis for endothelial function, in contrast, is unknown and was studied here.

Methods & Results: To induce a loss of cholesterol synthesis in endothelial cells, POR and Cyp51 knockout was performed in human umbilical vein endothelial cells (HUVECs) and an endothelial-specific tamoxifen-inducible POR knockout mouse (ecPOR^-/-) was generated.

Knockout of POR in HUVEC led to an accumulation of the Cyp51 substrate lanosterol, whereas its product, desmosterol was reduced. Functionally, this was associated with increased basal and VEGF-stimulated angiogenic sprouting. Similarly, endothelial sprouting from aortic segments was increased in ecPOR^-/- mice as compared to control mice. Importantly, this effect was also observed in vivo as retina angiogenesis was increased after endothelial-specific deletion of POR.

Cellular cholesterol levels are sensed by the SREBP2 (sterol regulatory element-binding proteins) system, and indeed, SREBP2 signaling was increased after deletion of POR in cultured cells as well as in vivo. Analysis of publicly available SREBP2 CHIP-seq data from statin treated mice show a high correlation between cholesterol metabolism and NOTCH signaling. RNAseq of whole aorta as well as isolated aortic endothelial cells revealed a significant decrease in Notch signaling genes (Notch2, Ep300, Rbpj and Psenen). Functionally, this should lead to increased proliferation and a tip cell phenotype in ecPOR^-/-, as we indeed observed.

Conclusion: Inhibition of the endothelial POR/CYP-axis improves endothelial cell angiogenic function through an activation of the SREBP2 system and subsequent tip cell specification.