Endothelial cytochrome P450 -derived cholesterol limits angiogenesis

The cytochrome P450 redox system is composed of a cytochrome P450 reductase (POR) and multiple CYP450 enzymes (CYP450). Of the CYP450 isoenzymes, CYP51A1 is essential for endogenous cholesterol biosynthesis. Elevated circulating cholesterol is a well-established risk factor for cardiovascular disease, however, the role of intracellular cholesterol synthesis in normal endothelial function remains unclear. To investigate this, we generated CRISPR/Cas9 knockouts of the cytochrome P450 reductase in primary human endothelial cells (EC) and studied an endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR^-/-). Deletion of POR led to the accumulation of lanosterol, the substrate of POR/CYP51A1, and a reduction in desmosterol. Functionally, POR deficiency promoted basal and VEGF-induced angiogenesis in spheroids and mouse aortic segments. Retinal angiogenesis was increased in ecPOR^-/- mice in vivo. Mechanistically, POR deletion activated the Sterol Regulatory Element Binding Transcription Factor (SREBP2) regulatory pathway, as shown by increased nuclear translocation of cleaved SREBP2 in EC and in en face-stained mouse aortae. Overexpression of nuclear SREBP2 in endothelial cells mimicked the angiogenic phenotype observed upon POR deletion. Conversely, double deletion of POR and SREBP2 normalized angiogenesis to levels of control cells. RNAseq of POR-deficient EC revealed an upregulation of PI3K-related signaling pathways and genes involved in cholesterol homeostasis, including enhanced expression of pro-angiogenic factors. In line with these findings, knockout of POR increased cellular PIP3 levels, AKT phosphorylation, and activation of downstream targets such as p70 S6 kinase. These findings demonstrate that inhibition of the endothelial POR/CYP51A1 axis impairs endogenous cholesterol synthesis, activates SREBP2, and enhances angiogenesis via PI3K/AKT/mTOR signaling, highlighting a critical and novel link between intracellular cholesterol metabolism and vascular growth.