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Endothelial cytochrome P450 -derived cholesterol limits angiogenesis [ext631_RNA-seq]

ABSTRACT: The cytochrome P450 reductase (POR) monooxygenase system 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 POR in primary human endothelial cells (EC) and studied a previously developed 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. Moreover, 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE326109 | GEO | 2026/07/22

REPOSITORIES: GEO

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Project description:Immunometabolism is a growing field that centers on a core paradigm: perturbations in metabolism alter a cell’s biological response in immunity and, likewise, inflammatory signals, such as cytokines and pathogens, can directly influence cellular metabolism. Although the carbons within cholesterol cannot be used for catabolic energy production, there is a growing appreciation that a similar relationship exists between cholesterol homeostasis and immunity. The majority of this literature is focused on cholesterol dynamics in the context of leukocyte immunobiology. However, the endothelium also plays an important role during the acute inflammatory response. Endothelial cells (ECs) rapidly respond to extrinsic signals, such as tissue damage or microbial infection, by upregulating factors to activate and recruit circulating leukocytes to the site of injury. Dysregulation or aberrant activation of ECs leads to disease, such as atherosclerosis. I studied the role of cholesterol and its master regulator, SREBP2, in the EC response to acute inflammatory stress. ECs treated with cytokines upregulated SREBP2 cleavage and classical cholesterol biosynthesis gene expression within the late phase of the acute inflammatory response. Furthermore, SREBP2 activation was dependent on NF-kB DNA binding and classical SCAP-SREBP2 processing. We used bacterial cytolysin probes to show that inflammatory stress significantly decreased accessible cholesterol, leading to dysfunctional sterol sensing and downstream SREBP2 cleavage. We also explored what role SREBP2 plays in the EC inflammatory response. Loss of SREBP2 in ECs treated with inflammatory cytokine altered EC phenotype, which was defined by decreased chemokine expression and increased type I inflammatory signaling. Interestingly, this effect on the EC inflammatory transcriptome could not be accounted by changes in cholesterol, but rather through SREBP2 binding to promoters of pro-inflammatory transcription factors. Preliminary results revealed that BHLHE40 and KLF6 are direct targets of SREBP2 and that loss of one of KLF6 could mimic the effect of SREBP2 knockdown on chemokine expression. This study is the first to provide an in-depth characterization of the relationship between SREBP2 and the endothelial acute inflammatory response.

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Endothelial cytochrome P450 -derived cholesterol limits angiogenesis [ext631_RNA-seq]

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