CYB5R4 sustains endothelial proliferation and ischemia-induced angiogenesis by maintaining RRM2-dependent nucleotide balance.

Angiogenesis is essential for revascularization in peripheral artery disease (PAD), yet pro-angiogenic therapies remain inconsistent. Here, we identify a cytosolic reductase, CYB5R4, as an intrinsic regulator of endothelial proliferation and ischemia-induced angiogenesis. In mice, global haploinsufficiency or inducible endothelial deletion of Cyb5r4 delayed perfusion recovery after femoral artery ligation and reduced capillary expansion. CYB5R4 is known to promote stearoyl-CoA desaturase (SCD) activity and is required for the de novo synthesis of monounsaturated fatty acids. In human endothelial cells, CYB5R4 silencing impaired proliferation with G1-S arrest that was not rescued by monounsaturated fatty acids and differed from the loss of SCD, indicating an SCD-independent mechanism. RNA sequencing with Bayesian network inference highlighted the ribonucleotide-reductase subunit RRM2 as a key downstream mediator. RRM2 overexpression partially restored proliferation. Integrated untargeted metabolomics and targeted nucleotide quantification revealed an imbalanced nucleotide pool in CYB5R4-deficient cells. These findings support a model in which CYB5R4 sustains endothelial proliferation and ischemia-driven angiogenesis by maintaining RRM2-dependent nucleotide balance. Targeting the CYB5R4-RRM2 axis may improve therapeutic angiogenesis in PAD.