Transsulfuration metabolism is essential for ferroptosis resistance in quiescent endothelial cells

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial process involved in both physiological and pathological contexts. During angiogenesis, quiescent endothelial cells (QECs) forming the vascular bed begin to proliferate and switch their metabolism to support anabolic and energetic needs in response to growth factors and hypoxic conditions. Recent research has demonstrated that ferroptosis, an iron-dependent form of cell death mediated by lipid peroxidation, can affect angiogenesis. Cysteine, a thiol-containing amino acid, is crucial for the synthesis of sulfur-containing biomolecules that control ferroptosis. Glutathione (GSH), a reducing tripeptide containing a cysteine residue, serves as a cofactor for the enzyme glutathione peroxidase 4 (GPX4) to donate electrons to peroxides of polyunsaturated fatty acyl phospholipids. Cysteine can be acquired from its extracellular oxidized form, cystine, via the glutamate-cystine antiporter (system xCT) or synthesized de novo via the transsulfuration pathway (TSP). However, whether proliferating ECs (PECs) and QECs differentially modulate the cysteine/GSH/GPX4 axis to protect themselves from ferroptosis is still unknown. Our findings revealed that PECs primarily utilize extracellular cystine to synthesize GSH, which is essential for avoiding ferroptosis. In contrast, QECs exhibit a resilient response to cystine starvation by activating the TSP. Interestingly, chronic and severe hypoxia induces ferroptosis resistance in PECs exposed to cystine limitation, mimicking the metabolic profile of QECs. Molecularly, QECs exhibit high NRF2 expression necessary to support TSP under cystine limitation and protect QECs from ferroptosis. In vivo experiments confirm the susceptibility of ECs to cell death by xCT inhibition in a retinal model of sprouting angiogenesis. These findings highlight differential regulation of cysteine metabolism in PECs and QECs and suggest that the cysteine/GSH/GPX4 axis could be a potential therapeutic target for diseases involving angiogenesis.