Changwen Fang, Xiaolong Du, Yuxing Wang, Yiqi Jin, W Q Wang
ABSTRACT Ferroptosis—a regulated, iron‐dependent cell death driven by lipid peroxidation—has been implicated in vascular pathology, yet how endoplasmic reticulum (ER) stress couples to ferroptotic execution in vascular smooth muscle cells (VSMCs) remains unclear. We investigated whether the PERK–EIF2α–ATF4–CHAC1 signalling axis links ER stress to ferroptosis in human aortic SMCs. Human aortic smooth muscle cells (HASMCs) and primary aortic smooth muscle cells (AoSMCs) were exposed to erastin (± ferrostatin‐1), with pharmacologic modulation using buthionine sulfoximine (BSO; glutamate–cysteine ligase inhibitor) and salubrinal (selective EIF2α dephosphorylation inhibitor). Viability (CCK‐8), ER‐stress markers (p‐PERK, p‐EIF2α, GRP78, ATF4, CHAC1) and ferroptosis effectors (GPX4, ACSL4, ALOX15) were quantified by Western blotting and densitometry. Immunofluorescence assessed GPX4 and p‐EIF2α/CHAC1 colocalization. Lipid peroxidation was evaluated by C11‐BODIPY 581/591 imaging and malondialdehyde (MDA) content; labile Fe 2+ was measured by calcein‐AM quenching; apoptosis was examined by TUNEL. Statistics used t ‐tests or one‐way ANOVA. Erastin reduced viability and activated the EIF2α–ATF4–CHAC1 pathway in both HASMCs and AoSMCs, as evidenced by increased p‐EIF2α/ATF4/CHAC1 and decreased GPX4, with concomitant upregulation of ACSL4 and ALOX15. GPX4 immunofluorescence declined, while C11‐BODIPY and MDA indicated robust lipid peroxidation, and calcein‐AM revealed expansion of the labile Fe 2+ pool; ferrostatin‐1 mitigated these changes. BSO further amplified p‐PERK→p‐EIF2α → ATF4 → CHAC1 signalling and deepened GPX4 loss, whereas salubrinal sustained EIF2α phosphorylation, augmented ATF4/CHAC1, and further depressed GPX4. Across conditions, effects were consistent in both cell types and reached statistical significance (most p < 0.05 to < 0.01). ER‐stress signalling via PERK–EIF2α–ATF4–CHAC1 constitutes a proximal driver of ferroptosis in human aortic SMCs by eroding glutathione tone and disabling GPX4, thereby promoting lipid peroxidation in an iron‐rich milieu. Pharmacologic tuning of EIF2α phosphorylation and glutathione biosynthesis modulates ferroptotic susceptibility, nominating EIF2α–ATF4–CHAC1, GPX4/GSH homeostasis and iron handling as actionable nodes to preserve VSMC integrity in oxidizing vascular environments.