3gaojunjiaConvergence

Convergence of ER stress and ferroptosis in diabetic vasculopathy: therapeutic protection by ferrostatin-1 Yue Ma a,1, Junjia Gao b,1, Yaqian Sun a, Yufan Gu a, Lu Zhang a, Yanru Zhen a, Hui Jia c, Yueyang Liu a,*, Qian Xu a,*, Ming-Sheng Zhou a,* a Shenyang Key Laboratory of Vascular Biology, Institute of Life Science, Shenyang Medical College, Shenyang 110034, China b Department of Cardiology, The 2nd Affiliated Hospital of Shenyang Medical College, 110002, China c School of Traditional Chinese Medicine, Shenyang Medical College, Shenyang 110034, China A R T I C L E I N F O Keywords: Diabetic vasculopathy Endoplasmic reticulum stress Endothelial insulin resistance Ferroptosis Oxidative stress A B S T R A C T Diabetic vascular complications are driven by endothelial dysfunction and insulin resistance, their underlying mechanisms remain incompletely understood. Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation, has been implicated in various diseases. However, its role in diabetic vas- culopathy is unclear. This study investigated the contribution of ferroptosis to diabetic endothelial injury and its interplay with oxidative and endoplasmic reticulum (ER) stress. In diabetic db/db mice, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reduced aortic thickness (− 65%), reactive oxygen species production (− 87.3%), and improved endothelium-dependent relaxation to acetylcholine (75.6%) and insulin (73.1%), independent of glycemic control. In human umbilical vein endothelial cells (HUVECs), high glucose induced ferroptosis, evi- denced by mitochondrial shrinkage, downregulation of glutathione peroxidase 4/ solute carrier family 7 member 1 (SLC7A11), and lipid peroxidation. Fer-1 co-treatment suppressed these effects, concurrently alleviating oxidative/ER stress and restoring insulin-stimulated phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase signaling. Mechanistically, quenching oxidative stress with N-acetylcysteine attenuated high glucose- induced ER stress, ferroptosis, and insulin resistance. Furthermore, inhibiting ER stress or silencing the pro- apoptotic transcription factor C/EBP homologous protein (CHOP), a known repressor of SLC7A11, attenuated high glucose-induced ferroptosis and partially restored insulin signaling. Our findings suggest the presence of a pathway in which high glucose-driven oxidative stress may initiate ER stress, leading to CHOP-mediated sup- pression of SLC7A11 and subsequent ferroptosis, thereby contributing to endothelial insulin resistance. These results raise the possibility that targeting ferroptosis could represent a promising therapeutic strategy for diabetic vascular complications, possible through mechanisms distinct from glucose-lowering. Abbreviations: ATF4, activating transcription factor 4; BCA, bicinchoninic acid; CCK-8, cell counting kit-8; CHOP, C/EBP homologous protein; DCFH-DA, 2′,7′- Dichlorodihydrofluorescein diacetate; DHE, dihydroethidium; DMSO, dimethyl sulfoxide; eIF2α, eukaryotic initiation factor 2α; eNOS, endothelial nitric oxide synthase; ER, endoplasmic reticulum; Fer-1, ferrostatin-1; FTH1, ferritin heavy chain 1; GPX4, glutathione peroxidase 4; GRP78, glucose-regulated protein 78; GSH, reduced glutathione; GSSG, oxidized glutathione disulfide; HBSS, hanks’ balanced salt solution; HE, hematoxylin and eosin; HG, high glucose; HRP, horseradish peroxidase; HUVECs, human umbilical vein endothelial cells; HMEC-1, Human Microvascular Endothelial Cell line-1; 4HNE, 4 Hydroxynonenal; INS, insulin; ITT, Insulin tolerance test; NAC, N-acetylcysteine; NG, normal glucose; Nrf2, nuclear factor erythroid 2-related factor 2; OS, osmotic stress; PBS, phosphate-buffered saline; PERK, Protein kinase RNA-like endoplasmic reticulum kinase; PI3K, phosphatidylinositol 3-kinase; PSS, physiological salt solution; PVDF, polyvinylidene fluoride; ROS, reactive oxygen species; SBP, systolic blood pressure; SDS-PAG