Objective. This study investigated whether the erythrocyte fraction in thrombi would be increased with serum iron via oxidative stress. Methods. This study retrospectively enrolled patients with acute ischemic stroke treated using endovascular treatment in a single stroke center from October to December 2019. We examined the relationship between serum iron and erythrocyte‐rich thrombi and the correlation of serum iron and the erythrocyte fraction in thrombi using clinical samples. Experiments in vivo and in vitro were performed to investigate the influence of oxidative stress on the correlation between serum iron concentration and erythrocyte fraction in thrombi. Results. We found from the clinical samples that serum iron concentration was related to erythrocyte‐rich thrombi and positively associated with the erythrocyte fraction in thrombi in vivo. Further, the tightness of the fibrin networks regulating the erythrocyte fraction in thrombi was increased with serum iron concentration in vivo. Additionally, the oxidative stress level was increased with serum iron concentration in vivo. Moreover, we found that the tightness of the fibrin networks increased with higher oxidative stress levels in vitro. Lastly, experiments in vivo with inhibiting oxidative stress showed that the erythrocyte fraction in thrombi and the tightness of fibrin networks significantly increased in the iron group than those in the iron with oxidative stress inhibitor group and control group. Conclusions. Oxidative stress played a role in the process that the erythrocyte fraction in thrombi was increased with serum iron by influencing fibrin networks.

Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between HDL, LDL and VLDL. Lipoprotein particles contain a neutral lipid core composed of CE and TG surrounded by a surface monolayer of phospholipids (PL), free cholesterol (FC), and apolipoproteins, most notably, apo B-100 in LDL and VLDL and apo A-I in HDL. An elevated level of LDL-cholesterol (LDL-C) and/or a low level of HDL-cholesterol (HDL-C) in human plasma are major risk factors for cardiovascular disease (CVD). Since increased CETP can reduce HDL-C concentration and CETP deficiency is associated with elevated HDL-C levels, CETP inhibitors, including torcetrapib, anacetrapib and dalcetrapib have been investigated in clinical trials for treating CVD. Despite the intense clinical interest in CETP inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. CETP is a hydrophobic glycoprotein of 476 amino acids (∼53 kDa, before posttranslational modification). Its crystal structure reveals a banana-shaped molecule with N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60 Å-long hydrophobic central cavity. Three CETP neutral lipid transfer hypotheses were proposed more than two decades ago: 1) a shuttle mechanism that involves CETP collecting CEs from one lipoprotein and delivering them through the aqueous phase to another lipoprotein; 2) a tunnel mechanism in which CETP bridges two lipoproteins forming a ternary complex, with lipids flowing from the donor to acceptor lipoprotein through the CETP molecule; and 3) a modified tunnel mechanism implicating a CETP dimer. One difficulty in investigating CETP mechanisms using structural methods is that interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We validated an optimized negative-staining electron microscopy (NS-EM) protocol in which flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. We applied this protocol to study the mechanisms by which CETP interacts with spherical HDL, LDL and VLDL. Three-dimensional (3D) reconstructions of CETP, free and HDL-bound, were obtained by single-particle techniques. In addition, we used inhibitory CETP antibodies to identify the regions of CETP that interact with HDL and LDL. Finally molecular dynamics (MD) simulation was used to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. We discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into HDL and its C-terminal domain interacting with LDL or VLDL. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Endothelial dysfunction and impaired mitophagy represent core pathological drivers of atherosclerosis progression, yet upstream molecular regulators governing this process in vascular endothelium remain largely uncharacterized. This study delineated the functional role and mechanistic basis of filamin C (FLNC) in atherosclerosis-related endothelial injury. We profiled FLNC expression in 20 paired human carotid plaque and adjacent normal vascular tissues collected from patients undergoing carotid endarterectomy. In vitro, CRISPRa/CRISPRi-stable HUVEC lines were challenged with ox-LDL, with pharmacological (Mdivi-1) and genetic (si-PINK1/si-Parkin) rescue assays used to verify causal signaling. In vivo, Tie2-driven endothelial-specific FLNC-overexpressing ApoE^−/− mice were fed a high-fat diet to induce atherosclerotic lesions. FLNC was significantly downregulated in human plaques, ox-LDL-treated HUVECs, and atherosclerotic mouse aortas. FLNC overexpression attenuated endothelial apoptosis and oxidative stress, while its knockdown exacerbated these injuries. Mechanistically, FLNC activated PINK1/Parkin-mediated mitophagy to restrict cytosolic mtDNA leakage and suppress subsequent activation of the cGAS-STING inflammatory cascade. Mitophagy inhibition or PINK1/Parkin silencing fully abrogated FLNC’s cytoprotection, and endothelial FLNC overexpression retarded plaque progression in vivo. Collectively, our findings identify FLNC as a novel endogenous endothelial mitophagy regulator, revealing an unreported regulatory mechanism and supporting FLNC as a promising therapeutic target for atherosclerotic disease.