Figures and data
Abnormal shear stress induces endothelial ferroptosis.
(A) Diagram of the microfluidic device and calculation of the shear stress. Three levels of shear stress (τ) are formed in the tunnel-like channel, depending on the varying cross-sectional area of flow: LSS = 0.99 dynes/cm2, MSS = 4.78 dynes/cm2, and HSS = 24 dynes/cm2. These values were calculated using a formula that includes the dynamic viscosity of the fluid (μ), flow rate (Q), channel width (W), cross-sectional area (A), wetted perimeter (pw), membrane bulge height (H), and a function of H/W (g(H/W)). (B) Fluorescent microscopic results of PI staining in ECs (The scale bar = 250 μm); (C) ELISA results of extracellular LDH in ECs; (D, E) Flow cytometry results of lipid peroxidation in ECs indicated by 4-HNE and C11 BODIPY. (F, G) Protein expression of SLC7A11 and intracellular CoQ10 level. (H) The results of Fer-1 (10 μM) treatment. Data are expressed as the mean ± standard deviation from three or more independent experiments. Statistical significance was determined using an unpaired t-test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
RNA-seq reveals LSS suppresses cholesterol biosynthesis and UPR.
(A) Volcano plot of DEGs (LSS vs. MSS;|log2FC|>0.6, Q<0.05); (B) KEGG pathways; (C) GO biological processes; (D) GSEA of steroid biosynthesis and UPR. NES: normalized enrichment score. FDR: false discovery rate. (E) Heatmap of DEGs in lipid synthesis and metabolism. (F) Heatmap of DEGs in UPR. (G) Protein expression of BiP, PERK, MVD, and IDI1 under different shear stress conditions. Data are expressed as the mean ± standard deviation from three or more independent experiments. Statistical significance was determined using an unpaired t-test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. KEGG: Kyoto Encyclopedia of Genes and Genomes, GO: gene ontology, GSEA: gene set enrichment analysis.
KLF6 regulates the UPR and mevalonate pathway.
(A) Heat map of the identified TF genes downregulated by LSS; (B) Predicted KLF6-binding motifs in promoters of PERK, HSPA5, MVD, IDI1 (JASPAR database; matrix ID: MA0472.1); (C) Protein expression of KLF6 under different shear stress conditions; (D) Validation of KLF6 overexpression by Western blot (OE: overexpression, NC: negative control); (E, F) KLF6-OE largely restores the expression of PERK, BiP, MVD, and IDI1 under LSS and HSS to levels seen under MSS. Data are expressed as the mean ± standard deviation from three or more independent experiments. Statistical significance was determined using an unpaired t-test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
KLF6 overexpression alleviates endothelial ferroptosis and early atherosclerotic events induced by LSS and HSS.
(A, E) Protein expression of SLC7A11; (B, F) ELISA results of extracellular LDH; (C, G) ELISA results of intracellular CoQ10; (D, H) Flow cytometry results of lipid peroxidation indicated by C11 BODIPY; (I, K) Fluorescent microscopic results of THP-1 cells (green label) adhering to the VSMC-EC bilayer cell architecture (The scale bar = 250 μm); (J, L) Lipid accumulation visualized by Oil Red O staining (The scale bar = 25 μm). Data are expressed as the mean ± standard deviation from three or more independent experiments. Statistical significance was determined using an unpaired t-test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
