Fig 2: The miR-25/KLF2 axis regulates the NFκB signaling pathway. (A,B) Bars show the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) in HUVECs transfected with the negative control (NC), KLF2 siRNA (siKLF2), or the KLF2-overexpressing vector (KLF2). (C) Bars show the mRNA expression of VCAM-1, ICAM-1, IL-6, and MCP-1 in HUVECs transfected with the NC or miR-25 mimics; or co-transfected with miR-25 (miR-25 mimics) and KLF2; or transfected with the NC or KLF2. (D,E) The protein expression of VCAM-1, ICAM-1, IL-6, and MCP-1 in HUVECs transfected with the NC or miR-25 mimics, or co-transfected with miR-25 mimics and KLF2 detected by western blotting and ELISA. (F) Bars show the mRNA expression of VCAM-1, ICAM-1, IL-6, and MCP-1 in HUVECs transfected with KLF2 and simultaneously treated with exosomes isolated from GES-1 cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: EC senescence affects the plaque characteristics. (A) En face analysis of the aorta in ApoE-KO and ApoE-KO/VEcad-TERF2DN-Tg mice fed a HCD for 8 weeks. Atherosclerotic plaques were quantitatively analyzed (n = 5 for ApoE-KO; n = 6 for ApoE-KO/TERF2DN-Tg). Bars: 1 mm. (B) Immunohistochemistry for CD68 in aortic sinus sections of ApoE-KO and ApoE-KO/VEcad-TERF2DN-Tg mice fed a HCD for 8 weeks. CD68-positive areas were quantified (n = 5 for ApoE-KO; n = 6 for ApoE-KO/TERF2DN-Tg). Bars: 100 μm. (C) H-E staining of aortic sinus sections of ApoE-KO and ApoE-KO/VEcad-TERF2DN-Tg mice fed a HCD for 8 weeks. Arrowheads indicate the necrotic core. The size of necrotic core was measured (n = 5 for ApoE-KO; n = 6 for ApoE-KO/TERF2DN-Tg). Bars: 100 μm. (D) Immunohistochemistry for VCAM-1 in aortic sinus sections of ApoE-KO and ApoE-KO/VEcad-TERF2DN-Tg mice fed a HCD for 2 weeks. Immunohistochemistry for vWF was performed using the serial sections to identify ECs. Arrowheads indicate the vWF-positive endothelium on the plaque surface. Bars: 100 μm. Data are presented as mean ± SEM. *p < 0.05.

Fig 4: The effect of exposure to PAHs on the protein expression of adhesion molecules in aortic tissue. (A) Relative protein expression of ICAM-1 (p = 0.117). (B) Relative protein expression of VCAM-1 (p = 0.210). (C) Representative Western blots are shown for ICAM-1, VCAM-1, and α/β-tubulin. Data are presented as the mean ± SEM. Welch’s ANOVA test. (Control, n = 4; 10 µg, n = 4; 30 µg, n = 4; 50 µg, n = 4).

Fig 5: NF-κB signaling is critically involved in the hyper-inflammability in senescent ECs. (A) Inhibition of NOS using L-NAME did not abolish the enhanced VCAM-1 induction in response to TNF-α in senescent ECs (n = 3 each). (B) Inhibition of NOS using L-NAME did not abrogate the enhanced monocyte adhesion in senescent ECs treated with TNF-α (n = 3 each). (C) Inhibition of NF-κB signaling using helenalin abolished the enhanced VCAM-1 induction in response to TNF-α in senescent ECs (n = 3 each). Data are presented as mean ± SEM. **p < 0.01 and ****p < 0.0001.