Fig 2: Effect of GSK3 inhibitors on the nuclear translocation of Nrf2, β-catenin, and the p65 subunit of NF-κB in LPS-stimulated SIM-A9 cells. Western immunoblots and derived graphical representation of the protein expression pattern for Nrf2 (a), NF-κB p65 (b), and β-catenin (c) are shown. Data is expressed as means ± SEM. Group comparisons were drawn using one-way ANOVA, followed by the Student–Newman–Keuls post hoc test; *P-value < 0.05 (relative to the LPS-stimulated group); $P-value < 0.05 (relative to negative [untreated] controls);; #P-value < 0.05 (BRD0705 at any given concentration relative to BRD3731 at the corresponding concentration)

Fig 3: APOE genotype and sex-dependent inflammation are mediated through p65 activation: (A) representative images for immunocytochemistry of p65 in control and LPS + IFNg treated APOE3 and APOE4, male and female PMG (scale bar = 100 µm). (B) Quantification of total p65 and (C) quantification of nuclear p65. N = 3 isolations. Each data point denotes the average p65 integrated density/cell density for an individual isolation. A denotes a significant increase compared to the E3 male control (p < 0.05). # denotes a significant increase compared to the respective genotype and sex control (p < 0.05). Data are represented as the mean ± standard deviation. * and **** indicate statistical significance for the comparison and denote p < 0.05 and 0.0001, respectively. ns = not statistically significant (p > 0.05).

Fig 4: Influence of VIB (V), hyper-g and iRPM-exposure on the gene expression of NF-?B signaling factors: (A,B) NFKB1, (C,D) NFKB2, (E,F) RELA, (G,H) NFKBIA, (I,J) NFKBIB, (K,L) IKBKG, (M,N) NFKBIE. n = 5. The data are given as mean ± standard deviation. * p < 0.05 vs. corresponding 1 g-controls. The dashed vertical line seperates two independent experiments.

Fig 5: Role of miR-15a-5p and miR-199a-3p in the human and experimental atherosclerosis. The levels of miR-15a-5p and miR-199a-3p were significantly increased in carotid from patients with advanced atherosclerosis (ACA) and in the aortas from ApoE -/- HFD. These decreases of both miRNAs provoke a significant increase of their potential targets’ expression, such as: IKKa, IKKß and p65 as well as their phosphorylation in consequence a higher I?Ba degradation and NF-?B activation, contributing to the inflammation present in advanced atherosclerosis. On the other hand, the decrease of miR-15a-5p and miR-199a-3p produce a significant increase of LOX-1 that might favour ox-LDL uptake in VSMCs. More importantly, in vascular cell lines, HUVECs and VSMCs, we demonstrated that miR-15a-5p or miR-199a-3p overexpression reduced protein levels of their studied targets in consequence the decline of NF-?B activation and ox-LDL uptake. Moreover, we isolated miRNAs from EVs (enriched exosomes) of patients with advanced atherosclerosis and healthy controls and a significant increase of both miRNAs in EVs from ACA was noted, whereas only miR-15a-5p might be useful as biomarker of advanced atherosclerosis.