Fig 1: pDCs and eNOS. The pDCs from db/db mice incubated with endothelial cells blunted eNOS phosphorylation in response to ATP compared to pDCs from db/het using kit assay (A) and Western blot analysis with cumulative data (B). Isolated MRA from db/het reactivity with and without pDCs from db/db mice. pDCs from db/db mice impair MRA endothelium-dependent relaxation but not contractility and endothelium-independent relaxation (C) *P < 0.05, **P < 0.01, ***P < 0.001, and ns, not significant, (n = 5); one-way ANOVA with Tukey's multiple comparisons for eNOS expression and unpaired t-test for MRA endothelium-dependent contraction and relaxation.
Fig 2: ER stress and eNOS phosphorylation in MRA. The ER stress (PERK, CHOP, Xbp1, and ATF6) induction in MRA from male (A) and female mice (B) Reduction in eNOS phosphorylation in the MRA from male and female db/db compared to db/het and db/db mice treated with anti-PDCA mAb using kit assay (C,D) and Western blot analysis with cumulative data (E,F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, not significant, (n = 5); one-way ANOVA with Tukey's multiple comparison.
Fig 3: Proposed mechanism of action of fenofibrate in irradiated HCAECs in-vitro.Irradiation reduced NO signalling via inactivation of the PI3K–AKT–eNOS pathway, whereas fenofibrate reactivated this pathway and restored NO production (A). Consistent with this, irradiation increased ROS generation, NOX activity, MDA levels, and 3-NT levels, while fenofibrate mitigated this effect (B). Irradiation also triggered an inflammatory response, which was counteracted by fenofibrate (C). The released cytokines contribute to inflammation, changes in cytoskeleton organisation and initiation of EndMT, and fenofibrate effectively attenuated the processes (D). Alterations in the pathways described above play a crucial role in the remodelling of vascular endothelial cells involved in the initiation and progression of atherosclerosis. Fenofibrate acts to reduce or restore the effects of irradiation on these pathways (E–F). Solid lines indicate correlations validated in this study, while dashed lines indicate unvalidated correlations.
Fig 4: Effect of different concentrations of NOR on eNOS phosphorylation in HUVEC (A). Effect of NOR on ICAM-1, VCAM-1 expression (B) (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle; ## p < 0.01, ### p < 0.001 vs. TNF-α.
Fig 5: Analysis of the PI3K-AKT-eNOs pathway.qPCR and ELISA analysis of the components of PI3K-AKT-eNOS pathway were performed in HCAECs (A–F). The expression level of mRNA was measured using qPCR, 2d (A) and 7d (B) after irradiation with and without fenofibrate (Feno). The expression levels of AKT and p-AKT both serine and threonine and eNOS and p-eNOS proteins were compared using ELISA after 2d (C and E) and 7d (D and F). The levels of NO produced in the HCAECs were measured at the 2d and 7d after irradiation (G). The error bars represent the standard deviation (±SD) (Two-way ANOVA, Tukey's multiple comparisons test; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).
Supplier Page from Abcam for Phospho-eNOS (S1177) and Total eNOS ELISA Kit