Fig 2: Activation of mTORc1 by RHEB expression is sufficient to phosphorylate eNOS S1177. (A) Representative western blot analysis of pAKT (S473), total AKT, pS6 (S235/235), total S6, pENOS (S1177), total eNOS and a loading control, ERK2, in control and RHEBS16H‐expressing HUVECs ± hVEGF. (B) Quantification of protein densitometry for pENOS (S1177) normalized to loading control ERK2 for control and RHEBS16H‐expressing HUVECs ± hVEGF. HUVECs were stimulated with hVEGF for 10 min. (C) Quantification of protein densitometry for pAKT (S473), pS6 (S235/236), and pENOS (S1177) for RHEBS16H‐expressing HUVECs +hVEGF +0.1 ng/mL rapamycin. Data are presented as percent change compared to rapamycin untreated conditions. The red dotted line is representative of untreated RHEBS16H protein levels. Graphs are presented as mean ± SEM, *=P < 0.05 N = 3. Data are representative of at least two independent infections and three western blot analyses.

Fig 3: Endothelial cell-specific loss of FABP3 promotes eNOS expression and activation. HUVECs were transfected with either scrambled control or siFABP3 for 24 h and treated for additional 24 h with LPS, and then protein and RNA were extracted to perform immunoblot and qPCR, respectively. A–C, immunoblotting for eNOS, p-eNOS, and GAPDH (A), and quantification for eNOS (B) and p-eNOS/eNOS ratio (C). D, E, F and H, immunoblotting for AKT, pAKT, and GAPDH and quantification for pAKT/AKT ratio (E), AKT (F), and pAKT (H). J and K, immunoblot (J) and (K) quantification for p21. G and I, qPCR was performed for AKT (G) and p21 (I). Difference between the means of groups were calculated using one-way ANOVA with Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus Scr Control. #p < 0.05, ##p < 0.01, ###qPCR, and data are represented as mean ± SD. AKT, protein kinase B; eNOS, endothelial nitric oxide synthase; FABP3, fatty acid–binding protein 3; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide.

Fig 4: Inhibition of EZH2 in rat aortic rings exposed to intermittent hyperglycemia reduces H3K27me3 levels and blocks inflammatory signaling ex vivo. (A–D) Immunoblotting for H3K27me3 ((A), n = 4), KLF2 ((B), n = 4), eNOS ((C), n = 4), and ICAM1 ((D), n = 4) in rat aortic rings challenged with the EZH2 inhibitor GSK126 (10 μM for 72 h) in combination with intermittent high glucose. Tissues continuously exposed to normal glucose (5.5 mM) were considered as the non-intermittent high glucose treatment condition. All analyzed data were normalized to the control treatment condition. Values represent the mean ± SD. * p < 0.05 and ** p < 0.01 by unpaired t test.

Fig 5: Genetic ablation of GPR35 (G-protein–coupled receptor 35) in aortic endothelial cells (ECs) improves cell functions.A, Representative fluorescent images showing characteristics of mouse aortic ECs (MAECs) by fluorescent staining of Dil-ac-LDL uptake and Ulex-lectin binding, CD31, VE-Cadherin, and eNOS (endothelial nitric oxide synthase). Scale bar, 100 μm. B, Real-time quantitative polymerase chain reaction (qPCR) confirmed the expression levels of Gpr35 in the GPR35WT and little to no expression in the GPR35KO MAECs. n=6–12, *P<0.05. C, Cell migration assay in GPR35WT and GPR35KO MAECs. n=6 per group. Scale bar, 400 μm. D, Three-dimensional (3D) tube formation assay in GPR35WT and GPR35KO MAECs. n=7 group. Scale bar, 400 μm. E, Cell proliferation assay in GPR35WT and GPR35KO MAECs. n=4 group. In B–E, Mann-Whitney U tests were used, *P<0.05 vs GPR35WT ECs, horizontal lines show mean±SD. DAPI indicates 4’,6-diamidino-2-phenylindole.