Fig 2: Western blotting of endothelial nitric oxide synthase (eNOS) and its phosphorylation. No significant difference was seen between four groups in eNOS/a-tubulin ratio (A: Young WT, n = 6; young GLO1 Tg, n = 6; mid-age WT, n = 10; mid-age GLO1 Tg, n = 14) or dimer/monomer ratio of eNOS (B: Young WT, n = 3; young GLO1 Tg, n = 3; mid-age WT, n = 12; mid-age GLO1 Tg, n = 16). NS indicates no significant difference by one-way ANOVA. (C) eNOS phosphorylation on Thr495 was elevated by aging in WT rats and was significantly reduced in GLO1 Tg rats compared with mid-age WT rats (C-1). Age-related increase in p-eNOS (Thr495) was significantly attenuated in GLO1 Tg rats (C-2). Young WT, n = 11; young GLO1 Tg, n = 6; mid-age WT, n = 16; mid-age GLO1 Tg, n = 16. Two-way ANOVA with post hoc Bonferroni correction was performed. The number of multiple comparison was 4. Phosphorylation on Ser1177 tends to decrease by aging in WT rats (D-1). Age-related dephosphorylation of eNOS (Ser1177) was significantly attenuated in GLO1 Tg rats. *P < 0.05 by t-test (D-2). Young WT, n = 10; young GLO1 Tg, n = 5; mid-age WT, n = 16; mid-age GLO1 Tg, n = 17. Myr-Akt serves as a positive control for phosphorylation of eNOS on Ser1177.

Fig 3: Effects of MPs from patients with ACS concurrent with T2DM on eNOS in the rat thoracic aorta. MPs from patients with ACS and/or T2DM (A) decreased eNOS and its phosphorylation at the Ser1177 site and (B) upregulated eNOS phosphorylation at T495 in the rat thoracic aorta. *P<0.05 vs. Blank and Control; #P<0.05 vs. ACS and T2DM. MP, microparticle; ACS, acute coronary syndrome; T2DM, type 2 diabetes mellitus; eNOS, endothelial nitric oxide synthase.

Fig 4: Effect of SFI on eNOS content (A) and eNOS mRNA expression (B). 10, 20, and 40 µl/ml of SFI and mevastatin significantly increased eNOS content (A), and 10 µl/ml of SFI obviously up-regulated eNOS mRNA expression, as mevastatin (B). Values are expressed as the mean ± SD, n = 6. * P < 0.05, ** P < 0.01 vs. Control.

Fig 5: Akt mediates tBHQ-induced eNOS phosphorylation and NO production in cultured endothelial cells.(A) HUVEC were infected with control or Akt siRNA for 48 hours. Then cells were exposed to tBHQ at 25 µM for 6 hours. Total cell lysates were analyzed by western blot for the indicated proteins. The blot is a representative of four blots obtained from four separated experiments. Data presented are means ± SEM from 4 independent experiments. *P < 0.05 vs. control group. #P < 0.05 vs. control siRNA alone. NS indicates no significance. (B–D) HUVEC infected with control or Akt siRNA for 48 hours. The NO production was determined by measuring the intensity of DAF fluorescence in (B) and quantitative analysis of NO amount was shown in (C). Total cell lysates were subject to assay eNOS activity (D). Data presented are means ± SEM from 4 independent experiments. *P < 0.05 vs. control siRNA group. NS indicates no significance.