Fig 2: GLO1 expression and activity in thoracic aorta. Immunohistochemistry shows the same localization of GLO1 in thoracic aorta both in mid-age WT and GLO1 Tg rats (A). Human GLO1 was detected in thoracic aortas of GLO1 Tg rats by polymerase chain reaction using specific primers for human GLO1 (B) and by Western blotting using an antibody directed against both rat and human GLO1 (C). GLO1 overexpression showed a 2.6- and 1.7-fold increase in GLO1 activity in thoracic aorta in young and mid-age rats, respectively (D). Young WT, n = 5; young GLO1 Tg, n = 4; mid-age WT, n = 6; mid-age GLO1 Tg, n = 5. Two-way ANOVA with post hoc Bonferroni correction was performed. The number of multiple comparison was 4. **P < 0.01, ***P < 0.001, vs. age-matched WT rats. NS indicates no significance.

Fig 3: GLO1 attenuates methylglyoxal (MG) modification (A) and oxidative stress markers (B, C). (A) Immunohistochemical analysis of the MG-modified protein, argpyrimidine, showed the cytoplasmic and nuclear expression patterns of argpyrimidine in endothelium and media. The number of argpyrimidine-positive cells per unit length of circumference was used to express the level of MG-modified protein in endothelium. Age-related increase in MG-modified protein in endothelium was attenuated in GLO-1 Tg rats. Young WT, n = 5; young GLO1 Tg, n = 6; mid-age WT, n = 12; mid-age GLO1 Tg, n = 9. Two-way ANOVA with post hoc Bonferroni correction was performed. The number of multiple comparison was 4. *P < 0.05, mid-age WT vs. GLO-1 Tg rats. NS indicates no difference between young WT and GLO1 Tg rats. (B) Glycative and oxidative stress marker, carboxymethyllysine, was decreased in mid-age GLO1 Tg rats compared with mid-age WT rats. n = 3, each. *P < 0.05 by t-test. (C) 8-OHdG levels in urine over 24 h were measured by ELISA. Results indicates significantly lower levels of oxidative stress in mid-age GLO1 Tg rats than WT rats. Mid-age WT, n = 10; mid-age GLO1 Tg, n = 12. *P < 0.05 by t-test.

Fig 4: GLO1 ameliorated phosphorylation of endothelial nitric oxide synthase (eNOS) (Thr495) induced by methylglyoxal (MG) in association with lowering of glycative stress in human aortic endothelial cells (HAECs). (A) GLO1 overexpression decreased argpyrimidine and phosphorylation of eNOS (Thr495). rAd-GLO1 and empty indicate HAECs infected with the recombinant adenovirus vector with or without GLO1, respectively. HAECs were infected with at a multiplicity of infection (MOI) of 20. (B) On the other hand, MG increased phosphorylation of eNOS (thr495) in a dose-dependent manner in association with glycative stress. After starvation for 24 h, HAECs were treated with MG at final concentrations of 0, 25, 265, and 420 μm, for 24 h. (C) A schema describes the mechanism of GLO1 attenuates age-related endothelial dysfunction.

Fig 5: Dose-dependent relaxation curve for (A) acetylcholine (ACh) and (B) sodium nitroprusside (SNP), and (C) nitrate and nitrite production induced by ex vivo ACh stimulation. (A, B) Four aortic rings from each rat were used for experiments, two E+ rings and two E− rings. All four rings were treated with ACh, SNP, and NG-nitro-l-arginine methyl ester (L-NAME). Young WT, n = 5; young GLO1 Tg, n = 6; mid-age WT, n = 12; and mid-age GLO1 Tg, n = 9. (A) Endothelium-intact vessels (E+) of mid-age rats showed impaired ACh-induced vasorelaxation compared with those of young rats; **P < 0.0001, ##P < 0.0001 vs. young WT/GLO1 Tg rats, respectively. Importantly, mid-age GLO1 Tg rats showed significantly better relaxation responses than mid-age WT (−62.2 ± 2.6% vs. −71.2 ± 2.1%, *P < 0.05). The ACh dose responses of endothelium-deprived vessels (E−) and E+ vessels pretreated with L-NAME were completely abolished in all groups. Two-way ANOVA with post hoc Bonferroni correction was performed. The number of multiple comparison was 112. (B) Endothelium-independent vasorelaxation by SNP was attenuated by aging in both WT and GLO1 Tg rats only at the concentration of 10−7 m, but the dose responses were not significantly different between WT and GLO1 Tg rats. Two-way ANOVA with post hoc Bonferroni correction was performed. The number of multiple comparison was 30. †P < 0.0001, young vs. mid-age rats in the same genotype group. (c) Nitrate/nitrite was used as an index of nitric oxide (NO) production by ACh stimulation. Aortic rings in Krebs–Henseleit buffer under continuous bubbling with 95% O2–5% CO2 were stimulated by 5 × 106 m ACh. Nitrate/nitrite in Krebs–Henseleit buffer was measured before and after ACh stimulation. Nitrate/nitrite was expressed as pmol mg−1 wet weight of aortic rings. Nitrate/nitrite significantly diminished in mid-age WT rats, whereas mid-age GLO1 Tg rats showed almost the same level of NO production as young WT rats. Young WT, n = 5; mid-age WT, n = 3; mid-age GLO1 Tg, n = 6. *P < 0.05 by t-test, young vs. mid-age WT rats. NS indicates no significance.