Fig 1.
The endothelial cells were exposed to the experimental condition for 3 days. They were grouped as follows: (1) constant normal glucose medium (5.5 mM:NG); (2) constant high glucose medium (22 mM: HG); and (3) alternating normal and high glucose media every 12 h (N/HG).
Fig 2.
Effect of high glucose on SA-β-gal activity in HUVECs.
NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. To confirm the effect of glucose in HUVECs, NG, HG and N/HG were cultured with each Stimulus for 3 days. HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in Fig 1. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A) SA-β-gal activity was evaluated cytochemically. The values of the three independent experiments are mean ± S.D. **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG. (B) SA- β-gal-positive cells (blue) can be detected via cytochemical staining.
Fig 3.
Effect of high glucose on p53, p21, p16INKa, and DNA ladder on Apurinic/apyrimidinic (AP) sites.
HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in Fig 1. Namely, N/HG was stimulated twice with HG (at 4-hour intervals) for a total of 4 hours daily (9 a.m. to 11 a.m., 3 p.m. to 5 p.m.), and was cultured in NG in other time of the total 4-hour HG stimulation. (A)-(D): Effect of high glucose on p53, p21, p16INKa protein. (E): Effect of continuous and intermittent high glucose on endothelial DNA damage on Apurinic/apyrimidinic(AP) sites. *p<0.05; **p<0.01 vs. NG; #p<0.05 vs. HG. The values of the three independent experiments are mean ± S.D.
Fig 4.
Effect of high glucose on telomerase activity and telomere length in HUVECs.
NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Telomerase activity was measured by the telomere repeat application protocol (trap) assay. The values of the three independent experiments are mean ± S.D. **p<0.01 vs. NG; ##p<0.01 vs. HG. (B) Telomere length was measured to evaluate the relationship to replicative senescence. The data shown represent the average of two independent experiments.
Fig 5.
eNOS activity in HUVECs exposed to high glucose.
NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Typical Western blots for total eNOS, Ser-1177 phosphorylated eNOS, and Thr-495 phosphorylated eNOS are shown. GAPDH served as the loading control. (B-D) Summary of quantification of densitometric measurement of the immunoblot data. Total eNOS expression and eNOS phosphorylation levels were normalized to GAPDH and total eNOS, respectively. (E) NO production by HUVECs, using the fluorescent dye DAF-2-DA. (F) The data of NOx (NO2- and NO3-), NO metabolites measured by HPLC.
Fig 6.
ROS and superoxide generation in HUVECs exposed to high glucose.
NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H2DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22phox protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22phox siRNA effectively eliminated p22phox protein expression. (E) Transfection of p22phox siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22phox siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22phox siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *p<0.05; **p<0.01; ***p<0.001 vs. NG; ###p<0.001 vs. HG.