Effect of high glucose on p53, p21, p16^INKa, and DNA ladder on Apurinic/apyrimidinic (AP) sites.

<p>HUVECs were cultured with constant high glucose (HG) and intermittent glucose (N/HG) introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123169#pone.0123169.g001" target="_blank">Fig 1</a>. 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, p16^INKa 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.</p

Research Design.

<p>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).</p

ROS and superoxide generation in HUVECs exposed to high glucose.

<p>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 p22^<i>phox</i> protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22^<i>phox</i> siRNA effectively eliminated p22^<i>phox</i> protein expression. (E) Transfection of p22^<i>phox</i> siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22^<i>phox</i> siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22^<i>phox</i> siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 vs. NG; ###<i>p</i><0.001 vs. HG.</p

Effect of high glucose on SA-β-gal activity in HUVECs.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123169#pone.0123169.g001" target="_blank">Fig 1</a>. 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.</p

Effect of high glucose on telomerase activity and telomere length in HUVECs.

<p>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. **<i>p</i><0.01 vs. NG; ##<i>p</i><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.</p

Role of eNOS in the effect of nifedipine on high glucose-induced cellular senescence in HUVECs.

<p><b>(A)</b> Influence of 1-NAME on the effect of 1 nM nifedipine on HG (22 mM glucose)-induced increase in SA-β-gal positive cells (n = 5). <b>(B)</b> Transfection of eNOS siRNA effectively eliminated eNOS protein expression (n = 3). <b>(C)</b> Influence of eNOS siRNA transfection on the effect of 1 nM nifedipine on HG-induced increase in SA-β-gal activity (n = 3). <b>(D)</b> Effect of 1 nM nifedipine on eNOS expression and phosphorylation at Ser-1177 under HG conditions (n = 6). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus HG alone.</p

Effects of atenolol, perindopril, and nifedipine on high glucose-induced cellular senescence in HUVECs.

<p><b>(A)</b> Cytochemical staining for SA-β-gal activity. Cells were incubated with 22 mM glucose for 3 days in the absence and presence of 10 µM atenolol, 10 µM perindopril, or 1 nM nifedipine. Bar = 100 µm. <b>(B)</b> A bar graph summarizes the results from 5 experiments shown in <b>A</b>. <b>(C)</b> Telomerase activity was measured by the telomere repeat application protocol (trap) assay (n = 4). ***<i>P</i><0.001 versus NG (5.5 mM glucose). ##<i>P</i><0.01, ###<i>P</i><0.001 versus HG (22 mM glucose) alone.</p

Additional file 2: Figure S2. of Critical role of endogenous histamine in promoting end-organ tissue injury in sepsis

Transcription levels of IL-1β, IL-6, and MCP-1 in lung, liver, and kidney tissues of H1R−/−/H2R−/− mice following CLP-induced sepsis. Tissues were harvested at 18 h after surgery (n = 5–8/group). The values were expressed as a fold increase above sham-operated WT normalized GAPDH. (PNG 10 kb

Additional file 3: Figure S3. of Critical role of endogenous histamine in promoting end-organ tissue injury in sepsis

Transcription levels of IL-1β, IL-6, and TNF-α in lung, liver, and kidney tissues of d-chlorpheniramine- and famotidine-treated mice following CLP-induced sepsis. Tissues were harvested 18 h after surgery (n = 7–15/group). The values were expressed as a fold increase above sham-operated control normalized GAPDH. (PNG 8 kb

ROS generation in HUVECs exposed to high glucose.

<p><b>(A)</b> Effects of 1 µM H₂O₂ on ROS generation in cells exposed to 22 mM glucose (HG) for 3 days (n = 4–6). The cells were stained with fluorescent probe CM-H₂DCFDA, and ROS were detected by flow cytometry. <b>(B)</b> Effects of 1 nM nifedipine, 220 µM H₂O₂, and 500 µM sodium nitroprusside (SNP) on superoxide production in cells exposed to HG for 3 days (n = 3–6). Superoxide detection was made using DHE. <b>(C)</b> Expression of p22<i>^phox</i> in cells incubated with 5.5 mM glucose (NG), HG, and HG in presence of 1 nM nifedipine. In the top trace, typical Western blots are shown. β-Actin served as loading control. In the bottom trace, a bar graph summarizes the results of 6 independent experiments. (D) Effect of 5 mM NAC on HG-induced SA-β-gal activity (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus NG. ##P<0.01, ###<i>P</i><0.001 versus HG alone.</p