C2CD4B Evokes Oxidative Stress and Vascular Dysfunction via a PI3K/Akt/PKCα–Signaling Pathway

Figures

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  Figure 1. Effect of C2CD4B gene silencing on mice mesenteric arteries exposed to hyperglycemic conditions. (A) mRNA expression of C2CD4B determined by quantitative reverse transcription polymerase chain reaction in mice mesenteric arteries treated at different time points (3, 6 h) under normal glucose (NG, 5 mM) or high glucose conditions (HG, 30 mM); (n = 3). (B) Representative western blot and densitometric analyses evaluating protein levels of C2CD4B in mice mesenteric arteries treated with normal glucose (NG) or high glucose (HG) at different time points (3, 6 h), pretransfected with either scramble siRNA (NG 3 h; NG 6 h; HG 3 h and HG 6 h), or specific siRNA against C2CD4B (Si−C2CD4B) before HG 6 h; (n = 4). Non-parametric Kruskal–Wallis test with Dunn’s correction was used. (C) Acetylcholine (ACh)–evoked vasorelaxation in mice mesenteric arteries exposed to NG or HG conditions and pretransfected with siRNA against C2CD4B (Si–C2CD4B) or scrambled siRNA (Si–SCR); (n = 3). (D) ROS levels were detected by DHE in mice mesenteric arteries treated with normal glucose (NG), high glucose (HG) alone, or pretransfected with siRNA against C2CD4B (Si–C2CD4B). Box plots show relative fluorescence…

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  Figure 2. Effect of recombinant C2CD4B on mice mesenteric arteries and human EC. (A) Acetylcholine (ACh)–evoked vasorelaxation in mice mesenteric arteries exposed to vehicle or recombinant C2CD4B; (n = 3). (B) Acetylcholine (ACh)–evoked vasorelaxation in mice mesenteric arteries exposed to vehicle or recombinant C2CD4B in presence or absence of the antioxidant agent tempol; (n = 3). (C) Cellular ROS levels assessed by determining dihydrorhodamine 123 (DHR) fluorescence intensity (AU, arbitrary units); (n = 4–6). (D) NADPH–induced lucigenin chemiluminescence (data are expressed as increase in chemiluminescence per minute in arbitrary units) in HUVECs treated with 100 ng/mL of C2CD4B for 1 h; (n = 4–6). Statistical analyses were performed using Student’s t-test, one-way or two-way ANOVA followed by Bonferroni post-hoc test. * p < 0.05; *** p < 0.001; # p < 0.05.

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  Figure 3. C2CD4B promotes endothelial dysfunction of mice mesenteric arteries through PI3K/Akt signaling pathway. (A,B) Representative western blot and densitometric analyses of three independent experiments evaluating protein levels of (A) phospho–PI3K and (B) phospho–Akt protein expression in HUVECs treated with vehicle (ctrl), or recombinant C2CD4B. (C) NADPH–induced lucigenin chemiluminescence (data are expressed as increase in chemiluminescence per minute in arbitrary units) in HUVECs treated with 100 ng/mL of C2CD4B in the presence or absence of wortmannin, or AKT inhibitor X; (n = 4–5). (D) Acetylcholine (ACh)–evoked vasorelaxation in mice mesenteric arteries exposed to vehicle or recombinant C2CD4B in the presence or absence of wortmannin (n = 3). (E) NO detection by DAF-FM in mice mesenteric arteries treated with vehicle, C2CD4B alone, or pre-treated with wortmannin. Box plot shows relative fluorescence intensity; (n = 3). (F) ACh–evoked vasorelaxation in mice mesenteric arteries exposed to vehicle or recombinant C2CD4B in the presence or absence of AKT inhibitor X; (n = 3). Statistical analyses were performed using Student’s t-test, one-way or two-way ANOVA followed by Bonferroni…

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  Figure 4. C2CD4B induces eNOS uncoupling in mesenteric arteries. ROS production detected by DHE in mice mesenteric arteries treated with vehicle, C2CD4B alone, or pre-treated with L-NAME. Box plot shows relative fluorescence intensity; (n = 4). Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test. ** p < 0.01, # p < 0.05.

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  Figure 5. Wortmannin prevents C2CD4B–induced eNOS uncoupling in EC. (A) Representative western blot and densitometric analyses of three independent experiments evaluating eNOS dimer (280 kDa) and eNOS monomer (140 kDa) protein expression in HUVECs treated with vehicle (ctrl), recombinant C2CD4B alone, or pre-treated with wortmannin. Dimer/monomer eNOS were examined by non-reducing low-temperature SDS-PAGE. (B) Representative western blot and densitometric analyses of three independent experiments evaluating protein levels of phosphorylated eNOS at Thr 495 (p-eNOST495), and total eNOS expression in HUVECs treated with vehicle (ctrl), recombinant C2CD4B alone, or pre-treated with wortmannin. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test. ** p < 0.001; ## p < 0.001.

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  Figure 6. Pharmacological inhibition of PKCα reverses eNOS uncoupling and prevents vascular dysfunction induced by C2CD4B. (A) Representative western blot and densitometric analyses of 3 independent experiments evaluating the expression of phosphorylated form PKCα in HUVECs treated with vehicle (ctrl), recombinant C2CD4B alone, or pre-treated with wortmannin. (B) Representative western blot and densitometric analyses of three independent experiments evaluating eNOS dimer (280 kDa) and eNOS monomer (140 kDa) protein expression in HUVECs treated with vehicle (ctrl), recombinant C2CD4B alone, or pre-treated with the pharmacological inhibitor of PKCα, Go6976. Dimer/monomer eNOS were examined by non-reducing low-temperature SDS-PAGE. (C) Acetylcholine (ACh)–evoked vasorelaxation in mice mesenteric arteries exposed to vehicle or recombinant C2CD4B in the presence or absence of the PKCα inhibitor, Go6976; (n = 3–4). (D) Dose–response curves to phenylephrine of mice mesenteric arteries exposed to vehicle or recombinant C2CD4B in the presence or absence of Go6976; (n = 3–4). Statistical analyses were performed using one-way or two-way ANOVA followed by Bonferroni post-hoc test. * p < 0.05; ** p <…