High-Carbohydrate Diet Enhanced the Anticontractile Effect of Perivascular Adipose Tissue Through Activation of Renin-Angiotensin System

Figures

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  FIGURE 1. Evaluation of (A) cumulative food intake, (B) body weight, and (C) adiposity index of mice fed standard or HC diets. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses or in the dispersion points in the graphs. Statistical significance was determined using (A,B) two-way ANOVA followed by Holm–Sidak’s post hoc test or (C) Student’s t-test. *p < 0.05 vs. control.

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  FIGURE 2. Histological analysis of thoracic aorta and PVAT of control and HC groups. Representative histological sections stained by (A,B) hematoxylin-eosin, or picrosirius under the incidence of (E,F) normal polychromatic light or (G,H) polarized light. The areas of (C) middle layer or (D) PVAT, and (I) the percentage of collagen fibers are represented in graphical bars with mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in the dispersion points in the graphs. Statistical significance was determined using Student’s t-test. *p < 0.05 vs. control. Arrows indicate the abundance of collagen fibers in the adventitious layer of blood vessels. Scale bars indicate (A,B) 500 μm or (E–H) 50 μm.

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  FIGURE 3. Vasodilator response induced by ACh in the presence or absence of PVAT in aortas of control and HC groups. Cumulative concentration-response curves for ACh in the presence or absence of PVAT in aortas of (A) control, (B) HC, and (C) overlapping curves of control and HC groups. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses in the graphs. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test.

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  FIGURE 4. Contractile response induced by PE in the presence or absence of PVAT in aortas of control and HC groups. Cumulative concentration-response curves for PE in the presence or absence of PVAT in aortas of (A) control, (B) HC, and (C) overlapping curves of control and HC groups. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses in the graphs. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test. *p < 0.05 vs. control PVAT; #p < 0.05 vs. HC PVAT represent the differences in the Emax values.

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  FIGURE 5. Involvement of RAS in the effect of HC diet on the PVAT control of vascular tone. Cumulative concentration-response curves for PE in the presence of PVAT in aortas of control and HC groups previously incubated or not with (A) captopril, (B) A779, or (C) PD123,319. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses in the graphs. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test. *p < 0.05 vs. control PVAT; #p < 0.05 vs. HC PVAT represent the differences in the Emax and pD2 values.

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  FIGURE 6. Immunofluorescence of Mas and AT2 receptors in the PVAT of control and HC groups. (A,D,H,K) Nuclear immunostaining with DAPI. Immunostaining for (B,E) Mas or (I,L) AT2 receptors. Overlap of immunostaining for (C,F) Mas receptors and DAPI or (J,M) AT2 receptors and DAPI. Fluorescence intensity emitted by binding the selective secondary antibody to (G) Mas (red fluorescence) or (N) AT2 (green fluorescence) receptors in the PVAT, respectively, were represented in graphical bars with mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in the dispersion points in the graphs. Statistical significance was determined using Student’s t-test. *p < 0.05 vs. control. Arrows indicate the location of the endothelial layer of blood vessels. Scale bars indicate 50 μm.

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  FIGURE 7. Involvement of PI3k-Akt-NOS pathway in the effect of HC diet on PVAT control of vascular tone and evaluation of NO availability. Cumulative concentration-response curves for PE in the presence of PVAT in aortas of control and HC groups previously incubated or not with (A) LY294,002 or (B) L-NAME. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test. *p < 0.05 vs. control PVAT; #p < 0.05 vs. HC PVAT represent the differences in the Emax and pD2 values. Representative sections of basal NO availability in the PVAT of (C) control and (D) HC groups exposed to DAF-2DA probe. Arrows indicate the location of the endothelial layer of blood vessels. Scale bars indicate 100 μm. (E) Quantification of NO production in the PVAT was expressed as fluorescence intensity in graphical bars with mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in the dispersion points in the graphs. Statistical significance was determined using Student’s t-test. *p < 0.05…

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  FIGURE 8. Implication of eNOS, iNOS, and nNOS in the effect of HC diet on control of vascular tone induced by PVAT. Cumulative concentration-response curves for PE in the presence of PVAT in aortas of control and HC groups previously incubated or not with (A) L-NNA, (B) 1,400 W, or (C) 7Ni. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses in the graphs. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test. *p < 0.05 vs. control PVAT; #p < 0.05 vs. HC PVAT; ∧p < 0.05 vs. control PVAT L-NNA or 1,400 W represent the differences in the Emax and pD2 values.

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  FIGURE 9. Immunofluorescence of eNOS, iNOS and nNOS in the PVAT of control and HC groups. (A,D,H,K,O,R) Nuclear immunostaining with DAPI. Immunostaining for (B,E) eNOS, (I,L) iNOS or (P,S) nNOS. Overlap of immunostaining for (C,F) eNOS and DAPI, (J,M) iNOS and DAPI or (Q,T) nNOS and DAPI. Fluorescence intensity emitted by binding the selective secondary antibody to (G) eNOS (red fluorescence), (N) iNOS (red fluorescence), or (U) nNOS (green fluorescence) in the PVAT, respectively, were represented in graphical bars with mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in the dispersion points in the graphs. Statistical significance was determined using Student’s t-test. *p < 0.05 vs. control. Arrows indicate the location of the endothelial layer of blood vessels. Scale bars indicate 50 μm.

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  FIGURE 10. Involvement of H2O2 and potassium channels in the effect of HC diet on PVAT control of vascular tone and evaluation of H2O2 availability. Cumulative concentration-response curves for PE in the presence of PVAT in aortas of control and HC groups previously incubated or not with (A) catalase or (E) TEA. Values represent mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in parentheses in the graphs. Statistical significance was determined using two-way ANOVA followed by Holm–Sidak’s post hoc test. *p < 0.05 vs. control PVAT; #p < 0.05 vs. HC PVAT; ∧p < 0.05 vs. control PVAT TEA represent the differences in the Emax and pD2 values. Representative sections of basal H2O2 levels in the PVAT of (B) control and (C) HC groups exposed to DCF-DA probe. Arrows indicate the location of the endothelial layer of blood vessels. Scale bars indicate 100 μm. (D) Quantification of H2O2 production in the PVAT was expressed as fluorescence intensity in graphical bars with mean ± SEM. The total number of animals required to carry out the experiments in each group (n) is presented in the dispersion points in the graphs. Statistical significance…

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  FIGURE 11. Proposed mechanisms underlying the anticontractile effect of PVAT during HC diet-induced obesity. Signaling cascade triggered by RAS through activation of Mas and AT2 receptors, PI3K, nNOS, or iNOS that lead to increased production of the vasodilator molecules NO and H2O2, and subsequently hyperpolarization by opening potassium channels.