ABC | Volume 110, Nº6, June 2018

Original Article Oishi et al Endothelial dysfunction and blood pressure in rats Arq Bras Cardiol. 2018; 110(6):558-567 130 120 110 100 90 80 70 60 50 40 30 20 10 0 0 6 12 18 24 VAT (grams) Time (Weeks) CT HFD *+ *+ *+ *+ Figure 1 – Visceral adipose fat (VAT) in control (CT) and high-fat diet (HFD) groups over the weeks. *P < 0.05, compared with CT; + p < 0.05, within-group comparison (0 vs. 6, 6 vs. 12, 12 vs. 18, 18 vs. 24 weeks). Seven rats from each group were compared at each time point. Statistical analysis The normality of distribution (of all quantitative and continuous variables) was checked using the Kolmogorov‑Smirnov test. A sample of 7 animals in each group was required to provide 85% statistical power with a two-tailed alpha of 0.05 for pD2 and 90% for all other variables analyzed in this study. Differences between the CT and HFD groups were compared using two-way repeated measures analysis of variance ANOVA. When differences were indicated, a Newman-Keuls post hoc analysis was used with a statistical significance set at p < 0.05. These data were expressed as mean ± SD (Statistica software 7.0, StatSoft. Inc, USA). Vascular reactivity data of pD2 and Emax were expressed as mean ± SD with a statistical significance set at p < 0.05 (Graphpad Prism 3.0). Pearson correlation was made between pD2 and the SBP, pD2 and VAT, blood pressure and VAT, IL-6 and pD2, TNF- α and pD2, CRP and pD2 and between adiponectin and pD2, with a statistical significance of 5%. Results Total visceral adipose tissue The sum of the weight of the retroperitoneal, visceral and epididymal adipose tissues – (VAT) was higher in HFD than in the CT group at 6 weeks. At 24 weeks, fat weight was 300% higher in the HFD than the CT group. VAT in the CT group increased at 12 weeks compared to 6 weeks, but remained unchanged for the rest of the experimental period (Figure 1). Inflammatory status The inflammatory cytokines IL-6, TNF- α and CRP were increased in serum of HFD animals in 6, 12, 18 and 24 weeks when compared to the CT group (Figure 2 A, B, C). On the other hand, the levels of serum adiponectin decreased in the HFD group after 6, 12, 18 and 24 weeks of the experimental protocol (Figure 2 D). In the CT group, no changes were found in these cytokine levels. Vascular reactivity No differences were found (Figure 3A) in the endothelium- dependent relaxation induced by acetylcholine (pD2) in the CT group over the entire experimental period. On the other hand, the pD2 was impaired in aortas of obese animals at 6, 12, 18 and 24 weeks compared to CT rats. Moreover, we observed a decrease in pD2 throughout the experimental period in HFD group (Figures 3B, C). No differences were observed in the maximum relaxant effect (MaxE) in both CT and HFD groups. In endothelium- denuded aortic rings, there were no differences in the pD2 and MaxE to endothelium-independent relaxation induced by SNP in the CT and HFD groups in all the weeks evaluated (Table 1). There was a strong negative correlation between pD2 and SBP (r = -0.722, p < 0.01). Moreover, we found a negative correlation between pD2 and VAT (r = -0.729, p < 0.01), between pD2 and inflammatory cytokines (pD2 and IL‑6, r = -0.74; pD2 and TNF- α , r = -0.86; pD2 and CRP, r = -069, p < 0.05) and a positive correlation between pD2 and adiponectin (r = 0.77, p < 0.01). Serum nitric oxide (NO) and aorta lipid peroxidation By quantification of serum NO metabolites, we observed that NO level decreased at 6 weeks in HFD rats and remained lower throughout the experimental period when compared to the CT group. The time of experiment had no effect on NO concentrations in CT and the HFD groups (Figure 4). Levels of lipid peroxidation in aorta increased at 12 weeks of a high-fat diet and remained high throughout the experimental period when compared to the CT group. In the HFD group, there was an increase in lipid peroxidation at 12 weeks when compared to 6 weeks (Figure 5). 560