Rosiglitazone, a Ligand to PPAR γ, Improves Blood Pressure and Vascular Function through Renin-Angiotensin System Regulation

Abstract

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has been reported to act as insulin sensitizer and exert cardiovascular actions. In this work, we hypothesized that RGZ exerts a PPARγ-dependent regulation of blood pressure through modulation of angiotensin-converting enzyme (ACE)-type 2 (ACE2)/angiotensin-(1-7)/angiotensin II type-2 receptor (AT₂R) axis in an experimental model of high blood pressure. We carried on experiments in normotensive (Sham) and aortic coarctation (AoCo)-induced hypertensive male Wistar rats. Both sham and AoCo rats were treated 7 days with vehicle (V), RGZ (5 mg/kg/day), or RGZ+BADGE (120 mg/kg/day) post-coarctation. We measured blood pressure and vascular reactivity on aortic rings, as well as the expression of renin-angiotensin system (RAS) proteins. We found that RGZ treatment in AoCo group decreases blood pressure values and improves vascular response to acetylcholine, both parameters dependent on PPARγ-stimulation. RGZ lowered serum angiotensin II (AngII) but increased Ang-(1-7) levels. It also decreased 8-hydroxy-2'-deoxyguanosine (8-OH-2dG), malondialdehyde (MDA), and improved the antioxidant capacity. Regarding protein expression of RAS, RGZ decreases ACE and angiotensin II type 1 receptor (AT₁R) and improved ACE2, AT₂R, and Mas receptor in AoCo rats. Additionally, an in silico analysis revealed that 5'UTR regions of RAS and PPARγ share motifs with a transcriptional regulatory role. We conclude that RGZ lowers blood pressure values by increasing the expression of RAS axis proteins ACE2 and AT₂R, decreasing the levels of AngII and increasing levels of Ang-(1-7) in a PPARγ-dependent manner. The in silico analysis is a valuable tool to predict the interaction between PPARγ and RAS.

Figure 1

Effect of rosiglitazone (RGZ) on noradrenaline (NA)-induced aortic reactivity in absence (a) or presence (b) of N(ω)-nitro-L-arginine-methyl-ester (L-NAME, 300μM). The values represent the mean ± SEM (n=6 animals per group). One-way analysis of variance (ANOVA) post Tukey. ⁺P < 0.05 sham-V vs. sham-RGZ, ^∗P < 0.05 sham-V vs. AoCo-V, ^#P < 0.05 AoCo-V vs. AoCo-RGZ.

Figure 2

Effect of rosiglitazone (RGZ) on vascular reactivity exerted by acethylcholine. The aortic reactivity was evaluated in sham-vehicle treated (a), aortic coarctated (AoCo)-vehicle treated (b), AoCo-RGZ treated (c), and AoCo-RGZ + bisphenol A diglycidyl ether (BADGE) treated (d) rats under basal conditions (●) or in presence of N(ω)-nitro-L-arginine-methyl-ester (L-NAME, 300μM) (■). The values represent the mean ± SEM (n=6 animals per group). One-way analysis of variance (ANOVA) post Tukey. ^∗P < 0.05 basal vs. L-NAME conditions.

Figure 3

Effect of rosiglitazone (RGZ) on aortic angiotensin converting enzyme (ACE) (a) and ACE2 (b) expression. The images show representative western blots. The protein expression was evaluated in homogenate of aorta from sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). The values represent the mean ± SEM (n=6 animals per group). One-way analysis of variance (ANOVA) post Tukey. ^∗P < 0.05 Sham-V vs. AoCo-V, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, ^%P < 0.05 Sham-RGZ vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE. ND: not detected.

Figure 4

Effect of rosiglitazone (RGZ) on aortic angiotensin II type 1 (AT₁R) (a), angiotensin II type 2 (AT₂R) (b), and Mas (c) receptor expression. The images show representative western blots. The protein expression was evaluated in homogenate of aorta from sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). The values represent the mean ± SEM (n=6 animals per group). One-way analysis of variance (ANOVA) post Tukey. ^∗P < 0.05 Sham-V vs. AoCo-V, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE. ND: not detected.

Figure 5

Effect of rosiglitazone (RGZ) on angiotensin II and angiotensin-(1-7) production. Values obtained from aorta's homogenate (a, c) and serum (b, d) from sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). The values represent the mean ± SEM (n=6 animals per group). One-way analysis of variance (ANOVA) post Tukey. ^∗P < 0.05 Sham-V vs. AoCo-V, ⁺P < 0.05 Sham-V vs. Sham-RGZ, ^%P < 0.05 Sham-RGZ vs. AoCo-RGZ, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE.

Figure 6

Effect of rosiglitazone (RGZ) on 8-hydroxy-2′-deoxyguanosine (8-OH-2-dG), a marker of oxidative stress-induced DNA damage in homogenate from aorta (a) and serum (b), and serum malondialdehyde (c) from sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). The values represent the mean ± SEM (n=6 animals of group). One-way analysis of variance (ANOVA) post Tukey. ^∗P < 0.05 Sham-V vs. AoCo-V, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE.

Figure 7

Effect of rosiglitazone (RGZ) on total antioxidant capacity of sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). The parameter was evaluated in serum. The values represent the mean ± SEM (n=6 animals of group). One-way analysis of variance (ANOVA) post Tukey. ⁺P < 0.05 Sham-V vs. Sham-RGZ, ^∗P < 0.05 Sham-V vs. AoCo-V, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE.

Figure 8

Effect of rosiglitazone (RGZ) on PPARγ activity (a) and PPARγ expression of sham or aortic coarctated (AoCo) rats treated with vehicle (V), rosiglitazone (5mg/kg, RGZ), or RGZ + bisphenol A diglycidyl ether (120 mg/kg, RGZ+BADGE). PPARγ activity was evaluated using a transcription factor assay kit (Cayman Chemicals, Ann Arbor, MI, USA). Western blot image is representative of 6 different experiments. Bars represent the mean ± SEM (n=6 animals of group). One-way analysis of variance (ANOVA) post Tukey. ⁺P < 0.05 Sham-V vs. Sham-RGZ, ^#P < 0.05 AoCo-V vs. AoCo-RGZ, and ^&P < 0.05 AoCo-RGZ vs. AoCo-RGZ+BADGE.

Figure 9

In silico analysis obtained by bioinformatic on line tools. The motifs (M) sequences in 5′ untranslated regions obtained by MEME program indicate the length and position regarding the transcription start signal (a). Transcription factors found through TESS database in motifs (b). Percentage of nucleotide presence on these motifs (c).