Pioglitazone treatment increases COX-2-derived prostacyclin production and reduces oxidative stress in hypertensive rats: role in vascular function

Abstract

Background and purpose: PPARγ agonists, glitazones, have cardioprotective and anti-inflammatory actions associated with gene transcription interference. In this study, we determined whether chronic treatment of adult spontaneously hypertensive rats (SHR) with pioglitazone alters BP and vascular structure and function, and the possible mechanisms involved.

Experimental approach: Mesenteric resistance arteries from untreated or pioglitazone-treated (2.5 mg·kg⁻¹ ·day⁻¹ , 28 days) SHR and normotensive [Wistar Kyoto (WKY)] rats were used. Vascular structure was studied by pressure myography, vascular function by wire myography, protein expression by Western blot and immunohistochemistry, mRNA levels by RT-PCR, prostanoid levels by commercial kits and reactive oxygen species (ROS) production by dihydroethidium-emitted fluorescence.

Key results: In SHR, pioglitazone did not modify either BP or vascular structural and mechanical alterations or phenylephrine-induced contraction, but it increased vascular COX-2 levels, prostacyclin (PGI₂) production and the inhibitory effects of NS 398, SQ 29,548 and tranylcypromine on phenylephrine responses. The contractile phase of the iloprost response, which was reduced by SQ 29,548, was greater in pioglitazone-treated and pioglitazone-untreated SHR than WKY. In addition, pioglitazone abolished the increased vascular ROS production, NOX-1 levels and the inhibitory effect of apocynin and allopurinol on phenylephrine contraction, whereas it did not modify eNOS expression but restored the potentiating effect of N-nitro-L-arginine methyl ester on phenylephrine responses.

Conclusions and implications: Although pioglitazone did not reduce BP in SHR, it increased COX-2-derived PGI₂ production, reduced oxidative stress, and increased NO bioavailability, which are all involved in vasoconstrictor responses in resistance arteries. These effects would contribute to the cardioprotective effect of glitazones reported in several pathologies.

Figure 1

External and internal diameter-intraluminal pressure (A, B) and wall : lumen-intraluminal pressure (C) in MRA from WKY rats and SHR untreated or treated with pioglitazone incubated in 0 Ca²⁺-KHS. (D) Representative transversal confocal projections of the vascular wall of MRA from WKY, SHR and pioglitazone-treated SHR. Vessels were pressure-fixed at 70 mmHg, stained with Hoechst 33342 and mounted intact on a slide. Projections were obtained from serial optical sections captured with a fluorescence confocal microscope (x40_oil immersion objective, zoomX2). Metamorph Image Analysis software was used to produce the transversal projection of the artery. Stress-strain relationship (E) and incremental distensibility-intraluminal pressure curves (F) in MRA from WKY, SHR and pioglitazone-treated SHR rats. *P < 0.05 versus WKY rats by two-way anova and Bonferroni post-test. n= 8–9 animals. Data are expressed as mean ± SEM.

Figure 2

(A) Quantitative RT-PCR assessment of PPARγ mRNA levels in mesenteric arteries from WKY rats and SHR untreated and treated with pioglitazone. (B) Representative Western blot with densitometric analysis for the inducible isoform of COX-2 protein expression (upper panel) and quantitative RT-PCR assessment of COX-2 mRNA levels (lower panel) in arteries from WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus WKY rats, ^#P < 0.05 versus untreated SHR by Student's t test. n= 5–12 animals. Data are expressed as mean ± SEM.

Figure 3

(A) Concentration–response curve to phenylephrine (Phe) in endothelium-intact (E+) MRA from WKY rats and SHR untreated or treated with pioglitazone. Effect of 1 µM NS 398 on the response to Phe in endothelium-intact (E+) MRA from WKY rats (B) and SHR untreated (C) or treated with pioglitazone (D) and in endothelium-denuded segments (E−) of MRA from pioglitazone-treated SHR (E). *P < 0.05 versus control by two-way anova and Bonferroni post-test. n= 6–10 animals. Data are expressed as mean ± SEM.

Figure 4

Effect of 10 µM SC 19220, 1 µM SQ 29,548, 1 µM furegrelate, 10 µM tranylcypromine, 1 µM RO 1138452 and the combination of SQ 29,548 and RO 1138452 on the concentration–response curve to phenylephrine (Phe) in endothelium-intact (E+) MRA from SHR treated with pioglitazone and effect of 1 µM SQ 29,548 on the response to Phe in endothelium-denuded segments (E−) of MRA from SHR treated with pioglitazone. *P < 0.05 versus control by two-way anova and Bonferroni post-test. n= 7–10 animals. Data are expressed as mean ± SEM.

Figure 5

(A) Levels of 6-keto-PGF_1α in the incubation medium after completion of the phenylephrine concentration–response curve in MRA from WKY rats and SHR untreated and treated with pioglitazone. (B) Representative record of the biphasic response elicited by iloprost (1 µM) in MRA precontracted with phenylephrine (Phe). (C) Contractile phase of the response to iloprost in MRA from WKY rats and SHR untreated or treated with pioglitazone and effect of 1 µM SQ 29,548, 1 µM RO 1138452 and 10 µM SC 19220 on the contraction induced by iloprost in pioglitazone-treated SHR. (D) Vasodilator phase of the response induced by iloprost in MRA from WKY rats and SHR untreated or treated with pioglitazone and effect of SQ 29,548, RO 1138452 and SC 19220 on the relaxation induced by iloprost in pioglitazone-treated SHR. (E) Quantitative RT-PCR assessment of TP and IP receptor mRNA levels in arteries from WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus WKY rats or versus control, ^#P < 0.05 versus SHR untreated rats by Student's t-test or by two-way anova and Bonferroni post-test. n= 5–7 animals. Data are expressed as mean ± SEM.

Figure 6

(A) Effect of 0.3 mM apocynin and 0.3 mM allopurinol on the concentration–response curve to phenylephrine (Phe) in MRA from WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus control by two-way anova and Bonferroni post-test. n= 7–12 animals. (B) Representative fluorescent photomicrographs of confocal microscopic sections of MRA from WKY rats and SHR untreated and treated with pioglitazone. Vessels were labelled with the oxidative dye dihydroethidium. Image size 375 × 375 µm. (C) Quantitative RT-PCR assessment of vascular NOX-1 mRNA levels and (D) plasma MDA levels in WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus WKY rats, ^#P < 0.05 versus SHR untreated rats by Student's t-test. n= 5–9 animals. Data are expressed as mean ± SEM.

Figure 7

(A) Representative Western blot and densitometric analysis for Cu/Zn-, Mn- and EC-SOD protein expression and (B) quantitative RT-PCR assessment of catalase mRNA levels in mesenteric arteries from WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus WKY rats, ^#P < 0.05 versus SHR untreated rats by Student's t-test. n= 5–6 animals. Data are expressed as mean ± SEM.

Figure 8

(A) Effect of 0.1 mM L-NAME on the concentration–response curve to phenylephrine (Phe) in WKY rats and SHR untreated and treated with pioglitazone. *P < 0.05 versus control by two-way anova and Bonferroni post-test. n= 6–7 animals. (B) Representative Western blot and densitometric analysis for eNOS protein expression in mesenteric arteries from SHR untreated and treated with pioglitazone. n= 5–6 animals. Data are expressed as mean ± SEM.