Peroxisome proliferator-activated receptors (PPAR) are a family of 3 nuclear hormone receptors, α, β/δ, and γ, that are members of the steriod receptor superfamily.1 Although PPAR-α is stimulated by natural ligands, such as fatty acids and eicosanoids, and by synthetic ligands, such as the lipid-lowering fibrates, activators of PPAR-γ are the insulin sensitizers known as thiazolidinediones or glitazones, including troglitazone, pioglitazone, and rosiglitazone.2
Until recently, the function of these receptors has concerned their ability to regulate energy balance.2 In addition, expression of PPAR-α and -γ has been described in the cardiovascular system.3,4 In vascular smooth muscle and endothelial cells, the activation of these nuclear receptors can suppress the production of proinflammatory cytokines and inhibit proliferation and cellular migration. Therefore, their activation seems to be associated with an antiinflammatory and antiatherosclerotic role.5,6 However, the contribution of these compounds in modulating vascular function remains unclear, as studies describe beneficial,7,8 innocuous,9 and prejudicial10 effects of PPAR agonists in the cardiovascular system.
Proinflammatory cytokines have been related to an upregulation of adhesion molecules on endothelial cells, activation of macrophages, smooth muscle cell proliferation, and vascular dysfunction.11-13 Moreover, in cardiovascular diseases such as hypertension, atherosclerosis, and heart failure, studies demonstrate an increased production of proinflammatory cytokines in blood vessels, which could result in vascular damage.14,15
The prolonged stimulation of β-adrenergic receptors with isoproterenol, a non-selective β-adrenergic agonist, is a well-known experimental model of heart “injury” in rats.16,17 In addition, Davel et al18 describe an increased vasoconstrictor response to phenylephrine and serotonin in the aorta of isoproterenol-treated rats. This change in vascular reactivity was related to altered endothelial modulation of the response to phenylephrine, mostly due to increased superoxide anion generation and reduced bioavailability of nitric oxide. In addition, we recently demonstrated that isoproterenol-treatment for 7 days enhanced RNA and protein expression of local pro-inflammatory factors in rat aortas.19 Thus, the aim of the present study was to investigate the effects of fenofibrate, a PPAR-α agonist, and pioglitazone, a PPAR-γ agonist, in the increased vasoconstrictor response to the α-adrenergic receptor agonist phenylephrine in aortic rings from 7-day isoproterenol-treated rats.
Three-month-old male Wistar rats (270 to 370 g) were obtained from colonies maintained at the Animal Quarters of the Institute of Biomedical Sciences of the University of Sao Paulo. The animals were housed 4 to 6 per cage at a constant room temperature and light cycle (12:12 hours light:dark). Food and water were allowed ad libitum to all animals. Care and use of laboratory animals and all experiments were conducted in compliance with the guidelines for biomedical research as stated by the Brazilian Society of Experimental Biology.
A group of rats was treated for 7 days with the nonselective β-adrenergic agonist, isoproterenol (0.3 mg·kg−1·day−1, SC, suspended in 0.1 mL of soy oil), and control rats received the same volume of vehicle.18,19
Simultaneously, both control and isoproterenol-treated rats received fenofibrate, PPAR-α agonist, (100 mg·kg−1·day−1, PO)20 or pioglitazone, PPAR-γ agonist, (2.5 mg·kg−1·day−1, PO).21,22 Fenofibrate was suspended in water23 while pioglitazone hydrochloride tablets (Actos, Abbott, Brazil) were transformed into dust and then suspended in carboximethylcelulosis 0.5%.8,9 These suspensions were administered by gavage to the animals. Thus, rats were randomly assigned to one of the following groups: control (CT), isoproterenol (ISO), control + fenofibrate (CTα), isoproterenol + fenofibrate (ISOα), control + pioglitazone (CTγ), and isoproterenol + pioglitazone (ISOγ).
After 7 days, all animals were weighed, anaesthetized, and killed by exsanguination. The thoracic aorta was dissected and divided into rings (4 mm), as described previously.24 To analyze the influence of the endothelium on vascular responses, the endothelial layer was mechanically removed in some experiments by rubbing the lumen with a needle.
The rings were mounted in an isolated organ bath system containing Krebs-Henseleit bicarbonate buffer (KHB). The buffer consisted of (in mM): NaCl 118, KCl 4.7, NaHCO3 25, CaCl2-2H2O 2.5, KH2PO4 1.2, MgSO4-7H2O 1.2, glucose 11, and EDTA 0.01. Thoracic aorta segments were subjected to tension of 1.0 g during a 45-minute equilibration period. Isometric tension was recorded by using an isometric force transducer (Letica TRI 210, Barcelona, Spain) connected to an acquisition system (Soft & Solution, SP, Brazil).
After 45 minutes of stabilization, the viability of vascular smooth muscle was assessed using KCl (75 mM). The maximal contraction of each ring was determined after 30 minutes of exposure to KCl (75 mM). Endothelial integrity was then tested by the response to acetylcholine (ACh, 10 μM) in segments precontracted with phenylephrine at a concentration (~10 μM) producing 50 to 70% of the contraction induced by KCl (75 mM). Concentration-response curves to phenylephrine (10−10 to 10−3 M) were determined in thoracic aortic rings with (E+) and without (E-) endothelium. The endothelium was considered intact if the aortic ring relaxed more than 90% to 10 μM ACh, and endothelial denudation was confirmed by less than 10% relaxation.
To evaluate the influence of fenofibrate or pioglitazone treatments on the role of nitric oxide and superoxide anion in the contractile response to phenylephrine, aortic rings were incubated with either the nonselective nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM) or the superoxide anion scavenger superoxide dismutase (SOD, 150 U/mL). Drugs were added 30 minutes before the concentration-response curve to phenylephrine was determined.
Phenylephrine hydrochloride, (-)-isoproterenol, acetylcholine chloride, fenofibrate, L-NAME dihydrochloride, and SOD (bovine erythrocyte) were purchased from Sigma (St Louis, MO). Pioglitazone was purchased from Abbott (Takeda Chemical Industries, Osaka, Japan; distributed by Abbott, Brazil).
Data were expressed as mean ± SEM and analyzed by Student t test and one-way or two-way ANOVA plus Tukey post-hoc test at P < 0.05 significance level.
The maximal contractile response to phenylephrine (Rmax) was expressed as percentage of the maximal contraction induced by KCl 75 mM. The negative log of the agonist concentrations resulting in 50% of the maximum response (pD2) was calculated from concentration-response curves by nonlinear regression analysis of the curve using computer-based fitting program Graphpad Prism (San Diego, CA).
Isoproterenol, PPAR agonists, or combined treatments did not significantly modify the body weight of the animals (CT: 340 ± 49 g, n = 20; ISO: 311 ± 25 g, n = 26; CTα: 333 ± 36 g, n = 13; ISOα: 294 ± 18 g, n =13; CTγ: 336 ± 43 g, n =13; ISOγ: 327 ± 26 g, n = 14; mean ± SD, P > 0.05, one-way ANOVA).
Vascular Contraction to KCl
Aortas from isoproterenol-treated rats showed reduced maximal contraction to KCl compared to aortas from the control group (CT: 1.82 ± 0.10 g, n = 17 versus ISO: 1.52 ± 0.09 g, n = 19; P < 0.05, t test) as previously described.18 This pattern of response was not modified by treatment with fenofibrate (CTα: 2.10 ± 0.23 g, n = 9 versus ISOα: 1.60 ± 0.11 g, n = 6; P < 0.05, t test) or pioglitazone (CTγ: 1.83 ± 0.10 g, n = 13 versus ISOγ: 1.50 ± 0.07 g, n = 14; P < 0.05, t test).
Endothelium-dependent Relaxation to ACh
To evaluate the endothelium-dependent relaxation response to ACh, aortic rings were precontracted with phenylephrine to a level reaching 50 to 70% of the maximal contraction to KCl (75 mM). As demonstrated in Table 1, the phenylephrine-induced plateau was not significantly different among the groups studied. In addition, there were no changes in ACh (10 μM)-induced relaxation between control and isoproterenol-treated rats (Table 1), as previously demonstrated.18 Fenofibrate or pioglitazone did not modify the ACh-induced relaxation in aortas from either the control or isoproterenol-treated rats (Table 1).
Effects of Fenofibrate and Pioglitazone Treatment in the Vasoconstrictor Response to Phenylephrine in Aortas from Control and Isoproterenol-treated Rats
Aortic rings from isoproterenol-treated rats showed a greater maximal vasoconstrictor response (Rmax) to phenylephrine than the control group (Table 2, Figure 1A) without significant changes in pD2 (Table 2). Neither fenofibrate (Figure 1B) nor pioglitazone (Figure 1C) treatment prevented the increased Rmax to phenylephrine induced by isoproterenol (Table 2).
In control rats, fenofibrate did not modify the Rmax or pD2 to phenylephrine (Table 2). However, pioglitazone per se increased the Rmax to this α-adrenoceptor agonist in aorta from control animals (Table 2). Treatment with PPAR agonists did not alter the pD2 to phenylephrine in any group studied (Table 2).
Effects of Fenofibrate and Pioglitazone on the Endothelial Modulation of the Vasoconstrictor Response to Phenylephrine in Aortas from Control and Isoproterenol-treated Rats
Removal of the endothelium enhanced the Rmax and pD2 to phenylephrine in aortic rings from all groups studied (Figure 2 and Table 2).
In the absence of the endothelium, there were no differences between the control and isoproterenol-treated groups (Table 2, Figures 2A and 2B), indicating that aortas from the isoproterenol-treated group show impaired endothelial modulation of phenylephrine-induced contraction in comparison to controls. Neither fenofibrate (Figure 2D) nor pioglitazone (Figure 2F) was able to reverse this effect of isoproterenol treatment on the endothelial modulation of phenylephrine-induced contraction.
In control animals, fenofibrate treatment did not induce changes in endothelial modulation of the vasoconstrictor response to phenylephrine (Table 2, Figures 2A and 2C). However, pioglitazone impaired the endothelial modulation in comparison to nontreated control animals (Table 2, Figures 2A and 2E).
Involvement of NO and Superoxide Anion in the Vasoconstrictor Response to Phenylephrine in Aortic Rings
When the vascular rings were incubated with L-NAME, it was observed that, in a similar manner to endothelial removal, L-NAME enhanced both pD2 and Rmax to phenylephrine in all groups studied (Table 2 and Figure 3). After L-NAME incubation, there were no differences in phenylephrine-induced contraction between aortas from control and isoproterenol-treated rats (Table 2, Figures 3A and 3B).
Neither fenofibrate nor pioglitazone was able to reverse the reduced effect of L-NAME on the contraction induced by phenylephrine in aortas of isoproterenol-treated animals (Figures 3D and 3F). Fenofibrate treatment did not significantly modify the influence of L-NAME in control animals (Table 2, Figures 3A and 3C). However, pioglitazone per se reduced the effect of L-NAME on phenylephrine-induced contraction (Table 2, Figures 3A and 3E).
SOD incubation had no effect in aortas from control animals (Table 2, Figure 4A), but it significantly reduced the Rmax to phenylephrine in the isoproterenol-treated group (Table 2, Figure 4B). SOD did not modulate the phenylephrine-induced contraction in control rats treated with fenofibrate (Table 2, Figure 4C). However, the increased Rmax to phenylephrine in control animals treated with pioglitazone was completely reversed by SOD (Table 2, Figure 4E). This indicates a pronounced superoxide-dependent effect in the phenylephrine-induced contraction of aortas from pioglitazone-treated animals. In addition, Rmax to phenylephrine in the presence of SOD was similar among ISO, ISOα, and ISOγ, suggesting that neither fenofibrate nor pioglitazone was able to modulate the generation of superoxide anions induced by isoproterenol-treatment of isolated rat aorta (Table 2, Figure 4).
We observed that fenofibrate and pioglitazone, PPAR-α and -γ agonists, respectively, could not reverse the enhanced vasoconstrictor response to the α-adrenoceptor agonist phenylephrine or the NO-superoxide anion imbalance present in aortas from 7-day isoproterenol-treated rats. In addition, pioglitazone treatment per se increased the vascular reactivity to phenylephrine and induced local oxidative stress in control aortas.
In certain cardiovascular diseases, such as essential hypertension and atherosclerosis, endothelial dysfunction is characterized by an increased response to vasoconstrictor stimulus and/or by impaired endothelium-dependent relaxation. This is also associated with an increased production of cytokines and other inflammatory mediators.14,15 As reported by Davel et al18,19 and in the present study, chronic β-adrenergic stimulation with isoproterenol for 7 days induces hyperreactivity to phenylephrine in thoracic aortic rings from rats without changes in the endothelium-dependent relaxation induced by ACh. In the absence of endothelium, contraction to the α-adrenergic agonist was similar between control and isoproterenol-treated groups, indicating that aortic rings from isoproterenol-treated animals exhibit reduced endothelial modulation in the response to phenylephrine. In addition, we have previously demonstrated that the enhanced contraction to phenylephrine and the endothelial dysfunction in isoproterenol-treated animals are associated with an incremented gene and protein expression of IL-1β and IL-6 via activation of the nuclear factor (NF)-κB in rat aortas.19
Previous studies suggest that PPAR-α and PPAR-γ ligands are able to inhibit synthesis of proinflammatory mediators.25-27 This effect of PPAR activation in vascular cells seems to be due, at least partially, to inhibition of NF-κB.28,29 In addition, some studies have shown that PPAR agonists can also prevent endothelial dysfunction.20,30,31
In the present study, we investigated whether treatment with PPAR-α and -γ agonists could ameliorate the impaired endothelial modulation of phenylephrine-induced contraction in aortas of isoproterenol-treated rats. We observed that co-treatment of isoproterenol with fenofibrate or pioglitazone did not reverse the altered vascular reactivity and impaired endothelial modulation observed in animals treated only with isoproterenol. In contrast, some studies have demonstrated a beneficial role for PPAR agonists in experimental models of cardiovascular diseases. Diep et al30 showed that treatment with the PPARα agonist docosahexaenoic acid for 7 days could prevent blood pressure elevation and improve vascular dysfunction in angiotensin II-infused rats. This effect was associated with a reduction of ROS generation and inflammation of the vascular wall. It was also reported by Diep et al32 that treatment with pioglitazone (10 mg·kg−1·day−1) or rosiglitazone (5 mg·kg−1·day−1) for 7 days attenuated the development of hypertension, corrected vascular structural abnormalities, and improved endothelial dysfunction induced by angiotensin II infusion. In the present study, rats were co-treated with isoproterenol and fenofibrate (100 mg·kg−1·day−1) or with pioglitazone (2.5 mg·kg−1·day−1) for 7 days, which is comparable to doses used in previous studies. However, we did not find any beneficial vascular effects of PPAR agonists in this experimental model.
Our results are in contrast to other results obtained for PPAR agonists in the cardiovascular system. This could be explained by differences in the experimental designs of each study, regardless of the presence of metabolic disorders or hypertension. It is possible that different kinds of PPAR ligands at different doses and used for different treatment periods could induce varying effects. Another feature that could explain the differing results is the route of PPAR agonist administration. Here, pioglitazone and fenofibrate were administrated by gavage, as in the study reported by Frantz et al.9 Others investigators7,32 have used a dietary supplement allowing different peak and steady-state concentrations.33
In line with our results, Frantz et al9 showed that pioglitazone treatment (20 mg·kg−1·day−1) was not able to decrease the vasoconstrictor response induced by phenylephrine or alter the impaired relaxation to ACh and sodium nitroprusside in isolated aortas from mice after coronary artery ligation. The authors also demonstrated that pioglitazone treatment did not change mortality, ventricular dilatation, and collagen deposition in infarcted animals. Similar to the myocardial infarction model induced by coronary artery ligation, chronic isoproterenol-treatment is accompanied by pronounced β-adrenergic stimulation without development of hypertension, suggesting similarities in the neurohumoral pathways involved in the mechanisms that induce vascular dysfunction in these models.
The increased vasoconstrictor response of aortic rings from isoproterenol-treated animals compared to controls was associated with reduced NO bioavailability, as incubation with L-NAME abolished the difference between control and isoproterenol-treated rats. Neither fenofibrate nor pioglitazone was able to change the effect of L-NAME in the isoproterenol-treated animals. Together, these data suggest that neither PPAR agonist was able to improve the reduced NO bioavailability induced by chronic isoproterenol treatment.
In contrast to the present results, it has been reported that PPAR-α and -γ agonists can increase NO bioavailability in cultured cells.34-36 In vivo, PPAR-α and -γ agonists have been shown to reduce superoxide generation, restore endothelial dysfunction, and improve vasorelaxation to ACh in aorta of diabetic rats.8,37 However, Blanco-Rivero et al10 describe how, despite increasing NO production, fenofibrate can induce endothelial dysfunction in a time-dependent manner. The authors observed that after 6 weeks of fenofibrate treatment (100 mg·kg−1·day−1), the endothelium-dependent relaxation to ACh were reduced in isolated rat aortic rings. The effects of chronic treatment with PPAR ligands in the cardiovascular system are therefore still controversial.
Incubation of aorta with SOD did not alter contraction to phenylephrine in the control group, but this contraction was found to be reduced in isoproterenol-treated rats. In addition, Davel et al18 demonstrated that these isoproterenol-treated animals show elevated levels of superoxide generation in the aorta. The increased superoxide generation could explain the reduced NO bioavailability because the O2− radical may have several effects either directly or indirectly through the generation of other radicals, such as ONOO−. This can lead to rapid inactivation of NO, leading to endothelial dysfunction.38,39 In the present study, neither fenofibrate nor pioglitazone were able to reverse the superoxide-dependent contraction induced by isoproterenol treatment. In contrast, some studies describe an increase in gene and protein expression for Cu/Zn-SOD and a suppression of NADPH oxidase by PPAR agonists in endothelial cells.40,41 Likewise, rosiglitazone and fenofibrate were able to prevent the vascular increase in superoxide anion production in DOCA-salt animals.7 However, there are some studies demonstrating oxidative and apoptotic effects of glitazones.42,43 In the present study, the results also suggest an oxidative effect of pioglitazone per se in aortas from isoproterenol-treated rats that seems to increase the vasoconstrictor response to phenylephrine. On the other hand, fenofibrate-treatment for 7 days did not significantly modify the vascular reactivity to phenylephrine in aorta from control animals, in line with recent results from Blanco-Rivero et al.10
The PROspective pioglitAzone Clinical Trial In macroVascular Events (PROACTIVE) recently showed that treatment with the PPAR-γ agonist results in a reduction in coronary and stroke events in diabetic and hypertensive patients. However, this benefit was counterbalanced by an increase in congestive heart failure as well as symptomatic edema.44 It is possible that, the beneficial vascular role of PPARs is more prominent in vascular diseases associated with lipid metabolism and energy balance disorders, such as atherosclerosis,45 diabetes mellitus,8,37 and obesity.46,47 In addition, FIELDS study showed nonsignificant alterations in thrombotic events and coronary and sudden deaths in patients treated with fenofibrate.44 A link between rosiglitazone and a significantly increased risk of myocardial infarction has also been demonstrated.48
It is known that many patients have baseline cardiovascular alterations and take fenofibrate and/or pioglitazone as anti-lipidemic and anti-diabetic drugs, respectively. Our findings reinforce the idea that PPAR protective effects cannot be uniformly observed and warn that extrapolation of the protective properties of those compounds to any clinical employment must be done with caution, and further study is required to clarify the effects of PPAR agonists in the cardiovascular system.
In summary, neither fenofibrate nor pioglitazone ameliorates the increased vasoconstrictor response to phenylephrine and the NO/O2− imbalance present in aorta from 7-day isoproterenol-treated rats. Moreover, pioglitazone treatment per se increased the contraction to phenylephrine in aortas, probably related to increased O2− levels associated with reduced NO bioavailability.
We gratefully acknowledge Natasha Hausman for helpful suggestions in the preparation of the article.
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