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Acute effect of rosiglitazone on relaxation responses in hypercholesterolemic corpus cavernosum

Abstract

Thiazolidinediones (TZDs) improve vascular endothelial dysfunction through non-genomic effects of peroxisomal proliferator-activated receptor γ. This study investigated the acute effect of one of the TZD, rosiglitazone, on endothelium-dependent relaxation response of corpus cavernosum (CC) in hypercholesterolemic rabbits. New Zealand rabbits were divided into two groups randomly as control and cholesterol groups. Hypercholesterolemia was induced by feeding rabbits with 2% cholesterol diet (w/w) for 6 weeks. Endothelium-dependent and -independent relaxation response of CC were evaluated in the presence of rosiglitazone by organ bath studies with cumulative doses of acetylcholine (Ach) and sodium nitroprusside (SNP). Maximal relaxation (Emax) response to Ach significantly decreased owing to hypercholesterolemia in CC tissues. However, in vitro incubation of rosiglitazone with different concentrations (0.1, 1 and 10 μm) did not improve the Ach-dependent Emax responses in hypercholesterolemic rabbit CC. Surprisingly, rosiglitazone caused a significant decrease in Ach-dependent relaxation in healthy CC. Emax responses to SNP did not differ in the presence of rosiglitazone in both the control and hypercholesterolemic groups. Rosiglitazone does not improve hypercholesterolemia-induced endothelial dysfunction in CC tissues while it dose-dependently impairs endothelium-dependent relaxation in healthy CC tissue.

Introduction

Erectile function is a hemodynamic process that is dependent on corpus cavernosum (CC) and related vascular smooth muscle relaxation primarily mediated by nitric oxide (NO) pathway. Alterations in the bioactivity/bioavailability of NO pathway cause endothelial dysfunction, which has a pivotal role in the development of erectile dysfunction (ED).1 Although the pathophysiology of ED is multifactorial, it mostly depends on penile vascular dysfunction.2 Hypercholesterolemia is a worldwide problem and has been identified as one of the major risk factor for the development of ED.3 Hypercholesterolemia causes an increase in oxidative stress and impaired NO bioavailability within the penis.4 In recent studies, peroxisomal proliferator-activated receptor (PPAR) agonists have been investigated in the treatment of hypercholesterolemia-induced endothelial dysfunction and atherosclerosis.5, 6

PPARs are nuclear receptors that act as ligand-activated transcription factors. PPAR γ activation improves insulin sensitivity in the skeletal muscle and liver and reduces hyperglycemia.7 PPAR γ receptors are activated by synthetic ligands as thiazolidinediones (TZDs). TZDs are mainly used as insulin-sensitizing drugs in patients with type 2 diabetes mellitus. It has been shown that TZDs not only improve insulin resistance but also improve endothelial dysfunction independent of their metabolic action.8 The effects of long-term oral TZD treatments on ED were investigated in clinical and experimental studies.9, 10 However, the acute effect of TZDs on endothelium-dependent relaxation via releasing NO in hypercholesterolemic CC tissue has not been studied. In order to investigate acute effect of TZDs on hypercholesterolemic CC tissues, we investigated the endothelium-dependent relaxation of CC tissues in hypercholesterolemic rabbits in the presence/absence of one of the PPAR γ agonist, rosiglitazone.

Materials and methods

Animals

This study was approved by the Dokuz Eylul University Medical School Animal Care and Investigational Committee (51/2009) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Adult New Zealand male rabbits weighing 2.5–3 kg were housed identically in individual cages. Rabbits were chosen randomly and divided into two groups as control and cholesterol groups and each group included n=5 animals. Hypercholesterolemia was induced by feeding rabbits with 2% cholesterol diet (CD) (w/w) for 6 weeks.11 No animal was excluded from the study during the feeding period. No one was blinded in the study.

Total cholesterol measurement

Blood samples were collected from ear vein to measure total cholesterol levels in all animals. Measurements were performed by enzymatic assays and expressed in mg per 100 ml.

In vitro organ bath studies

The animals were anesthetized with 60 mg kg–1 thiopental and killed by exsanguination from the common carotid artery in the seventh week of the experiment. Penis was rapidly removed and placed in modified Krebs–Henseleit solution that consisted of (mM) NaCl 136.9, KCl 2.7, KH2PO4 0.5, CaCl2 1.8, MgSO4 0.6, NaHCO3 11.9 and glucose 11.5; pH: 7.4. Tunica albuginea and corpus spongiosum were cleaned off and CC were prepared as strips for organ bath studies. Four-to-five cavernosal strips of approximately equal size (3 × 3 × 4 mm3) were obtained from each penis. CC strips were suspended in organ baths containing 10 ml of Krebs–Henseleit solution under 1.5 g tension. The solution was kept at 37 °C and continuously gassed with 95% O2 and 5% CO2 at pH 7.35. Tissues were washed with Krebs solution every 15 min during 90 min.

After equilibration period, according to our protocol cumulative doses of acetylcholine (Ach) 0.01–10 μM were applied to the tissues precontracted with 3 μm phenylephrine (Phe) in order to evaluate endothelium-dependent relaxation responses in both the control and hypercholesterolemic groups. Endothelium-independent relaxation responses were also evaluated with cumulative doses of sodium nitroprusside (SNP) at 0.001–1 μM in both groups. In order to evaluate the effect of rosiglitazone on endothelium-dependent relaxation responses, the dose–response curve to Ach was carried out in the absence or presence of rosiglitazone,12 which was added to the organ bath 30 min prior to contraction with 3 μM Phe in both groups. Isometric tensions were recorded with an amplifier system (MP30 Biopac systems, Santa Barbara, CA, USA) on a computer by using the BIOPAC computer program. All data were expressed as mean±s.e.m. Relaxation responses to Ach and SNP were calculated according to the percentage of Phe contraction.

Chemicals

The following chemicals were used: Cholesterol, Ach, Phe, and SNP were purchased from Sigma Aldrich (St Louis, MO, USA) and rosiglitazone was obtained from Cayman Germany. Phe and Ach were dissolved in distilled water. Rosiglitazone was dissolved in dimethyl sulfoxide (DMSO). All working solutions were prepared fresh from stocks.

Statistical analysis

All data are expressed as mean±s.e.m. Relaxation responses to Ach and SNP were calculated according to the percentage of Phe contraction. The normality distribution of the data was determined by using Kolmogorov–Smirnov test. Statistical analysis was performed by one-way analysis of variance followed by Tukey–Kramer post-hoc test and Student’s t test (Graphpad, San Diego, CA, USA). P-values of <0.05 were considered to be statistically significant.

Results

Animal weights and serum total cholesterol measurements

Total cholesterol levels were significantly increased after 6 weeks of feeding with 2% CD (Table 1, P<0.05). There were no significant differences in body weights between the beginning and at the end of the experimental procedure in each group (data not shown).

Table 1 Serum cholesterol levels

Effects of CD on endothelium-dependent and -independent relaxation responses in CC tissues

To investigate the influence of hypercholesterolemia on endothelium-dependent relaxation response of CC, ACh-induced relaxation responses were compared between hypercholesterolemic and control rabbits. Maximal relaxation (Emax) of Ach was significantly attenuated in the CC of hypercholesterolemic rabbits when compared with control rabbits (35.40±4.9%, 57.32±6.4, P<0.05, respectively; Figure 1). SNP-induced relaxation responses in the CC of hypercholesterolemic rabbits were similar to that in the CC from control rabbits (Figure 2).

Figure 1
figure1

Endothelium-dependent relaxation responses of corpus cavernosum in the hypercholesterolemic (HC) and control groups. Dose-dependent relaxation responses of acetylcholine (Ach). Data are expressed as mean±s.e.m. Student's t-test was used in comparison of two groups as the control and HC groups (n=7 strips). *P<0.05 vs control group. Phe, phenylephrine.

Figure 2
figure2

Endothelium-independent relaxation responses of corpus cavernosum in the hypercholesterolemic (HC) and control groups. Dose-dependent relaxation responses of sodium nitroprusside. Data are expressed as mean±s.e.m. Student's t-test was used in comparison of two groups. Control group vs HC group (n=8–10 strips), P>0.05. Ach, acetylcholine; Phe, phenylephrine.

Effect of rosiglitazone on endothelium-dependent relaxation responses of CC tissue in the control and hypercholesterolemic groups

In vitro incubation of rosiglitazone with different concentrations (0.1, 1 and 10 μM) did not improve Emax responses of Ach in CC of hypercholesterolemic rabbits when compared with dose–response curves of Ach in the presence of DMSO (Figure 3). The final DMSO concentration in the bath was <0.01% and Emax in response to Ach alone and in the presence of DMSO was not significantly different in both groups (data not shown).

Figure 3
figure3

Endothelium-dependent relaxations in the presence of rosiglitazone (Ros) in hypercholesterolemic corpus cavernosum (CC) tissues. Concentration response curves of acetylcholine (Ach) on CC tissues obtained from hypercholesterolemic rabbits in the presence of varying concentrations of Ros and vehicle dimethyl sulfoxide (DMSO; 0.01%) (n=7–9). Data are expressed as mean±s.e.m. Statistical analysis was performed by one-way analysis of variance followed by Tukey–Kramer post-hoc test, P>0.05. Phe, phenylephrine.

In vitro incubation with rosiglitazone significantly decreased Emax responses to Ach at 0.1, 1 and 10 μM concentrations in CC tissue of control when compared with Emax response of Ach in the presence of DMSO (Figure 4).

Figure 4
figure4

Endothelium-dependent relaxation responses in the presence of rosiglitazone (Ros) in healthy corpus cavernosum (CC) tissues. Concentration response curves of acetylcholine (Ach) on CC tissues obtained from untreated age-matched rabbits in the presence of varying concentrations of Ros and vehicle dimethyl sulfoxide (DMSO; 0.01%) (n=7–12 strips). Data are expressed as mean±s.e.m. Statistical analysis was performed by one-way analysis of variance followed by Tukey–Kramer post-hoc test. *P<0.05, compared with vehicle. Phe, phenylephrine.

Effect of rosiglitazone on endothelium-independent relaxation response of CC tissues in the control and hypercholesterolemic groups

Incubation of CC tissues with 10 μM rosiglitazone did not alter Emax responses to SNP when compared with Emax responses of SNP in the presence of DMSO in both the groups (Table 2). There were no significant differences between Emax values in response to SNP alone and in the presence of DMSO in both the groups (data not shown).

Table 2 Emax values of SNP in the hypercholesterolemic and control groups

Discussion

The present study was designed to evaluate the acute effect of rosiglitazone on endothelium-dependent relaxation responses in a rabbit model of hypercholesterolemia with vascular dysfunction. Our data showed that rosiglitazone did not improve endothelium-dependent relaxation in hypercholesterolemic rabbit CC. Interestingly, rosiglitazone impaired endothelium-dependent relaxation in a dose-dependent manner in healthy CC. However, rosiglitazone had no effect on endothelium-independent relaxation responses in both the groups.

Hypercholesterolemia has been shown to cause ED through dysregulation of NO pathway.13 In the present study, we have shown a reduction in NO-dependent endothelium relaxation responses with 2% CD feeding for 6 weeks, which were consistent with the literature and our previous study.11, 14, 15 In recent studies, therapeutic interventions that are capable of preserving the NO pathway and improving endothelial function are extensively studied in the treatment of ED.16

PPARs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. PPAR γ is expressed in adipose tissue, pancreatic beta cells, vascular endothelium, macrophages and CC,17, 18 which are thought to be involved not only in diabetes but also in obesity, atherosclerosis and cancer.19 In addition, effects of long-term oral treatment of TZDs on aging, nerve injury and diabetes mellitus-induced ED have been investigated.20, 21, 22 However, there is no research investigating a rapid effect of single-dose treatment with TZDs on CC tissues. In long-term therapies, it has been shown that pioglitazone and rosiglitazone had protective effects on the corporal smooth muscle by inhibition of Rho-kinase, activation of NO–mediated pathway and ameliorating fibrosis and oxidative stress.10, 18, 20, 21

Long-term oral treatment with TZDs have been reported to restore vascular endothelial dysfunction in different types of pathological conditions, such as hypertension, hypercholesterolemia, metabolic syndrome hyperhomocysteinemia and diabetes mellitus.23, 24, 25, 26, 27 There are also studies indicating that acute administrations of TZDs improve vascular endothelial dysfunction through different mechanisms at higher concentrations through non-genomic effects of PPAR γ. Majithiya et al.27 reported that at higher concentrations (>1 μM) pioglitazone incubation increased NO-dependent relaxation responses in both healthy and diabetic rat aortas. Similar to this study, incubation with 10 μM rosiglitazone improves endothelial dysfunction in hypertensive mice carotid artery and cumulative addition of rosiglitazone caused a relaxation at higher concentration (>10 μM) with a different mechanism from PPAR γ activation and NO release.23 Moreover, preincubation of aortic rings with rosiglitazone (0.1, 0.3, 1 μM) reversed NO-dependent relaxation in a dose-dependent manner by suppressing oxidative stress in homocysteine thiolactone-induced endothelial dysfunction.26 Mendizabal et al.12 reported that incubation with pioglitazone or rosiglitazone (10 μM) improved endothelium-dependent relaxations to Ach and the endothelial modulation of Phe contractions by reducing the production of vasoconstrictor prostanoids from endothelial cells in spontaneous hypertensive rats. Interestingly Llorens et al.28 reported that rosiglitazone and pioglitazone have a dual action on the endothelium through increasing both production of vasoconstrictor prostanoids and NO in spontaneous hypertensive rats.

In the current study, we evaluated acute effect of rosiglitazone on CC tissues of hypercholesterolemic rabbits. Different from vascular function studies, dose-dependently incubation of CC tissues with rosiglitazone did not cause an improvement in endothelium-dependent relaxation in hypercholesterolemic rabbits. We evaluated 0.1, 1 and 10 μM concentrations of rosiglitazone on endothelium-dependent relaxation responses. Although acute effects of rosiglitazone were observed at a 10-μM concentration in other studies, rosiglitazone had no effect at these concentrations in the present study. Furthermore, an interesting finding of our study was that 10 μM concentration of rosiglitazone incubation had an inhibitory effect on NO-dependent relaxation responses in healthy CC. On the other hand, 10 μM rosiglitazone had no effect on SNP-induced relaxation in both hypercholesterolemic and control rabbit CC tissues similar to other studies.12, 23, 27 We may speculate that acute effects of rosiglitazone may be tissue specific. At lower concentrations, rosiglitazone may increase the release of endothelium-dependent vasoconstrictors or decrease the release of NO and lead to a functional imbalance in CC.

In conclusion, rosiglitazone does not improve hypercholesterolemia-induced endothelial dysfunction in CC tissues; contrarily, it dose-dependently impairs endothelium-dependent relaxation in healthy CC tissue. Additional studies are needed to identify the underlying mechanism of rosiglitazone and other TZDs.

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Akdag, H., Murat, N., Evcim, S. et al. Acute effect of rosiglitazone on relaxation responses in hypercholesterolemic corpus cavernosum. Int J Impot Res 28, 110–113 (2016). https://doi.org/10.1038/ijir.2016.11

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