Propylthiouracil, independent of its antithyroid effect ... - Atherosclerosis

Atherosclerosis 196 (2008) 383–390

Propylthiouracil, independent of its antithyroid effect, produces endothelium-dependent vasodilatation through induction of nitric oxide bioactivity Wei-Jan Chen a,∗,1 , Wan-Jing Ho a,1 , Gwo-Jyh Chang c , Szu-Tah Chen b , Jong-Hwei S. Pang c , Shih-Hsuan Chou d , Pei-Kwei Tsay e , Chi-Tai Kuo a a

e

First Cardiovascular Division, Department of Cardiology, Chang Gung Memorial Hospital, Fu-Shin Road no. 5, Kwei-Shan, Tao-Yuan 333, Taiwan b Department of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan c Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Tao-Yuan, Taiwan d Department of Physiology, Chang Gung University College of Medicine, Tao-Yuan, Taiwan Department of Public Health & Center of Biostatistics, Chang Gung University College of Medicine, Tao-Yuan, Taiwan Received 14 April 2006; received in revised form 14 July 2006; accepted 12 November 2006 Available online 18 December 2006

Abstract Objective: Propylthiouracil (PTU), independent of its antithyroid effect, is recently found to have a potent antiatherosclerotic effect. The aim of this study is to investigate whether PTU has a beneficial effect on endothelial function. Methods and results: Ninety patients with a history of hyperthyroidism receiving either PTU (n = 45) or methimazole (MMI) (n = 45) during the euthyroid status were enrolled in this study. Brachial artery endothelium-dependent (flow-mediated dilatation [FMD]) and endotheliumindependent (nitroglycerin-mediated dilatation) responses were assessed by high-resolution ultrasound image. Data for these two groups were compared with those of 41 healthy control subjects. The FMD values were significantly increased in patients maintained on PTU versus those in the MMI and control groups (9.3 ± 4.4%, 3.4 ± 2.5%, and 3.6 ± 3.4%, respectively; P < 0.01). Nitroglycerin-mediated dilatation had no significant difference between the PTU, MMI, and control groups (17.4 ± 7.5%, 15.9 ± 6.1%, and 17.5 ± 6.8%, respectively; P = 0.455). On multivariate analysis, no significant relationship was found between the FMD and thyroid hormone index levels. To further elucidate whether PTU has a direct effect on endothelial function, the effect of PTU on isolated segments of Sprague–Dawley rat aorta was studied. Vasodilatation induced by PTU was endothelium-dependent and could be blocked by pretreatment with nitric oxide (NO) inhibitors. PTU also increased NO formation in aortic segments. Conclusions: This study demonstrated that PTU produced endothelium-dependent vasodilatation through thyroid-independent and NOmediated mechanisms that may contribute to its beneficial effect on atherosclerosis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Endothelial function; Flow-mediated dilatation; Nitric oxide; Propylthiouracil

Endothelial dysfunction is an initial step in the atherosclerotic process and has predictive value for future cardiovascular events [1–3]. The pathogenesis of endothelial dysfunction is generally accepted as a decrease in the bioactivity of endothelium-derived nitric oxide (NO) [1–3]. Endothelium∗ 1

Corresponding author. Tel.: +886 3 3281200; fax: +886 3 3271192. E-mail address: [email protected] (W.-J. Chen). The first two authors contribute to this work equally.

0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.11.018

derived NO, synthesized from l-arginine by endothelial nitric oxide synthase (eNOS), stimulates guanylyl cyclase in vascular smooth muscles cells leading to vasodilatation [4]. NO is not only involved in regulating vascular tone via its vasodilatation effect but also in modulating vascular permeability, platelet adhesion/aggregation, leukocyte-endothelium adhesion, and smooth muscle cells proliferation, all early events in atherosclerotic cardiovascular diseases [5]. Ultrasonic assessment by flow-mediated dilatation (FMD) in the

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W.-J. Chen et al. / Atherosclerosis 196 (2008) 383–390

brachial arteries has emerged as a well-established technique to evaluate endothelium-dependent function [6]. Endothelial dysfunction is considered as a systemic disease process; therefore, endothelium-dependent function detected in the peripheral arteries may reflect systemic endothelial condition and risk for developing atherosclerosis [7]. Propylthiouracil (PTU), having its antithyroid effect by inhibiting iodide oxidation, monoiodotyrosine iodination and coupling steps in thyroxine production, as well as the peripheral conversion of thyroxine (T4 ) to triiodothyronine (T3 ) [8], is widely used in treating hyperthyroidism patients. Beyond the antithyroid effect, our recent study found that PTU, independent of its antithyroid effect, may inhibit the development of atherosclerosis in the aortas of rabbits fed a high cholesterol diet [9]. Because endothelial dysfunction plays a critical role in promoting atherosclerosis, we hypothesize that PTU may have beneficial effects on endothelial function. However, studies have demonstrated that endothelial function may also be influenced by thyroid status [10–14]. The present study is, therefore, designed to examine whether PTU affects endothelium-dependent function, and if so, whether this effect is associated with its effect on thyroid function. Specifically, this study sought to characterize endotheliumdependent function by measuring FMD in patients with euthyroid status who were taking PTU. Furthermore, to totally exclude the antithyroid effect of PTU, the second aim of this study was to determine whether acute treatment with PTU can induce endothelium-dependent vasodilatation and affect vascular NO production in isolated rat aorta.

1. Materials and methods 1.1. Study subjects A consecutive 90 patients with hyperthyroidism, receiving either PTU (n = 45) or methimazole (MMI) (n = 45) antithyroid agents for at least 6 weeks, were referred for evaluating endothelial function. All of these patients had to be in the euthyroid status during this procedure. Their duration of antithyroid therapy was 2.4 ± 0.8 months in the PTU group and 2.5 ± 1.3 months in the MMI group (range 1.5–8 months). Patients who had concomitant diseases such as hypertension, hypercholesterolemia (>200 mg/dL), diabetes mellitus, congestive heart failure, and renal insufficiency with the possibility of affecting endothelial function were excluded from this study. Data for the two groups were compared with those of 41 control subjects who underwent this procedure during routine health examination. The protocols were approved by the local research ethics committees. Each patient provided written informed consent. 1.2. Non-invasive assessment of endothelial function Non-invasive assessment of endothelium-dependent flowmediated dilatation and endothelium-independent vasodi-

latation with nitroglycerin was performed using a well-established and validated technique [6]. Briefly, all measurements were detected by a high-resolution Acuson ultrasound system and a 7.0 MHz linear array transducer (Acuson, Mountain View, CA, USA). Patients underwent the procedure after an overnight fasting. Studies were performed in a quiet and deep-light setting. The room temperature was maintained at 23–25 ◦ C. Patients were studied in a supine position with EKG monitoring. Blood pressure was taken on the right arm after resting for 10 min. The ultrasonic transducer was placed at 3–5 cm proximal to the antecubital fossa in the left arm. After a straight segment of the brachial artery was visualized, baseline brachial artery diameter and blood flow (by pulsed-Doppler signal) were determined. Reactive hyperemia was then induced by inflating the blood pressure cuff to 200–250 mmHg for 5 min at the forearm distal to the scanning site. Maximal peak flow was recorded within 15 s after cuff release. Reactive hyperemia was calculated as the ratio of maximal flow after cuff release to that at baseline. Brachial artery diameter was also measured from 45 to 60 s after cuff release. A second resting scan was recorded 15 min later and followed by administration of 400 ␮g sublingual glycerol trinitrate (GTN) spray. The final scan was acquired 4 min later. Arterial diameter was measured from the anterior to the posterior “M” line (the interface between media and adventitia) at the end-diastole using an R-wave trigger. Four cardiac cycles were analyzed and measurements were averaged. Images under each condition were stored for subsequent blinded measurements. All measurements were performed by the same observer who was unaware of any clinical details. The extent of FMD and the GTN-induced dilatation was expressed as the percentage of diameter increase from that at baseline. 1.3. Reproducibility We assessed reproducibility of our imaging study in 30 subjects. Intraobserver and interobserver variability for measuring brachial arterial diameter were compared by two baseline arterial diameter determinations. Correlation coefficients were 0.996 and 0.994, respectively. Average arterial diameters were 3.65 ± 0.65 and 3.64 ± 0.65 mm. Mean differences between the measurements were 0.04 ± 0.05 and 0.06 ± 0.06 mm (1.17 ± 1.40% and 1.60 ± 1.67% of the vessel diameter). 1.4. Organ chamber study All procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital. Male Sprague–Dawley rats weighting 250–300 g from the National Laboratory Animal Center were anesthetized with i.p. injection of 50 mg/kg sodium pentobarbital and sacrificed. The thoracic aorta was immediately isolated and placed into cold Krebs’ solution with the following composition


(mmol/L): NaCl 118.2, KCl 4.7, MgSO4 1.2, NaHCO3 25, KH2 PO4 1.2, CaCl2 1.9, and dextrose 11.7. The aorta was cut into rings about 5 mm in length. Each ring was suspended in an organ bath containing 5 mL Krebs’ solution and constantly gassed with 95% O2 + 5% CO2 at 37 ± 0.5 ◦ C. Two parallel stainless hooks were inserted into the aortic lumen; one was fixed in the organ bath and the other was connected to a force transducer. Contractions were recorded isometrically via a force–displacement transducer (FORT 10, WPI Co., Sarasota, FL, USA) connected to a Power Lab/4sp recorder fitted with a bridge amplifier (AD Instruments, Castle Hill, Australia). For creating a denuded aorta, the endothelium was removed by rubbing the intimal surface with a cotton stick and the absence of acetylcholine-induced dilatation was taken as an indicator of successful denudation. Vessels were precontracted with phenylephrine (PE), and data were expressed as a percentage of reduction in contraction obtained with PE. 1.5. NO determination Production of NO was measured from the organ bath following treatment with the indicated compounds. Acetylcholine was used as a positive control. Samples (100 ␮L) from the organ bath were collected within 1 min after the vessels completed dilatation in tracing, and stored in −80 ◦ C. Samples (5 ␮L) were then injected into a nitrogen purge chamber containing 0.8% VCl3 in 1 mol/L HCl. All NO metabolites were liberated as gaseous NO and reacted with ozone to form activated nitrogen dioxide that is luminescent in red and infrared spectra. The chemiluminescence was detected using a Nitric Oxide Analyzer (NOA 280i, Sievers Instruments, Boulder, CO, USA). Levels of NO were calculated from the area under the curve using NaNO3 as a standard, expressed as ␮mol/L. 1.6. Biochemical measurement Thyroid hormone index (T3 , T4 , and thyrotropin [TSH]) was obtained using the Automated Chemiluminescence System (Centaur, Bayer). Serum PTU levels were measured by high-performance liquid chromatography as described previously [9]. 1.7. Chemicals All chemicals were purchased from Sigma (St. Louis, MO). 1H-[1,2,4]oxadiazolo-[4,3,-␣]quinoxalin-1-one (ODQ) and PTU were dissolved in dimethysulphoxide (DMSO). Final concentration of DMSO in the organ bath was less than 0.1%, which had no effect on vascular reactivity. 1.8. Statistical analysis For multiple comparisons, one-way ANOVA followed by Tukey’s multiple comparisons test was used to compare continuous variables between groups. Differences between

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two groups were compared by Student’s t-test for continuous variables and Chi-square test for categorical variables. Concentration–response curves of PTU-induced vasodilatation in isolated rat aorta were analyzed by non-linear curve fitting using Sigmaplot software (Version 9.0, Systat Software Inc., Point Richmond, CA, USA). Stepwise multiple linear regression models were used to identify individual and joint factors affecting FMD values in the PTU and MMI groups. Correlations between variables were calculated by Pearson’s coefficient. A P-value less than 0.05 was considered statistically significant.

2. Results 2.1. PTU increases FMD in patients taking PTU As shown in Table 1, 90 patients receiving either PTU or MMI were enrolled in this study with 45 patients in each group and 41 subjects in the control group. This study showed no significant difference in age, sex, body height, body weight, body mass index, heart rate, systolic blood pressure, fasting serum glucose, and triglyceride levels between the PTU, MMI, and control groups. Euthyroid status of all patients taking PTU and MMI was confirmed by normal T3 and T4 levels, although their levels were still slightly higher than those in the control group. Because it may take more than 1 year to return the TSH level to its normal range in treating hyperthyroidism patients with antithyroid agents [15], most patients in the PTU and MMI groups had low TSH levels during the procedure. However, the difference in TSH levels between the PTU and MMI groups did not reach statistical significance (P = 0.236). There was no difference in the pretreatment T3 and T4 levels between the PTU and MMI groups. Patients receiving PTU had higher alanine aminotransferase (ALT) levels than those in the MMI group, consistent with a deleterious effect of PTU on liver function [8]. The proportion of patients who used ␤-blockade did not differ between the PTU and MMI groups. However, more patients in the MMI group received thyroxine supplements. Cholesterol levels and diastolic blood pressure were lower in the PTU and MMI groups than in the control group, possibly due to the residual effects of hyperthyroidism [15]. Vessel size of the left brachial artery and reactive hyperemia was not significantly different between the PTU, MMI, and control groups. Vessel size increased during the hyperemic stage and differed significantly between the PTU and MMI treatment groups. Accordingly, FMD levels in the PTU group were significantly higher than those in the MMI and control group (9.3 ± 4.4%, 3.4 ± 2.5%, and 3.6 ± 3.4%, respectively; P < 0.01). However, the response to GTN spray (nitroglycerin-mediated dilatation [NMD]) was not significantly different between these three groups (17.4 ± 7.5%, 15.9 ± 6.1%, and 17.5 ± 6.8%, respectively; P = 0.455). Fig. 1 shows the differences for FMD and NMD values between the PTU, MMI, and control groups.

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Table 1 Clinical parameters and vascular characteristics in the PTU, MMI, and control groups PTU

MMI

Control

P-value

Number Age (years) Sex (female) Body height (cm) Body weight (kg) Body mass index (kg/m2 ) Smoke Heart rate (beat/min) Atrial fibrillation Systolic pressure (mmHg) Diastolic pressure (mmHg) ALT (U/L) Glucose (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/mL)

45 39.2 ± 10.8 82.2% 158.6 ± 8.9 57.9 ± 10.9 22.9 ± 3.1 8.9% 75 ± 13 6.7% 112 ± 12 67 ± 10* 17.9 ± 5.8* 87.6 ± 12.9 155.1 ± 29.9* 87.9 ± 33.4

45 40.2 ± 12.1 88.9% 157 ± 6 56.7 ± 8.7 22.9 ± 2.9 8.9% 72 ± 15 4.4% 115 ± 13 70 ± 10† 10.4 ± 5.8† 86.2 ± 15.2 158.5 ± 26.4* 90.7 ± 27.6

41 40.7 ± 12.1 87.8% 157.7 ± 7.5 58.7 ± 9.2 23.6 ± 3.5 2.4% 74 ± 9 2.4% 116 ± 15 75 ± 10# 17.1 ± 7.3* 86 ± 8 169.5 ± 22.4† 89.6 ± 24.9

0.826 0.618 0.624 0.603 0.521 0.400 0.538 0.644 0.297 0.004