Endothelial Dysfunction Is Related to Aldosterone Excess and Raised Blood Pressure

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1 Endocrine Journal 29, 56 (), Endothelial Dysfunction Is Related to Aldosterone Excess and Raised Blood Pressure Kyoichiro Tsuchiya, Takanobu Yoshimoto and Yukio Hirata Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan Abstract. Primary aldosteronism (PA) is a secondary hypertension characterized by autonomous aldosterone hypersecretion from adrenocortical adenoma and/or hyperplasia. Recently it has been suggested that aldosterone excess is directly involved in the development of cardiovascular injury in PA independent of its hypertensive effect. The present study was designed to examine the relationship between aldosterone excess and endothelial dysfunction in PA patients. 25 PA patients were studied for vascular endothelial function by ultrasound measurement of flow-mediated vasodilation (FMD), and 1 PA patients were re-evaluated 3 months after surgical or medical treatment; 1 age-, gender-, and blood pressurematched hypertensive patients served as control subjects. Percent (%) FMD in PA patients (.6±2.%) was significantly (p <.1) lower than that in the control subjects (7.9±2.%). %FMD showed significant (p <.5) negative correlations with systolic blood pressure (SBP) (r=-.), brachial-ankle pulse wave velocity (r=-.52), plasma aldosterone concentration (PAC) (r=-.2), and aldosterone-renin ratio (ARR) (r=-.2), while SBP showed a positive correlation with PAC (r=.7). Percent FMD, SBP, PAC, and ARR significantly (p <.5) improved after surgical and medical treatment, although the changes of %FMD did not correlate with those of SBP, PAC or ARR. In conclusion, the present study has demonstrated that PA patients have endothelial dysfunction, which is related to aldosterone excess and raised blood pressure, and reversible after treatment, suggesting that aldosterone excess contributes to the development of endothelial dysfunction due to its hypertensive effect and/or its direct effect on the cardiovascular system. Key words: Primary aldosteronism, Endothelial dysfunction, Flow-mediated vasodilation (Endocrine Journal 56: , 29) Received Jan. 15, 29; Accepted Mar. 6, 29 as K9E-1 Released online in J-STAGE as advance publication Apr. 7, 29 Correspondence to: Kyoichiro Tsuchiya, M.D., Ph.D., Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Yushima, Bunkyo-ku, Tokyo , Japan. ktsuchiya.cme@tmd.ac.jp Declaration of competing interests: none to declare. PRIMARY aldosteronism (PA) is a secondary hypertension characterized by autonomous aldosterone hypersecretion due to adrenocortical adenoma and/or hyperplasia. Recently, accumulating lines of evidence reveal its higher prevalence (about 5-15%) among unselected hypertensive patients than previously thought [1, 2, 3]. Moreover, several clinical studies have shown that PA patients have higher incidence of proteinuria, cerebral hemorrhage, and left ventricular hypertrophy than did age- and sex-matched patients with essential hypertension despite higher blood pressure and longer duration [, 5, 6]. These results suggest that aldosterone is directly involved in the development of cardiovascular injury by the mechanism(s) other than its sodium-water retention and hypertensive effect. However, the mechanism(s) by which excess aldosterone per se directly induces cardiovascular injury remains undetermined yet. Endothelial dysfunction is considered as an initial event in the development of cardiovascular disease [7]. Ultrasound measurement of flow-mediated vasodilation (FMD) of a brachial artery has been widely used as a non-invasive method for evaluating endothelial function based on reactive hyperemia-induced endothelial nitric oxide (NO) release []. It has recently been reported that hypertensive patients with high plasma aldosterone concentrations (PAC) had greater impairment of FMD than those with normal PAC [9]. Furthermore, acute administration of aldosterone has been shown to inhibit acetylcholine-induced vasodila-

2 55 TSUCHIYA et al. tion in healthy humans [1]. However, it remains unknown whether endothelial function is impaired in PA patients, and whether the correction of aldosterone excess could reverse endothelial dysfunction. To address these issues, the present study was designed to investigate the relationship of endothelial function to PAC and other clinical parameters in PA patients before and after correction of aldosterone excess by surgical and medical treatment. Subjects and Methods Study population and patient characteristics This study was approved by the Ethics Committee of our institute, and written informed consent was provided from all subjects before enrollment. We studied 25 PA patients who consulted our clinics. They were treated with calcium channel blockers and/or α1- blockers during endocrinological examination. They were diagnosed as PA based on suppressed plasma renin activity (PRA) ( 1. ng/ml/h) and elevated PAC ( ng/dl), greater PAC to PRA ratio (aldosteronerenin ratio: ARR) ( 2), and suppression of PRA (< 1. ng/ml/h) after stimulation with furosemide-upright. For localization of PA, adrenal CT and/or selective adrenal venous sampling (AVS) were performed in all 25 patients to differentiate between aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). Ten age-, gender- and blood pressure-matched hypertensive control subjects were recruited and studied. The clinical characteristics of PA patients and control subjects are shown in Table 1. Sixteen patients were diagnosed as APA and 9 patients as IHA by the localization studies. There were no significant differences of SBP and DBP between the two groups, since most of the PA patients (n=23) have received antihypertensives for blood pressure control. PAC, ARR, 2-h urinary aldosterone excretion, and bapwv in PA group were significantly (p <.5) higher, and PRA was lower than the control group. There were no significant differences of gender, age, smoking, lipid profile, hemoglobin A1c, fasting plasma glucose, serum potassium, and carotid IMT between the two groups. Six APA patients had unilateral adrenalectomy and four IHA patients were treated with a mineralocorticoid receptor antagonist (spironolactone). Age, gen- der, blood pressure, PAC, PRA, ARR, and %FMD before treatment did not differ among the 25 patients. Measurements of hormonal and vascular functional parameters PRA and PAC were measured by radioimmunoassay kit using Renin-Riabead (Dinabot, Tokyo, Japan) and SPAC-S Aldosterone (TFB, Tokyo, Japan), respectively. Flow-mediated vasodilation (FMD) of brachial artery was measured in all patients and control subjects using Sonos 55 (Philips Medical Systems, Andover, MA, USA) as described [11]. Percent flow-mediated vasodilation (%FMD) and %nitroglycerin (NTG)- mediated vasodilation (%NMD) were expressed as the %increases in the maximal diameter from the baseline after cuff deflation within sec, and 3 min after administration of NTG (.3 mg), respectively. NTG was not administered in 9 PA patients because of their cardiovascular complications. Ten PA patients were reevaluated for FMD and blood pressure 3 months after surgical or medical treatment. Carotid artery intima-media thickness (IMT) and brachial-ankle pulse wave velocity (bapwv) were measured as described previously [11] using ultrasound (Sonos 55) and Form PWV/ABI (Omron Healthcare, Kyoto, Japan.), respectively. Statistical analysis Data are expressed as means ± standard deviation (SD) or median (interquartile range of 25-75%). A log transformation was performed for variables that did not follow a normal distribution. The χ 2 test was used for comparing proportions of subjects in the two groups. Comparison between groups was made using non-paired t test. Linear relationships between two continuous variables were tested by Pearson s correlation coefficient. Difference of values between pre- and post-treatment in PA patients was analyzed using paired t test. All statistical analyses were performed using Windows software Prism 5. (GraphPad Software, La Jolla, CA, USA). Results As shown in Figure 1, %FMD in PA patients (.6±2.%) was significantly (p <.1) lower than

3 ENDOTHELIAL DYSFUNCTION IN PA 555 Table 1. Clinical characteristics of patients with primary aldosteronism (PA) and control subjects. PA Control Number (APA / IHA) 25 (16 / 9) 1 Men / Women 1 / 11 5 / 5 Age (years) 57.3± ±11. Body mass index (kg/m 2 ) 22.± ±1. Systolic blood pressure (mmhg) 136±17 11±2 Diastolic blood pressure (mmhg) 3±1 ±15 Brachial-ankle pulse wave velocity (m/sec) 17.±2.1** 15.3±2. Carotid intima-media thickness (mm).79±.16.77±.15 Low-density lipoprotein cholesterol (mg/dl) 17±27 1±21 High-density lipoprotein cholesterol (mg/dl) 6±2 57±23 Triglyceride (mg/dl) 17±56 115±39 Hemoglobin A1c (%) 5.±.5 5.±.6 Fasting plasma glucose (mg/dl) 11±25 11±1 Insulin (μu/ml) 5.±3. 3.7±1.9 HOMA-R 1.1±. 1.±.7 Serum potassium (meq/l) 3.±.5.1±.3 Plasma aldosterone concentration (ng/dl) 2.1±1.* 1.5±6. Plasma renin activity (ng/ml/h).3 [.1-.5]** 1. [.3-2.9] Aldosterone-renin ratio (ARR) [6-253]*** 9.5 [ ] 2-h urinary aldosterone excretion (μg/day) 13.7±7.7* 7.9±. APA: aldosterone-producing adenoma, IHA: idiopathic hyperaldosteronism, HOMA: Homeostasis model assessment insulin resistance index. Data means ± SD or median [interquartile range].*p <.5, **p <.1 and ***p <.1 vs. control group. A %FMD 15 B %NMD *** 3 1 (%) (%) Control subjects Primary aldosteronism Control subjects Primary aldosteronism Fig. 1. Endothelium-dependent and -independent vasodilation in patients with primary aldosteronism (PA) and control subjects. (A) %FMD and (B) %NMD in ( ) 1 control subjects and 25 PA patients ( : APA, : IHA). ***p <.1.

4 556 TSUCHIYA et al. A r = -. B r = -.52 C r = -.2 %FMD(%) %FMD(%) %FMD(%) SBP (mmhg) bapwv (m/s) 2 6 PAC (ng/dl) D r = -.2 E r =.7 %FMD (%) PAC(ng/mL) Log ARR SBP (mm/hg) Fig. 2. Correlations between endothelium-dependent vasodilation and vascular and hormonal parameters in PA patients. Univariate correlations between %FMD and (A) systolic blood pressure (SBP), (B) brachial-ankle pulse wave velocity (bapwv), (C) plasma aldosterone concentration (PAC), and (D) Log aldosterone-renin ratio (ARR), and (E) between PAC and SBP, are shown. The present study has demonstrated for the first time that PA patients had endothelial dysfunction as assessed by FMD, which was significantly correlated with bapwv, PAC and ARR as well as blood pressure, and reversible after treatment for PA. In the present study, we have employed a non-invasive ultrasound technique for detection of endothelial function in PA patients. Measurement of brachial FMD by ultrasound has been widely accepted as a reliable method for evaluating endothelial function [7]. Previous reports have demonstrated impaired brachial FMD in patients with cardiovascular risk facin control group (7.9±2.%), whereas %NMD was similar between two groups; there was no significant difference of %FMD between APA (.±2.2%) and IHA (.3±1.6%). To evaluate the association between %FMD and vascular and hormonal parameters in PA patients, univariate correlation analysis was performed. As shown in Figure 2, %FMD showed significant (p <.5) negative correlations with SBP (r=-.), bapwv (r=-.52), PAC (r=-.2), and ARR (r=-.2), while SBP significantly (p <.5) correlated with PAC (r=.7). There were no significant correlations between %FMD and serum potassium, PRA, and 2-h urinary aldosterone excretion (data not shown). To elucidate whether correction of aldosterone excess and/or action could reverse endothelial dysfunction, the changes of various hormonal and vascular parameters before and after surgical (n=6) or medical (n=) treatment were examined in 1 PA patients. As shown in Figure 3, SBP, PAC, and ARR significantly (p <.5) decreased, and %FMD significantly (p <.5) increased after the treatment. However, the changes of %FMD did not show significant correlation with those of SBP, PAC, or ARR. Discussion

5 ENDOTHELIAL DYSFUNCTION IN PA 557 A B C D * SBP (mm/hg) 1 ** PAC (ng/dl) 2 *** Log ARR *** %FMD (%) Fig. 3. Changes of vascular and hormonal parameters in PA patients before and after treatment. Changes of (A) SBP, (B) PAC, (C) Log ARR, and (D) %FMD in 1 PA patients ( ) before and ( ) 3 months after surgical (n=6) or medical (n=) treatment are shown. PAC and ARR were re-evaluated in 6 APA patients treated with surgery. Each point with bars indicates mean±sd. *p <.5, **p <.1 and ***p <.1 vs. before treatment.. ent study, suggesting that aldosterone excess may also contribute to the development of endothelial dysfunction in PA independent of raised blood pressure. Our results are in accordance with those of a previous study showing that hypertensive patients with high PAC showed lower %FMD than those with normal PAC [9], although PA was not confirmed in these patients. Another study has also shown that patients with low renin-essential hypertension had impaired methacholine-induced vasodilation [22]. The negative correlations between %FMD and blood pressure, PAC and ARR in our PA patients suggest that endothelial dysfunction was attributable not only to hypertension, but also to aldosterone excess per se in PA patients. The mechanism(s) by which aldosterone impairs endothelium-dependent vasodilation remains unknown. It has been shown that aldosterone directly inhibits NO production by decreasing endothelial NO synthase activity [23], and increases superoxide anion generation in endothelial cells [2]. Impaired endothelium-dependent vasodilation has been associated with enhanced degradation of NO by superoxide anion [25]. In addition, oxidative stress induced by aldosterone has been shown to be involved in the development of cardiovascular injury in aldosterone-induced hypertensive rats [26]. Thus, it is plausible to speculate that aldosterone-induced decrease in NO bioavailability and increase in ROS generation are involved in endothelial dysfunction in PA. The improvement of %FMD after correction of aldosterone excess by surgical removal of adrenal tutors, such as essential hypertension [], diabetes [13], obesity [1], and dyslipidemia [15]. In addition, brachial FMD has been shown to reflect coronary artery endothelial function [16] and predict cardiovascular events [17, 1]. Therefore, the measurement of brachial FMD is clinically useful for detecting early endothelial dysfunction not only in cardiovascular diseases, but also in endocrine and metabolic disorders. In the present study, bapwv in PA patients was higher than that in the control group, and there was a negative correlation between %FMD and bapwv. Endothelial dysfunction detected as a decreased brachial FMD is recognized as an early and reversible vascular change due to impairment of endothelial NO production and/or its bioavailability [7]. In contrast, increased bapwv reflects arterial stiffening due to functional and structural changes of the vasculature [19]. Since endothelial NO has been shown to regulate arterial elasticity [2, 21], the negative correlation between %FMD and bapwv in this study could represent vascular changes resulting from impaired endothelial NO availability in PA. Recent studies have shown that hypertension is one of the major cardiovascular risk factors causing endothelial dysfunction []. In fact, positive correlations between %FMD and SBP as well as SBP and PAC in the present study suggest that aldosteroneinduced raised blood pressure could be involved in endothelial dysfunction in PA patients. However, %FMD in PA patients was lower than that in age- and blood pressure-matched control subjects in the pres-

6 55 TSUCHIYA et al. mor in APA or its action by receptor blockade in IHA by spironolactone as demonstrated in this study suggests that endothelial dysfunction associated with PA is reversible. Although we failed to observe significant correlations between the changes of %FMD and those of blood pressure, PAC and ARR, possibly due to small sample numbers, the present findings suggest that improvement in both raised blood pressure and aldosterone excess could equally contribute to the improvement of endothelial dysfunction. The limitation of the present study is the relatively small number of PA patients and control subjects both in baseline and follow-up study, which prevented multivariate analysis from drawing a definite conclusion that aldosterone excess per se affects endothelial function independent of blood pressure. Further study using larger sample size of PA patients before and after treatment is required. In conclusion, the present study has demonstrated that PA patients have endothelial dysfunction, which was related to aldosterone excess and raised blood pressure and reversible after treatment, suggesting that aldosterone excess contributes to the development of endothelial dysfunction due to its hypertensive effect and/or its direct effect on the cardiovascular system. Acknowledgment This study was supported in part by Grants-in- Aids from the Ministry of Education, Science, Sports, Culture and Technology, and the Ministry of Health, Labor and Welfare, of Japan. References 1. Mulatero P, Stowasser M, Loh KC, Fardella CE, Gordon RD, Mosso L, Gomez-Sanchez CE, Veglio F, Young WF Jr (2) Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents. J Clin Endocrinol Metab 9: Rossi GP, Sacchetto A, Chiesura-Corona M, De Toni R, Gallina M, Feltrin GP, Pessina AC (21) Identification of the etiology of primary aldosteronism with adrenal vein sampling in patients with equivocal computed tomography and magnetic resonance findings: results in 1 consecutive cases. J Clin Endocrinol Metab 6: Omura M, Saito J, Yamaguchi K, Kakuta Y, Nishikawa T (2) Prospective study on the prevalence of secondary hypertension among hypertensive patients visiting a general outpatient clinic in Japan. Hypertens Res 27: Rossi GP, Sacchetto A, Visentin P, Canali C, Graniero GR, Palatini P, Pessina AC (1996) Changes in left ventricular anatomy and function in hypertension and primary aldosteronism. Hypertension 27: Halimi JM, Mimran A (1995) Albuminuria in untreated patients with primary aldosteronism or essential hypertension. J Hypertens 13: Takeda R, Matsubara T, Miyamori I, Hatakeyama H, Morise T (1995) Vascular complications in patients with aldosterone producing adenoma in Japan: comparative study with essential hypertension. The Research Committee of Disorders of Adrenal Hormones in Japan. J Endocrinol Invest 1: Deanfield JE, Halcox JP, Rabelink TJ (27) Endothelial function and dysfunction: testing and clinical relevance. Circulation 115: Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE (1992) Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 3: Nishizaka MK, Zaman MA, Green SA, Renfroe KY, Calhoun DA (2) Impaired endothelium-dependent flow-mediated vasodilation in hypertensive subjects with hyperaldosteronism. Circulation 19: Farquharson CA, Struthers AD (22) Aldosterone induces acute endothelial dysfunction in vivo in humans: evidence for an aldosterone-induced vasculopathy. Clin Sci (Lond) 13: Tsuchiya K, Nakayama C, Iwashima F, Sakai H, Izumiyama H, Doi M, Hirata Y (27) Advanced endothelial dysfunction in diabetic patients with multiple risk factors; importance of insulin resistance. J Atheroscler Thromb 1: Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE (199) Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 323: Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA (1996) Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 27: Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G, Baron AD (1996) Obesity/insulin re-

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