International Journal of Cardiology 176 (2014) 1399–1401

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Letter to the Editor

Decreased levels of serum Angiotensin-(1–7) in patients with pulmonary arterial hypertension due to congenital heart disease Hailong Dai a,b,c,⁎,1, Yongfei Gong a,1, Zhicheng Xiao b,c,1, Xuefeng Guang a,⁎, Xiaolong Yin a,⁎ a b c

Department of Cardiology, Yan'an Affiliated Hospital of Kunming Medical University, Yunnan Cardiovascular Hospital, Kunming, PR China Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming, PR China Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia

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Article history: Received 30 July 2014 Accepted 2 August 2014 Available online 8 August 2014 Keywords: Pulmonary arterial hypertension Congenital heart disease Angiotensin-(1–7)

Pulmonary arterial hypertension (PAH) is a progressive disease with poor survival outcome. The presence of PAH increases morbidity and reduces survival in patients with congenital heart disease (CHD) [1]. The field of CHD–PAH has seen dramatic progress over the last two decades with improved survival and quality of life for patients, however, this remains a deadly and debilitating disease [2]. The exact pathogenesis of CHD–PAH is poorly understood, therapeutic options for those patients are limited. The angiotensin (Ang) converting enzyme 2 (ACE2)-Ang-(1–7)-Mas receptor axis is an important component of the renin-angiotensin system, ACE2 coverts Ang II into Ang-(1–7), which exerts both vasodilatory and anti-proliferative effects [3,4]. Recent studies have demonstrated the therapeutic effects of Ang-(1–7) in monocrotaline-induced PAH model [5,6]. Our previous study showed that serum ACE2 levels and activity were decreased in the patients with CHD–PAH [7,8]. In this study, we explored the hypothesis that serum Ang-(1–7) levels was also decreased in the patients with CHD–PAH. The study population consisted of 90 consecutive CHD patients presenting to our institution and 30 healthy subjects adjusted for age and sex between August and October 2013. Exclusion criteria were coronary artery disease, hypertension, heart valve diseases, diabetes

⁎ Corresponding authors at: Department of cardiology, Yan'an Affiliated Hospital of Kunming Medical University, 245 of East Renming Road, Kunming 650051, China. Tel.:+86 871 6321 1384; fax: +86 871 6321 1021. E-mail addresses: [email protected] (H. Dai), [email protected] (X. Guang), [email protected] (X. Yin). 1 These three authors contributed equally to this study.

http://dx.doi.org/10.1016/j.ijcard.2014.08.021 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

mellitus, autoimmune diseases, bleeding tendency, liver or renal insufficiency, pulmonary disease such as chronic obstructive pulmonary disease, pulmonary embolism, and HIV infection. The CHD patients were assigned into 3 groups according to mean pulmonary arterial pressure (mPAP) which was measured by right heart catheterization: CHD without PAH group (mPAP ≤ 25 mm Hg), CHD with mild to moderate PAH group (25 mm Hg N mPAP ≤ 50 mm Hg), CHD with severe PAH group (mPAP N 50 mm Hg). The ethics committee of Yan'an Hospital of Kunming Medical University approved this study and an informed consent was given by each participant. Baseline demographic and clinical characteristics were collected. Fasting blood samples were analyzed for total cholesterol, triglyceride, liver function such as alanine aminotransferase, aspartate aminotransferase and kidney function in a routine diagnostic analyzer. 4 ml blood samples were collected in glass tubes without additive and allowed them to clot at room temperature for 60 min. Serum was separated by centrifugation at 3000 r/min for 15 min. Serum aliquots of 1.50 ml were obtained and stored at − 80 °C until use. The serum Ang-(1–7) levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, USCN Life Science Inc. Wuhan, China). All steps were performed according to the instruction. Right heart catheterization was done in the catheterization lab. With local anesthesia under continuous electrocardiographic monitoring, a 6 French pigtail catheter was advanced into the pulmonary artery through the right femoral vein by placement of a 6 or 7 French vascular sheath. The catheter was flushed with heparinized normal saline. Correct catheter positioning was verified by fluoroscopy. Transducers were positioned at the midaxillary line and zeroed at the atmospheric pressure. The patients were equilibrated for about 10 min before pulmonary arterial pressure measurements. Categorical variables were reported as percentage and continuous variables as means ± SD. Proportions were compared using the chisquare test. Continuous Gaussian distributed variables with ANOVA and non-Gaussian distributed variables with the Kruskal–Wallis test. Correlation between two continuous variables was assessed by Spearman's rank correlation test. Statistical significance was set at p b 0.05. Data were analyzed using SPSS 17.0 software (SPSS, Chicago, USA). The study included 90 CHD patients (31 men and 59 women; 61 atrial septal defect (ASD), 29 ventricular septal defect (VSD)) and 30 normal control group (group A). The 90 CHD patients were assigned into three groups: 27 cases of CHD without PAH group (group B), 42 cases of CHD with mild to moderate PAH group (group C), and 21

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H. Dai et al. / International Journal of Cardiology 176 (2014) 1399–1401

cases of CHD with severe PAH group (group D). Demographic and general clinical features are presented in Table 1. There were no significant differences in sex, age, alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, total cholesterol and triglyceride between the study groups. The levels of Ang-(1–7) in plasma measured by ELISA were 17.39 ± 1.33 pg/ml, 24.57 ± 2.19 pg/ml, 18.84 ± 1.59 pg/ml and 12.52 ± 1.85 pg/ml in groups A, B, C and D, respectively. The level of Ang-(1–7) in group B was higher than in group A (p b 0.001), the level in groups C and D was lower than in group B (p b 0.001), the level in group D was lower than in groups A and C (p b 0.001), and the level in group C was higher than in group A (p b 0.001) (Fig. 1). Through correlation and regression analysis, there was a negative correlation between mPAP and the levels of Ang-(1–7) (r = –0.841, p b 0.001) (Fig. 2). The present study demonstrated that serum levels of Ang-(1–7) are decreased in the patients with CHD–PAH, the mPAP is negatively correlated with serum level of Ang-(1–7). CHD–PAH is largely due to left-to-right shunt, which triggers a series of changes in the vascular structure and function of the pulmonary arteries [9]. The pathophysiological mechanisms responsible for the development of CHD–PAH are not completely known. Pulmonary vascular injury, inflammatory reaction and endothelial dysfunction [10] may all play an important role in this condition. ACE2 coverts Ang II into Ang-(1–7). Ang-(1–7) mediates vasodilation, antiproliferation, anti-apoptosis and antifibrosis, which antagonizes the actions of Ang II via the receptor Mas. Recent studies have indicated that an imbalance between the vasoconstrictive (ACE– Ang II-AT1R axis) and vasodilator (ACE2–Ang-(1–7)–Mas axis) mechanisms involving the pulmonary circulation leading to the development of PAH [3,5,11–16] Ang-(1–7) overexpression or injection via osmotic minipumps, induces a beneficial pulmonary outcome in monocrotalineinduced PAH model [5,6]. Our previous study showed that serum ACE2 levels and activity were decreased in the patients with CHD– PAH [7,8]. In this study, serum Ang-(1–7) levels in CHD patients with nonpulmonary hypertension was significantly higher than in the group of normal controls. This result are consistent with our previous findings, which showed that the levels of ACE2 protein contents in CHD patients with nonpulmonary hypertension was significantly higher than in the group of normal controls. This phenomenon may be due to ACE2 being increased in CHD patients before the onset of PAH in order to prevent the development of PAH, generating more Ang-(1–7). There are a number of limitations in this study. The relatively small number of patients was studied. The amount of left-to-right shunt in the various conditions of atrial and ventricular septum defects is not analyzed. In summary, this study is the first to demonstrate that the serum level of Ang-(1–7) is decreased in patients with CHD–PAH. We

Fig. 1. Serum levels of Ang-(1–7) in the study population. *p b 0.001 vs. control; &p b 0.001 vs. control; &p b 0.001 vs. CHD without PAH; #p b 0.001 vs. CHD with mild to moderate PAH.

Fig. 2. Ang-(1–7) levels correlated to pulmonary arterial pressure.

speculate that the decrease in Ang-(1–7) shifts the balance of the RAS towards the ACE–Ang II-AT1R axis, resulting in increases in vascular remodeling, fibrosis and PAH in CHD patients. So Ang-(1–7) may be a target for the treatment of CHD–PAH. Further studies are necessary to substantiate this conclusion.

Table 1 Clinical characteristics of the study groups. Characteristics

Control (n = 30)

CHD without PAH (n = 27)

CHD with mild to moderate PAH (n = 42)

CHD with severe PAH (n = 21)

Male gender Age, years Atrial septal defect Ventricular septal defect mPAP, mm Hg Alanine aminotransferase, U/L Aspartate aminotransferase, U/L Urea nitrogen, mmol/L Creatinine, μmol/L Total cholesterol, mmol/L Triglyceride, mmol/L

11 (36.7) 34.97 ± 1.70 – – – 21.73 ± 2.28 20.50 ± 1.99 5.03 ± 0.27 73.77 ± 2.62 4.58 ± 0.16 1.53 ± 0.13

13 (48.1) 34.19 ± 2.37 21 (77.8) 6 (22.2) 22.70 ± 0.40⁎ 23.26 ± 3.34 23.00 ± 2.80 4.28 ± 0.26 69.81 ± 2.86 4.53 ± 0.14 1.26 ± 0.09

11 (26.2) 31.95 ± 2.20 30 (71.4) 12 (28.6) 40.14 ± 0.79⁎ 20.95 ± 2.64 21.43 ± 1.21 4.19 ± 0.18 67.71 ± 1.95 4.57 ± 0.12 1.28 ± 0.09

7 (33.3) 36.38 ± 3.59 10 (47.6) 11 (52.4) 76.29 ± 3.64⁎ 19.86 ± 1.50 21.86 ± 1.58 4.38 ± 0.32 72.24 ± 1.87 5.14 ± 0.30 1.70 ± 0.25

Data are means ± SD or absolute numbers with percentages in parentheses.*p b 0.001 vs. CHD without PAH, CHD with mild to moderate PAH and CHD with severe PAH groups compared with each other; all other comparisons between control and the other 3 groups were nonsignificant.

H. Dai et al. / International Journal of Cardiology 176 (2014) 1399–1401

Conflicts of interest All authors have no conflicts of interest to disclose. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81360037), Natural Science Foundation of Yunnan (Nos. 2012FB009, 2013FZ284), Dr. Academic Award of Yunnan, the Talent Program, Yunnan Province, China and Monash Professorial Fellowship, Monash University, Australia. The authors of this manuscript have certified that they comply with the principles of ethical publishing in the International Journal of Cardiology. References [1] Simonneau G, Gatzoulis MA, Adatia I, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2013;62(25 Suppl.):D34–41. [2] D'Alto M, Diller GP. Pulmonary hypertension in adults with congenital heart disease and Eisenmenger syndrome: current advanced management strategies. Heart 2014; 100(17):1322–8. [3] Ferreira AJ, Shenoy V, Yamazato Y, et al. Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension. Am J Respir Crit Care Med 2009;179(11):1048–54. [4] Shenoy V, Qi Y, Katovich MJ, Raizada MK. ACE2, a promising therapeutic target for pulmonary hypertension. Curr Opin Pharmacol 2011;11(2):150–5. [5] Shenoy V, Ferreira AJ, Qi Y, et al. The ACE2/Ang-(1–7)/MAS axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension. Am J Respir Crit Care Med 2010;182(8):1065–72.

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