Association between circulating angiotensin-converting enzyme 2 and cardiac remodeling in hypertensive patients

Accepted Manuscript Title: Association between circulating angiotensin-converting enzyme 2 and cardiac remodeling in hypertensive patients Authors: Shichao Li, Zhijun Wang, Xiuhong Yang, Bo Hu, Yuling Huang, Sujing Fan PII: DOI: Reference:

S0196-9781(17)30048-7 http://dx.doi.org/doi:10.1016/j.peptides.2017.02.007 PEP 69746

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Please cite this article as: Li Shichao, Wang Zhijun, Yang Xiuhong, Hu Bo, Huang Yuling, Fan Sujing.Association between circulating angiotensinconverting enzyme 2 and cardiac remodeling in hypertensive patients.Peptides http://dx.doi.org/10.1016/j.peptides.2017.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Association between Circulating Angiotensin-Converting Enzyme 2 and Cardiac Remodeling in Hypertensive Patients

Shichao Li1, Zhijun Wang1*, Xiuhong Yang2*, Bo Hu3, Yuling Huang1, Sujing Fan2

1

Department of Cardiology, North China University of Science and Technology Affiliated

Hospital, 57 Jianshe South Rd, Tangshan City, Hebei Province 063000, China. 2

Department of Physiology, School of Basic Medical Sciences, North China University of

Science and Technology, 57 Jianshe South Rd, Tangshan City, Hebei Province 063000, China. 3

School of Public Health, North China University of Science and Technology, 57 Jianshe

South Rd, Tangshan City, Hebei Province 063000, China.

* Corresponding author: E-mail: [email protected] (ZJW); E-mail: [email protected] (XHY).

1

Highlights:

 Serum ACE2 concentration levels are increased in patients with essential hypertension.  There were significant associations between serum concentration of ACE2 and some echocardiographic parameters in hypertensive patients.  This study provided an avenue for further investigations in order to improve our understanding of the clinical utility and significance of targeting ACE2 for new therapeutic regimens.

Abstract Background: Angiotensin-converting enzyme 2 (ACE2) plays a vital role in the pathogenesis of hypertension-induced cardiac remodeling and exhibits cardioprotective properties in hypertensive animal models. Evidence that ACE2 is an important regulator of hypertensive cardiac remodeling in humans has not been addressed directly yet. Methods: A total of 161 patients with essential hypertension and 47 age- and sex-matched normotensive healthy subjects were consecutively recruited. Serum concentration levels of ACE2 were determined by enzyme-linked immunosorbent assay. Cardiac structural and functional parameters were measured by echocardiography. Results: Serum ACE2 concentrations were higher in hypertensive patients compared to healthy subjects (170.31 [83.50–707.12] pg/ml in patients versus 59.28 [39.71–81.81] pg/ml in healthy subjects, P<0.001). After adjustment for confounders, including age, sex, body mass index, snoring, smoking, duration of hypertension, comorbidities, medication use, mean 2

arterial pressure and N-terminal pro-brain natriuretic peptide, serum ACE2 concentrations were positively correlated with left atrial diameter, left ventricular end-diastolic diameter and left ventricular mass in hypertensive patients. Moreover, multiple regression analyses adjusting for covariates revealed that serum ACE2 concentrations were also independently associated with left ventricular ejection fraction and late diastolic filling velocities of the mitral inflow. Conclusions: This study reveals an elevated serum concentration of ACE2 and independent associations between serum ACE2 and echocardiographic parameters in hypertensive patients.

Keywords: angiotensin-converting enzyme 2, renin-angiotensin system, cardiac remodeling, hypertensive heart disease, hypertension

1. INTRODUCTION Hypertension is a major public health concern and one of the most important cardiovascular risk factors, responsible for up to one-third of deaths due to cardiovascular disease [1,2]. The latest survey shows that the prevalence of hypertension among Chinese adults is approximately 32.5%, but the awareness, treatment and control rates of hypertension in China are lower compared with western countries [2]. Hypertension-induced cardiac remodeling is defined as abnormalities in size, geometry, shape, composition and function of the heart resulting from hypertension [3]. Hypertensive heart disease (HHD) is a constellation of perturbations that is characterized by left ventricle 3

hypertrophy (LVH), left atrial (LA) enlargement and left ventricle (LV) dysfunction, leading to arrhythmias, myocardial ischemia and heart failure (HF) [4-6]. Angiotensin-converting enzyme 2 (ACE2) was identified as a human homologue of ACE and was found to be highly expressed in the heart, particularly in the endothelium of coronary vessels, cardiomyocytes and cardiac myofibroblasts [7-9]. A soluble form of ACE2, lacking the transmembrane and cytosolic domains, was found in the circulation [10]. A disintegrin and metalloproteinase 17 (ADAM17), as a sheddase, accounts for the proteolytic cleavage and shedding of ACE2 [10]. Soluble ACE2 is catalytically active and plays a pivotal role under pathological settings [10,11]. ACE2 catalyzes the conversions of angiotensin (Ang) II to Ang(1-7) and Ang I to Ang(1-9) [12,13]. Previous animal data have demonstrated that ACE2 is a crucial regulator of both blood pressure (BP) and HHD. The ace2 gene maps to a defined quantitative trait locus (QTL) on the X chromosome in salt-sensitive Sabra hypertensive rats, spontaneous hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP) [14]. ACE2 activators decrease BP, reverse cardiac remodeling and improve cardiac function [15]. Overexpression of ACE2 in SHRSP reduces BP and alleviates LVH [16]. Further, inhibition of ACE2 by MLN-4760 accelerates progression of cardiac hypertrophy and fibrosis in the (mRen2)27 transgenic hypertensive rats [17]. Hearts of ace2 mutant mice exhibit a severe reduction in cardiac contractility [14]. Moreover, administration of Ang II results in worsening hypertension, cardiac fibrosis and hypertrophy, and diastolic dysfunction in ACE2 knockout mice compared with wild-type mice [18]. On the contrary, some studies have shown conflicting results. Sustained overexpression of ACE2 in the heart leads to cardiac fibrosis 4

that is concomitant with cardiac dysfunction [19]. ACE2 does not modulate cardiac structure and function in Ang II-dependent hypertension mice model [20]. There are, however, only limited additional clinical studies. Urinary ACE2 level is positively correlated with systolic BP (SBP) in hypertensive patients [21]. Genetic variation in ace2 is associated with hypertension and increased LV mass (LVM) [22]. To date, evidence that ACE2 is an essential regulator of hypertensive cardiac remodeling in humans has not yet been directly addressed. Herein, in the current study, we sought to identify serum ACE2 concentrations in hypertensive patients and healthy controls, and to determine the relationships between circulating ACE2 and echocardiographic parameters of hypertensive cardiac remodeling.

2. MATERIAL AND METHODS 2.1 Study Population Patients with essential hypertension and age- and sex-matched normotensive healthy subjects were consecutively recruited in North China University of Science and Technology Affiliated Hospital from March 2015 to December 2015. Patients were considered to be hypertensive when SBP was above 140 mm Hg (1 mm Hg=0.133 kPa) and/or diastolic BP (DBP) was above 90 mm Hg at three different occasions or when receiving antihypertensive therapy. Exclusion criteria included secondary hypertension, hypertensive crisis, acute myocardial infarction, HF, cardiomyopathy, myocarditis, congenital heart disease, valvular heart disease, rheumatism, pulmonary disease, renal impairment, diabetes mellitus, thyroid disease, anemia, acute cerebrovascular accidents, infectious disease, liver disease, bone disease, malignant disease, collagen disease, pregnant women and athletes. Healthy 5

individuals with normal cardiac morphology were recruited from participants in annual health examinations. The study has been conducted according to the principles of the Declaration of Helsinki, and approved by the ethical committee of North China University of Science and Technology Affiliated Hospital. Written informed consent has been obtained from all of the subjects.

2.2 Data Collection A standardized questionnaire was completed for all subjects. Baseline clinical data, including general variables, risk factors for cardiovascular events (snoring and smoking status), history of diseases and current medication were collected. Anthropometric measurements were taken according to the standard protocol. Current smoking was defined as smoking one cigarette per day for at least half a year. Current medication use, including ACE inhibitor (ACEI), Ang II receptor blocker (ARB), β-blocker, calcium channel blocker (CCB) and diuretics, was defined as taking drugs daily for at least half a year at the time of enrolment. BP was measured for each subject in a seated position after a 5-minute rest using an electronic sphygmomanometer (HEM-7051; Omron) with an appropriately sized cuff at 8 AM. Triplicate measurements on the right arm were taken with an interval greater than 2 minutes between successive readings, and the average BP was used for analysis. After an overnight fasting of at least 12 hours, peripheral venous blood samples were obtained while each subject was supine at 6 AM. Blood samples were incubated for 30 minutes at room temperature. Serum fractions were separated by centrifugation (1,500 g, 15 minutes) and were analyzed immediately or stored at –80 °C until analysis. All biochemical 6

variables were measured on the Beckman Coulter AU5800 Analyzer. Serum creatinine (μmol/L) was measured using an enzymatic method. Estimated glomerular filtration rate (ml/min/1.73m2) was calculated according to the Cockcroft-Gault formula. Serum creatine kinase-MB, cardiac troponin I, lipid profile (total cholesterol, triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol), sodium and potassium levels were also determined.

2.3 Measurement of Serum ACE2 Concentration Serum ACE2 concentration was measured with commercially available enzyme-linked immunosorbent assay kits (catalog No. KA4223; Abnova) according to the protocol that was provided by the supplier. The limit of detection is 10 pg/mL. Intra-assay coefficient of variation from the three serum samples is between 4.5% and 5.2%. Inter-assay coefficient of variation from the three serum samples is between 6.8% and 7.8%.

2.4 Measurement of Circulating Biomarkers Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) (reference range, <450 pg/mL) and procollagen III N-terminal peptide (PIIINP) (a biomarker of collagen synthesis) (reference range, <120 ng/mL) concentrations were measured using commercially available kits (Hotgen Biotech Co., Ltd). The sensitivity of the assays is 5 pg/mL for NT-proBNP and 2 ng/mL for PIIINP. The intra- and inter-assay variations are 15% for both.

2.5 Echocardiography Transthoracic echocardiograms were performed on each subject using a echocardiograph (Vivid E9, GE) with 2–4 MHz M5S transducers by a trained, experienced sonographer blinded to the subject’s clinical data. Images were obtained with the patient lying in the 7

supine or left lateral decubitus position and reported according to American Society of Echocardiography guidelines [23]. The sonographer performed a comprehensive examination including a complete two-dimensional and color flow Doppler valvular assessment. The echocardiographic data represented the mean of three measurements on sequential cardiac cycles. Measurements included LA diameter (LAD), LV end-diastolic diameter (LVEDD), interventricular septal thickness (IVST) and LV posterior wall thickness (LVPWT). LAD was normalized for body surface area (BSA) and expressed as the LAD index (LADI). LVM was calculated

using

Devereux’s

formula

[24]:

LVM

(g)=0.8×1.04[(IVST+LVPWT+LVEDD)3–LVEDD3]+0.6. LVM was also normalized for BSA and expressed as the LVM index (LVMI). The relative wall thickness (RWT) was calculated as 2×LVPWT/LVEDD. Furthermore, LV ejection fraction (LVEF) was calculated in the parasternal long-axis view. Pulse Doppler echocardiography was used to assess LV diastolic function. Peak velocities of the early diastolic filling (E wave) and atrial filling (A wave) of the mitral inflow in an apical view were recorded and the E-to-A ratio was calculated.

2.6 Statistical Analysis Continuous variables with close to normal distributions are summarized as mean±standard deviation. Continuous variables with skewed distributions are summarized as median (interquartile range). Categorical variables are expressed as percentages. Differences between the two groups were assessed by the independent sample t test for continuous data with normal distributions or the Wilcoxon rank sum test for continuous data with skewed distributions. Characteristics were compared between groups using  2 test for categorical 8

variables. Non-normally distributed variables (ACE2, NT-proBNP and PIIINP) were logarithmically transformed (the base of logarithm was 10) for analyses when appropriate. Simple and multiple linear regression models were constructed to identify the associations between serum ACE2 concentration and echocardiographic parameters. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) 13.0 software. Two-tailed P values of <0.05 were considered statistically significant.

3. RESULTS 3.1 Subject Baseline Characteristics We enrolled 161 patients with essential hypertension and 47 healthy controls, and they were well matched for sex and age. The baseline characteristics of the subjects are shown in Table 1. Compared to healthy subjects, patients displayed higher body mass index (BMI) and lower levels of high density lipoprotein cholesterol (both P<0.05). The differences in levels of serum creatinine and potassium had no clinical significance.

BMI, body mass index; ACEI, Angiotensin-converting enzyme inhibitor; ARB, Angiotensin II receptor blocker; CCB, calcium channel blocker; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglycerides; HDL-c, high density lipoprotein cholesterol; LDL-c, low density lipoprotein cholesterol; CK, creatine kinase; cTnI, cardiac troponin I; eGFR, estimated glomerular filtration rate according to the Cockcroft-Gault formula. *

P<0.05, **P<0.001, hypertensive patients vs. healthy controls.

3.2 Cardiac Remodeling in Hypertension As shown in Table 2, hypertensive patients had significantly increased LAD, LADI, 9

LVEDD, IVST, LVPWT, LVM, LVMI and peak A wave compared to healthy individuals (all P<0.05). No significant differences were observed for RWT, LVEF, peak E wave and E/A ratio.

3.3 Serum ACE2 Concentration in Patients and Controls The hypertensive patients had higher levels of serum ACE2 concentration (absolute values) when compared with age- and sex-matched healthy subjects (170.31 [83.50–707.12] versus 59.28 [39.71–81.81] pg/ml, P<0.001) (Fig 1). No significant sex-related differences were detected for serum ACE2 concentration in either group (both P>0.05). No significant differences in serum ACE2 concentration were observed by the use of antihypertensive agents, including ACEI, ARB, β-blocker, CCB and diuretics (all P>0.05).

3.4

Correlations

Between

Serum

ACE2

Concentration

and

Echocardiographic Parameters of Hypertensive Patients In simple linear regression analyses, serum ACE2 concentration (absolute values) was positively associated with LAD (coefficient of determination [R2]=0.032, P=0.022), LVEDD (R2=0.087, P<0.001), LVM (R2=0.080, P<0.001) and E/A ratio (R2=0.054, P=0.003), while being negatively associated with LVEF (R2=0.087, P<0.001) and peak A wave (R2=0.045, P=0.008), but it was not correlated with IVST, LVPWT, RWT and peak E wave in hypertensive patients. However, no correlations were present between serum ACE2 concentration and echocardiographic parameters in healthy subjects (data not shown). In multiple regression analyses (forward stepwise), correlations between serum ACE2 10

concentration (absolute values) and LAD, LVEDD, LVM, LVEF, E/A ratio and peak A wave persisted after adjustment of age, sex, BMI, snoring, current smoker, duration of hypertension, coronary heart disease, medications (ACEI, ARB and CCB), mean arterial pressure, log NT-proBNP and log PIIINP (available exclusively to the analyses of E/A ratio and peak A wave) in hypertensive patients (Table 3). Similar correlations were not found in healthy subjects (data not shown).

Adjusted for continuous variables (age [years old], BMI [Kg/m2], duration of hypertension [years], MAP (mm Hg), log NT-proBNP and log PIIINP [available exclusively to the analyses of E/A ratio and peak A wave]) and binomial classification variables (sex [0=male, 1=female], snoring [0=no, 1=yes], current smoker [0=no, 1=yes], coronary heart disease [0=no, 1=yes], ACEI use [0=no, 1=yes], ARB use [0=no, 1=yes], CCB use [0=no, 1=yes]). Mutiple linear regression models for: LAD: F=8.128, P<0.001, R2=0.243, adjusted R2 (Ra2)=0.213; LVEDD: F=17.531, P<0.001, R2=0.313, Ra2=0.295; LVM: F=20.175, P<0.001, R2=0.344, Ra2=0.327; LVEF: F=7.974, P<0.001, R2=0.134, Ra2=0.117; E/A ratio: F=6.816, P=0.001, R2=0.083, Ra2=0.071; Peak A wave: F=8.304, P<0.001, R2=0.099, Ra2=0.087.

4. DISCUSSION The novel finding of this study is that serum concentration levels of ACE2 were significantly elevated in patients with essential hypertension. We further demonstrate that circulating levels of ACE2 were independently associated with cardiac structure and function in hypertensive patients. The role of ACE2 in hypertension has been reported in several recent studies. Brain 11

ACE2

overexpression

attenuates

oxidative

stress

and

neuroinflammation

in

deoxycorticosterone acetate-salt hypertension models, thereby blunting the development of neurogenic hypertension [25]. In SHR, recombinant human ACE2 inhibits oxidative stress and extracellular signalregulated kinase 1/2 pathway in hearts, and decreases hypertension [26]. Serum ACE2 activity is higher in hypertensive patients compared to healthy individuals [27]. Our data are consistent with previous research. Here we have found for the first time that compared to healthy subjects, serum ACE2 concentrations were increased in hypertensive patients. Higher circulating ACE2 levels may be due to more shedding of membrane-bound ACE2 [28], which implies a compensatory response to the hypertension [29]. It is interesting that medications have no influences on serum ACE2 levels. Similarly, another study also showed that urinary ACE2 levels in hypertensive patients are not changed by either ACEI or ARB excepting olmesartan [21]. Expression and release of ACE2 are regulated by proteolytic processing, transcriptional and post-transcriptional mechanisms [29], and therefore effects of drugs may be offset. In addition, there was no significant difference in serum ACE2 levels between men and women, which can be attributed to multiple regulatory mechanisms of ACE2 expression and (or) shedding. These confused results need further study. Further investigation into the association between serum ACE2 and cardiac structure is warranted. There is increasing evidence that ACE2 plays a crucial role in protection against hypertension-induced cardiac remodeling. Xanthenone, an ACE2 activator, causes improvements in cardiac function and reversal of myocardial fibrosis in the SHR [15]. Vascular ACE2 overexpression in SHRSP exhibits an attenuated development of LVH [16]. 12

On the other hand, inhibition of ACE2 by MLN-4760 exacerbates cardiac hypertrophy and fibrosis in the (mRen2)27 transgenic hypertensive rats [17]. Infusion of Ang II results in worsening hypertension, cardiac fibrosis and pathological hypertrophy, and diastolic dysfunction in ACE2 knockout mice compared with wild-type mice [18]. The increase in BP, LVM and LVPWT is detectable in ACE2 knockout mice when compared with its wild-type littermates [30]. More importantly, absence of ACE2 facilitates Ang II-mediated myocardial hypertrophy and pathological injury, chamber dilation and cardiac systolic dysfunction [30]. In this regard, it may be speculated that the independent associations of serum ACE2 and cardiac structural parameters, observed in this study, represent a causal relation. We showed that hypertensive cardiac remodeling included enlarged chamber, thickened wall and hypertrophic LV. After adjustment for covariates, serum ACE2 concentrations were found to be positively correlated with LAD, LVEDD and LVM in hypertensive patients. The above results implied that serum ACE2 concentration was predominantly associated with dilated LA and LV in hypertensive patients. Chamber dilation is characterized by an increase in cardiomyocyte length, which gradually results in HF with reduced ejection fraction [31]. Because growing evidence exists for increased plasma ACE2 activity in human HF [27, 29, 32], hypertensive patients with systolic dysfunction were excluded from our study. Nonetheless, we still suggested that increasing serum ACE2 concentration was strongly correlated with worsening LVEF, implicating that ACE2 modulates cardiac function starting from the chronically compensated state of HF. In fact, it is now well recognized that hypertension is more likely to develop HF with preserved ejection fraction at initial stages of disease [3]. The late diastolic filling velocity (A 13

wave) was mildly increased in hypertensive patients compared with normal subjects, which was mainly related to LA enlargement. In addition, serum ACE2 concentration displayed close correlations with peak A wave and E/A ratio in hypertensive patients rather than healthy individuals, which suggests that ACE2 is responsible for regulating diastolic function. This study has several limitations that should be considered. First, the study was an observational design without data regarding change in serum ACE2 levels and clinical outcomes during follow-up. Second, sample size was relatively small, limiting statistical powers. Third, variability of serum ACE2 levels in hypertensive patients was higher when compared with healthy controls for the heterogeneity of the former. Larger clinical trials are needed to confirm the relationship between peripheral ACE2 and hypertensive heart disease.

5. CONCLUSION To sum up, we demonstrate an elevated level of serum ACE2 concentration and an independent association between circulating ACE2 and cardiac remodeling in patients with essential hypertension. Our data provide an avenue for further investigations in order to improve our understanding of the clinical utility and significance of targeting ACE2 pathways for new therapeutic regimens.

ACKNOWLEDGEMENTS Funding: This work was supported by the National Natural Science Foundation of China (grant no. 81372029); the Key Project of Medical Science Research of Hebei Province (grant no. 20150501); and the Science and Technology Support Program of Hebei Province (grant 14

no. 13202001D).

DISCLOSURES None.

REFERENCES 1. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart disease and stroke statistics—2016 update: a report from the American Heart Association. Circulation. 2016;133: e1-e323. doi: 10.1161/CIR.0000000000000350 2. Lewington S, Lacey B, Clarke R, Guo Y, Kong XL, Yang L, et al. The burden of hypertension and associated risk for cardiovascular mortality in china. JAMA Intern Med. 2016;176: 524-532. doi: 10.1001/jamainternmed.2016.0190 3. Santos M, Shah AM. Alterations in cardiac structure and function in hypertension. Curr Hypertens Rep. 2014;16: 1-10. doi: 10.1007/s11906-014-0428-x 4. Ganau A, Devereux RB, Roman MJ, de Simone G, Pickering TG, Saba PS, et al. Patterns of left ventricular hypertrophy and geometric remodeling in essential hypertension. J Am Coll Cardiol. 1992;19: 1550-1558. 5. Muiesan ML, Salvetti M, Monteduro C, Bonzi B, Paini A, Viola S, et al. Left ventricular concentric geometry during treatment adversely affects cardiovascular prognosis in hypertensive

patients.

Hypertension.

2004;43:

731-738.

doi:

10.1161/01.HYP.0000121223.44837.de 6. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of 15

echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med. 1990;322: 1561-1566. 7. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human homolog of angiotensin-converting enzyme cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem. 2000;275: 33238-33243. 8. Danilczyk U, Penninger JM. Angiotensin-converting enzyme II in the heart and the kidney. Circ Res. 2006;98: 463-471. doi: 10.1161/01.RES.0000205761.22353.5f 9. Guy JL, Lambert DW, Turner AJ, Porter KE. Functional angiotensin-converting enzyme 2 is expressed in human cardiac myofibroblasts. Exp Physiol. 2008;93: 579-588. doi: 10.1113/expphysiol.2007.040139 10. Lambert DW, Yarski M, Warner FJ, Thornhill P, Parkin ET, Smith AI, et al. Tumor necrosis factor-α convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute

respiratory

syndrome-coronavirus

(SARS-CoV)

receptor,

angiotensin-converting enzyme-2 (ACE2). J Biol Chem. 2005;280: 30113-30119. doi: 10.1074/jbc.M505111200 11. Jia HP, Look DC, Tan P, Shi L, Hickey M, Gakhar L, et al. Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia. Am J Physiol Lung Cell Mol Physiol. 2009;297: L84-L96. doi:10.1152/ajplung.00071.2009 12. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, et al. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277: 14838-14843. doi: 10.1074/jbc.M200581200 13. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, et al. A novel 16

angiotensin-converting enzyme–related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res. 2000;87: e1-e9. doi: 10.1161/01.RES.87.5.e1 14. Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature. 2002;417: 822-828. 15. Hernández Prada JA, Ferreira AJ, Katovich MJ, Shenoy V, Qi Y, Santos RA, et al. Structure-based identification of small-molecule angiotensin-converting enzyme 2 activators as novel antihypertensive agents. Hypertension. 2008;51: 1312-1317. doi: 10.1161/HYPERTENSIONAHA.107.108944 16. Rentzsch B, Todiras M, Iliescu R, Popova E, Campos LA, Oliveira ML, et al. Transgenic angiotensin-converting enzyme 2 overexpression in vessels of SHRSP rats reduces blood pressure and improves endothelial function. Hypertension. 2008;52: 967-973. doi: 10.1161/HYPERTENSIONAHA.108.114322 17. Trask AJ, Groban L, Westwood BM, Varagic J, Ganten D, Gallagher PE, et al. Inhibition of angiotensin-converting enzyme 2 exacerbates cardiac hypertrophy and fibrosis in Ren-2 hypertensive rats. Am J Hypertens. 2010;23: 687-693. doi: 10.1038/ajh.2010.51 18. Zhong JC, Basu R, Guo D, Chow FL, Byrns S, Schuster M, et al. Angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction.

Circulation.

2010;122:

717-728.

doi:

10.1161/CIRCULATIONAHA.110.955369 19. Masson R, Nicklin SA, Craig MA, McBride M, Gilday K, Gregorevic P, et al. Onset of experimental

severe

cardiac

fibrosis

is

mediated

by

overexpression

of 17

angiotensin-converting

enzyme

2.

Hypertension.

2009;53:

694-700.

doi:

10.1161/HYPERTENSIONAHA.108.122333 20. Gurley SB, Allred A, Le TH, Griffiths R, Mao L, Philip N, et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J Clin Invest. 2006;116: 2218-2225. doi: 10.1172/JCI16980 21. Furuhashi M, Moniwa N, Mita T, Fuseya T, Ishimura S, Ohno K, et al. Urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin II receptor blocker. Am J Hypertens. 2014;28: 15-21. doi: 10.1093/ajh/hpu086 22. Patel SK, Wai B, Ord M, MacIsaac RJ, Grant S, Velkoska E, et al. Association of ACE2 genetic variants with blood pressure, left ventricular mass, and cardiac function in Caucasians with type 2 diabetes. Am J Hypertens. 2012;25: 216-222. doi: 10.1038/ajh.2011.188 23. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, et al. Recommendations for chamber quantification: a report from the American Society of Echocardiography’s

Guidelines

and

Standards

Committee

and

the

Chamber

Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr. 2005;18: 1440-1463. doi: 10.1016/j.echo.2005.10.005 24. Devereux RB, Alonso DR, Lutas EM, Gottlieb GJ, Campo E, Sachs I, et al. Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings. Am J Cardiol. 1986;57: 450-458. 18

25. Sriramula S, Xia H, Xu P, Lazartigues E. Brain-targeted angiotensin-converting enzyme 2 overexpression

attenuates

cyclooxygenase-mediated

neurogenic

inflammation.

hypertension

Hypertension.

by

2015;65:

inhibiting

577-586.

doi:

10.1161/HYPERTENSIONAHA.114.04691 26. Lo J, Patel V B, Wang Z, et al. Angiotensin‐ converting enzyme 2 antagonizes angiotensin II‐ induced pressor response and NADPH oxidase activation in Wistar–Kyoto rats and spontaneously hypertensive rats[J]. Exp Physiol, 2013, 98(1): 109-122. doi: 10.1113/expphysiol.2012.067165 27. Úri K, Fagyas M, Siket IM, Kertész A, Csanádi Z, Sándorfi G, et al. New perspectives in the renin-angiotensin-aldosterone system (RAAS) IV: Circulating ACE2 as a biomarker of systolic dysfunction in human hypertension and heart failure. PLoS One. 2014;9: e87845. doi:10.1371/journal.pone.0087845 28. Patel VB, Clarke N, Wang Z, Fan D, Parajuli N, Basu R, et al. Angiotensin II induced proteolytic cleavage of myocardial ACE2 is mediated by TACE/ADAM-17: a positive feedback mechanism in the RAS. J Mol Cell Cardiol. 2014;66: 167-176. doi: 10.1016/j.yjmcc.2013.11.017 29. Patel V B, Zhong J C, Grant M B, et al. Role of the ACE2/Angiotensin 1–7 Axis of the Renin–Angiotensin System in Heart Failure[J]. Circ Res, 2016, 118(8): 1313-1326. doi: 10.1161/CIRCRESAHA.116.307708 30. Song B, Zhang ZZ, Zhong JC, Yu XY, Oudit GY, Jin HY, et al. Loss of angiotensin-converting enzyme 2 exacerbates myocardial injury via activation of the CTGF-fractalkine

signaling

pathway.

Circ

J.

2013;77:

2997-3006.

doi: 19

10.1253/circj.CJ-13-0805 31. Drazner MH. The progression of hypertensive heart disease. Circulation. 2011;123: 327-334. doi: 10.1161/CIRCULATIONAHA.108.845792 32. Epelman S, Tang WH, Chen SY, Van Lente F, Francis GS, Sen S. Detection of soluble angiotensin-converting enzyme 2 in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system. J Am Coll Cardiol. 2008;52: 750-754. doi:10.1016/j.jacc.2008.02.088

20

Fig 1. Comparison of serum ACE2 concentration levels for hypertensive patients and healthy controls. Data are shown as logarithmically transformed ACE2 (log ACE2) in box plots. Horizontal lines within boxes, boxes and error bars represent median, interquartile range and range respectively. *P<0.001, hypertensive patients vs. healthy controls.

21

Table 1. Baseline clinical and biochemical characteristics of the subjects. Patients with hypertension (n=161)

Healthy controls (n=47)

P value

59±8

57±7

0.327

80/81 (49.7/50.3)

28/19 (59.6/40.4)

0.250

27.1±3.1

25.0±3.6

<0.001**

Snoring, n(%)

108 (67.1)

24 (51.1)

0.058

Current smokers, n(%)

38 (23.6)

9 (19.1)

0.521

Duration of hypertension, y

9 (3–10)

—

—

Coronary heart disease, n(%)

74 (46.0)

0

—

Stroke, n(%)

50 (31.1)

0

—

23 (14.3)

0

—

ARB, n(%)

22 (13.7)

0

—

β-blocker, n(%)

35 (21.7)

0

—

CCB, n(%)

66 (41.0)

0

—

8 (5.0)

0

—

General variables Age, y Sex, F/M (%/%) BMI, Kg/m2 Risk factors

Medical variables

Medications ACEI, n(%) Medications

Diuretics, n(%)

22

Cardiac variables SBP, mm Hg

152±22

129±12

<0.001**

DBP, mm Hg

89±14

76±11

<0.001**

Heart rate, beats/min

70±11

70±10

0.691

TC, mmol/L

4.86±1.11

5.18±1.14

0.085

TG, mmol/L

1.43 (1.08–1.95)

1.41 (1.05–1.79)

0.488

HDL-c, mmol/L

1.33±0.33

1.45±0.40

0.033*

LDL-c, mmol/L

2.76±0.72

2.88±0.73

0.318

CK-MB, U/L

10 (8–12)

10 (8–12)

0.679

0.36 (0.21–0.52)

0.39 (0.24–0.51)

0.584

74±19

68±12

0.012*

eGFR, ml/min/1.73m2

96.44±28.02

93.08±21.90

0.451

Sodium, mmol/L

141.74±2.02

141.97±1.71

0.477

3.89±0.36

4.04±0.28

0.011*

Biochemical variables

cTnI, μg/L Creatinine, μmol/L

Potassium, mmol/L

Values are given as mean±SD or as median (25th, 75th percentile) for continuous variables, and percentage for categorical data (n[%]).

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Table 2. Echocardiographic characteristics of hypertensive patients and healthy controls. Patients with hypertension (n=161)

Healthy controls (n=47)

P value

LAD, mm

36.44±4.90

33.34±3.75

<0.001**

LADI, cm/m2

2.05±0.29

1.97±0.22

0.042*

LVEDD, mm

48.77±5.50

44.63±3.83

<0.001**

IVST, mm

8.97±1.21

8.42±1.04

<0.001**

8.50 (8.08–9.54)

8.41 (7.60–9.05)

0.030*

RWT

0.36±0.05

0.38±0.05

0.063

LVM , g

151.7±45.1

121.0±29.0

<0.001**

81.6 (70.4–93.1)

70.0 (65.1–75.6)

<0.001**

LVEF, %

67 (63–71)

68 (65–74)

0.081

E wave, cm/s

71.9±18.6

73.2±20.0

0.676

A wavea, cm/s

87.5±19.3

81.3±16.5

0.047*

0.76 (0.67–0.90)

0.81 (0.73–1.15)

0.053

LVPWT, mm

LVMI, g/m2

E/A ratioa

Values are given as mean±SD or as median (25th, 75th percentile). LAD, left atrial diameter; LADI, left atrial diameter index; LVEDD, left ventricular end-systolic diameter; IVST, interventricular septal thickness; LVPWT, left ventricular posterior wall thickness; RWT, relative wall thickness; LVM, left ventricular mass; LVMI, left ventricular mass index; LVEF, left ventricular ejection fraction. a

E/A ratio could not be calculated for 2 patients owing to atrial fibrillation.

*

P<0.05, **P<0.001, hypertensive patients vs. healthy controls.

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Table 3. Factors associated with echocardiographic parameters of hypertensive patients in multivariate analyses. Std. Error

Standardized coefficients

t

P value

ACE2, pg/ml

0.001

0.198

2.743

0.007

Sex (male vs female)

0.721

–0.270

–3.682

<0.001

Age, y

0.045

0.166

2.194

0.030

BMI, Kg/m2

0.115

0.229

3.203

0.002

CCB (yes vs no)

0.731

0.177

2.431

0.016

log NT-proBNP

0.725

0.246

3.287

0.001

ACE2, pg/ml

0.001

0.267

3.963

<0.001

Sex (male vs female)

0.740

–0.376

–5.589

<0.001

BMI, Kg/m2

0.121

0.175

2.603

0.010

log NT-proBNP

0.735

0.235

3.482

0.001

ACE2, pg/ml

0.005

0.254

3.857

<0.001

Sex (male vs female)

5.939

–0.387

–5.896

<0.001

BMI, Kg/m2

0.975

0.194

2.945

0.004

log NT-proBNP

5.898

0.283

4.290

<0.001

ACE2, pg/ml

0.001

–0.289

–3.856

<0.001

MAP, mm Hg

0.043

–0.152

–2.025

0.045

log NT-proBNP

1.256

–0.176

–2.351

0.020

Variables LAD, mm

LVEDD, mm

LVM, g

LVEF, %

25

E/A ratio ACE2, pg/ml

<0.001

0.224

2.869

0.005

Age, y

0.003

–0.167

–2.134

0.034

ACE2, pg/ml

0.003

–0.200

–2.583

0.011

MAP, mm Hg

0.099

–0.233

–3.007

0.003

A wave, cm/s

LAD, left atrial diameter; ACE2, serum ACE2 concentration (absolute values); BMI, body mass index; CCB, calcium channel blocker; NT-proBNP, N-terminal pro-brain natriuretic peptide, common log transformed; LVEDD, left ventricular end-systolic diameter; LVM, left ventricular mass; LVEF, left ventricular ejection fraction; MAP, mean arterial pressure.

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