Hypertension Research (2015) 38, 208–212 & 2015 The Japanese Society of Hypertension All rights reserved 0916-9636/15 www.nature.com/hr ORIGINAL ARTICLE Higher circulatory level of endothelin-1 in hypertensive subjects screened through a cross-sectional study of rural Bangladeshi women Shamima Akter1,2,3, Subrina Jesmin1,2,3, Yoshio Iwashima4, Sakuramoto Hideaki1, Md Arifur Rahman2, Md Majedul Islam1,2, Masao Moroi3, Nobutake Shimojo1, Naoto Yamaguchi5, Takashi Miyauchi1, Satoru Kawano1, Taro Mizutani1 and Yuhei Kawano4 Endothelin-1 (ET-1) is a potential marker of the endothelial dysfunction, which has been shown to be elevated in hypertensive subjects. No previous study has investigated the circulatory level of ET-1 and hypertension in a South Asian country. The present study assessed the circulating levels of ET-1 in subjects with or without hypertension and further examined the association of ET-1 with clinical and metabolic parameters. A total of 2543 rural Bangladeshi women with a mean age of 44.5 years were studied using a cross-sectional survey. Multiple regressions were used to examine the association between the circulatory ET-1 levels and hypertension. The prevalence of hypertension was 29.3%. The ET-1 levels were significantly higher in the hypertensive (mean 3.08 pg ml–1, s.e. (0.19)) than in the non-hypertensive subjects (mean 2.01 pg ml–1, s.e. (0.03)) (P = 0.001). After adjusting for age, the ET-1 level had significant positive associations with the diastolic blood pressure (P = 0.002), systolic blood pressure (P = 0.001), mean arterial pressure (P = 0.002) and fasting blood glucose (P = 0.002). In a tertile analysis, we found that hypertension in the subjects was significantly increased as the levels of ET-1 increased (P for the trend = 0.001). In a stepwise multiple regression analysis, after adjusting for age and all other potential variables, we found that the mean arterial pressure and the fasting plasma levels have significant associations with the ET-1 level. The present study demonstrates that there is a higher concentration of ET-1 among the hypertensive subjects in an apparently healthy population of Bangladeshi rural women. The relationship between ET-1 and hypertension requires further investigation to define the clinical utility and predictive value of serum ET-1 levels for hypertension for a South Asian population. Hypertension Research (2015) 38, 208–212; doi:10.1038/hr.2014.160; published online 13 November 2014 Keywords: endothelin-1; hypertensive; rural women; South Asian INTRODUCTION High blood pressure is an important worldwide public health challenge because of its high frequency and its concurrent risks of stroke and cardiovascular and renal diseases.1,2 Worldwide, 26% of the adult population were hypertensive in 2000, and 29% are projected to have this condition by 2025.3 In Bangladesh, nearly 39% of the adults have hypertension, and this figure appears to be higher than among other South Asian populations.4 The identification of risk factors and the control of hypertension are of the utmost importance in this region. The importance of blood pressure as a modifiable risk factor for cardiovascular disease is well recognized. Although many effective and inexpensive blood pressure-lowering treatments are available, other clinical approaches for controlling hypertension need to be explored. Endothelin (ET) is a potent endothelial cell-derived venous and arterial vasoconstrictor peptide5 that consists of a family of 21 amino acid peptides (ET-1, ET-2 and ET-3).6 Among the family of peptides, ET-1 is the most abundant isoform and most potent vasoconstrictor in humans and contributes to the maintenance of the basal vascular tone.7 In animal models of hypertension, ET-1 is overexpressed in the vascular wall,8 and mice with an ET-1 gene that has been inactivated exhibit a slight elevation in blood pressure.9 In addition, ET-1 is involved in a variety of other physiological processes in both animals and humans, including cell proliferation,10 fibrosis,11 endothelial dysfunction,12 arterial stiffness13 and cardiac hypertrophy;14 all of which may contribute to the development and maintenance of hypertension. In this context, it is of interest to investigate the associations between ET-1 and blood pressure in humans. 1 Institute of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2Health & Disease Research Center for Rural Peoples (HDRCRP), Mohammadpur, Dhaka, Bangladesh; 3National Center for Global Health and Medicine (NCGM), Toyama, Shinjuku-ku, Tokyo, Japan; 4National Cerebral and Cardiovascular Center, Suita City, Osaka, Japan and 5Center for Health Science, Ibaraki Prefectural University, Ami, Ibaraki, Japan Correspondence: Assistant Professor S Jesmin, Graduate School of Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. E-mail: jsubrina@gmail.com Received 13 March 2014; revised 20 July 2014; accepted 25 July 2014; published online 13 November 2014 Endothelin-1 and hypertension in Bangladesh S Akter et al 209 To date, the possible relationship of ET-1 with essential hypertension has been limited to experimental studies,8,15 and few clinical studies in humans conducted in the developed countries have found elevated levels of ET-1 among hypertensive subjects,16–20 whereas other studies have found no significant difference21,22 compared with the healthy controls. To our knowledge, only one study has investigated the association between ET-1 and hypertension among an apparently healthy population.23 Moreover, no previous studies on this topic have been conducted in South Asian populations or populations from low-income countries. As racial differences exist in the plasma levels of ET-1,24 more research addressing the association between ET-1 and hypertension among other races requires investigation. As such, we aimed to examine the associations between the circulating ET-1 levels and hypertension among Bangladeshi rural women. MATERIALS AND METHODS Study procedure The present study is a community-based cross-sectional study performed in women from rural Bangladesh between 2009 and 2010. A total of 2543 rural Bangladeshi women with a mean age of 44.5 years were studied using a crosssectional survey. Females aged ⩾ 15 years were selected using the stratified multistage random sampling method. This sample size was sufficient to test all of our formulated research hypotheses at the 5% level of significance with a power of 80% (β = 0.20). We used the World Health Organization’s STEPwise approach to surveillance (STEPS) approach (modified), which entails a stepwise collection of the risk factor data based on standardized questionnaires covering demographic characteristics, somatic illnesses, somatic and mental symptoms, medications, lifestyle and health-related behaviors (step 1), basic physical measures (step 2) and basic biochemical investigations, such as blood glucose and cholesterol (step 3). The women were recruited from the village communities of the Gaibandha district and Noagon district, covering two of the divisions of Bangladesh (Rajshahi and Rangpur). (A division is the largest administrative tier in Bangladesh, and there are a total of seven divisions in Bangladesh.). The respondents were selected randomly after selecting the division, district, Upazila and villages and were recruited through local announcements at the community level and by house-to-house visits. The details of the study area have been described before in our previous study.25 The participants’ data were obtained through interviews and clinical examinations at mobile examination centers, where blood samples were also collected. The study was approved by the Ethical Committee of the Health and Disease Research Center of Rural Peoples, Dhaka, Bangladesh, Shahid Ziaur Rahman Medical College, Bogra, Bangladesh and conforms to the principles outlined in the Helsinki Declaration. All of the participants gave their written informed consent prior to inclusion in the study. Study subjects From a total of 2720 recruited women, we excluded the subjects with chronic illnesses such as hypothyroidism, pregnant women, those on hormone replacement therapy, those with known illnesses such as ischemic heart disease and those with diabetes. After the exclusions, a total of 2543 subjects remained in this study. Anthropometric and other variables The anthropometric measurements of these individuals while they were wearing light clothing and no shoes were obtained by well-trained examiners as follows: height was measured to the nearest 0.1 cm using a portable stadiometer (Seca, Hamburg, Germany); weight was then measured in an upright position to the nearest 0.1 kg using a calibrated balance beam scale; body mass index was calculated as the body weight (kg) divided by the square of the body height (m2); and waist circumference measurements were taken at the end of a normal expiration to the nearest 0.1 cm by measuring from the narrowest point between the lower borders of the rib cage and the iliac crest. Blood pressure measurement Blood pressure was measured twice in the right arm with the subject in a sitting position using a standard mercury manometer and cuff, to the nearest 2 mm Hg, with the initial reading taken at least 5 min after the subject was made comfortable, and again after an interval of 15 min. The average systolic and diastolic blood pressures were then estimated. Hypertension was defined as a systolic blood pressure ⩾ 140 mm Hg, a diastolic blood pressure ⩾ 90 mm Hg or the use of antihypertensive medication. The subjects who were determined to be hypertensive through the initial screening were followed up for 2 months, with four additional blood pressure measurements. Biochemical analysis Blood for the biochemical analysis was obtained from the participants after a 10–12 h overnight fast. The blood sample was collected using the standard blood sample collection procedure. Immediately after the collection of blood and labeling the blood vials, the samples were transported to the National Centre for Global Health and Medicine, Japan, for the biochemical assessment. For the analysis, the serum was immediately separated from the blood by centrifugation to evaluate the plasma concentration of the lipids. The triglycerides levels were measured using the lipoprotein lipase method (Wako Chemicals, Tokyo, Japan), high-density lipoprotein cholesterol levels were measured using the Determiner-L kit (Kyowa, Tokyo, Japan) and the fasting plasma glucose levels were measured using the Hexokinase G-6-PDH kit (Wako Pure Chemical Industries, Osaka, Japan). Enzyme-linked immunosorbent assay for plasma ET-1 level The concentration of the ET-1 in the plasma was determined using a Quantikine ET-1 Enzyme Immuno Assay Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocol. A 4.5 h solid-phase enzyme-linked immunosorbent assay was used, which contained synthetic ET-1 and antibodies raised against synthetic ET-1. This immunoassay has been shown to accurately quantitate synthetic and naturally occurring ET-1. The standards and samples were pipetted into the wells, and if present, the ET-1 antigen was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific to ET-1 was added to the wells. Following a wash to remove any unbound antibodyenzyme reagent, a substrate solution was added to the wells, and the color developed in proportion to the amount of ET-1 bound in the initial step. The color development was then stopped, and its intensity was measured. The ET-1 concentration of each sample was calculated with a standard curve constructed by plotting the absorbance of each standard solution. Statistical analysis The differences in the clinical characteristics between the hypertensive and nonhypertensive subjects were assessed by the t-test and the Mann–Whitney test for normal and skewed continuous variables, respectively. The mean ± s.e. and the median (interquartile range) are presented, where appropriate. A linear regression analysis was used to evaluate the association between the ET-1 plasma levels and the clinical/metabolic parameters for the unadjusted and ageadjusted models. To identify the independent determinants of ET-1 and hypertension, a stepwise multiple regression analysis was performed in forward direction with the significance level for additions to the model set to 0.20. The trend association between the tertiles of the ET-1 levels and the percentage of subjects with hypertension was tested using linear regression analysis. Receiver operator characteristic curves and the corresponding area under the curves were used to evaluate the diagnostic values of the tertiles of the ET-1 for the hypertensive status. The optimal cut-off point was selected based on the receiver operator characteristic curves providing the maximum diagnostic efficiency (the maximum value of specificity % plus sensitivity %). Twosided P values o0.05 were considered statistically significant. All of the analyses were performed using Stata version 12.0 (Stata Corp, College Station, TX, USA). Hypertension Research Endothelin-1 and hypertension in Bangladesh S Akter et al 210 RESULTS The individuals screened in this study as hypertensive were unaware that they had hypertension. This screening was conducted as part of a non-communicable disease screening of apparently healthy women in rural Bangladesh. Table 1 shows the clinical characteristics of the subjects according to their hypertensive and non-hypertensive status. The hypertensive subjects were relatively older, and many of their cardiometabolic factors, including systolic blood pressure, diastolic blood pressure, fasting blood glucose, triglycerides levels and waist circumference were significantly higher than their non-hypertensive counterparts. However, the high-density lipoprotein cholesterol levels were significantly lower in the hypertensive subjects than in the non-hypertensive subjects (Po0.05 for all). The plasma ET-1 levels were significantly higher in the hypertensive subjects than in the non-hypertensive subjects (P = 0.001) (Figure 1). Table 2 shows the association between the ET-1 levels and the metabolic or other clinical parameters. After adjusting for age, the ET-1 levels were significantly associated with the systolic blood Table 1 Clinical characteristics of subject according to hypertensive status Characteristics Subjects (prevalence) Non-hypertensive Hypertensive 1798 (70.7) 745 (29.3) P-valuea 40.52 ± 1.34b 48.85 ± 1.21 o0.001 22.67 (20.00–25.00) 22.89 (19.74–25.73) 0.799 Age (years) BMI (kg m–2)c Waist circumference (cm) Triglycerides (mg dl–1)c 75.48 ± 0.75 159.65 81.49 ± 1.03 179.78 HDL cholesterol (mg dl–1) (100.35–176.96) 42.06 ± 1.36 (128.44–232.01) 36.50 ± 1.45 Systolic blood pressure (mm Hg) 106.95 ± 1.49 151.57 ± 1.96 o0.001 Diastolic blood pressure (mm Hg) 71.26 ± 0.79 89.56 ± 1.01 o0.001 MAP (mm Hg) o0.001 o0.001 0.006 83.18 ± 0.98 110.24 ± 1.21 o0.001 Fasting blood glucose (mmol l–1)c 6.20 (5.20–6.80) 6.60 (5.60–7.95) o0.001 Insulin (μU ml–1)c 6.55 (2.66–9.74) 6.70 (3.78–13.64) 0.359 Abbreviations: BMI, body mass index; HDL, high-density lipoprotein, MAP, mean arterial pressure. aBased on t-test for normal continuous variables, Mann–Whitney test for non-normal continuous variables. bMean ± s.e. for normal variables (all such values. cMedian (interquartile range) for non-normal variables (all such values). P=<0.001 Plasma endothelin-1 (pg/ml) 3.5 3.08 3.0 2.5 2.01 2.0 1.5 1.0 0.5 0.0 Non-hypertensive Hypertensive Figure 1 Mean levels of plasma endothelin-1 according to hypertensive and non-hypertensive status. Hypertension Research pressure (P = 0.001), diastolic blood pressure (P = 0.002), mean arterial pressure (MAP) (P = 0.001), fasting plasma glucose level (P = 0.002) and plasma high-density lipoprotein levels (P = 0.03). Other variables, including the body mass index, waist circumference, triglycerides and fasting insulin levels, were not associated with the ET-1 levels. A stepwise multiple regression analysis considering all of the variables presented in Table 1 revealed that the mean arterial pressure and fasting plasma glucose levels were independent determinants of the plasma ET-1 levels (Table 3). In addition, when we performed multiple logistic regressions for the hypertension status and the other predictors, we found that an elevated plasma ET-1 level had a strong association with hypertension (Table 4). Figure 2 shows the association between the tertiles of the ET-1 levels and the percentage of hypertension. As shown in the figure, hypertension was significantly increased in subjects as the levels of ET-1 increased (P for trend = 0.001). Figure 3 shows the receiver operator characteristic curve for hypertension and the tertiles of ET-1. The area under the curves values were moderately higher (0.758; 95% CI = 0.687–0.829; s.e. = 0.036; Po0.001), demonstrating the sensitivity and 1-specificity of the prediction of the hypertension risk for the different levels of ET-1. The cut-off point for the tertiles of ET-1 was found to be T3, as the ET-1 levels of T3 were 2.38 or more. The sensitivities and specificities of ET-1 were 0.563 and 0.866, respectively. DISCUSSION In this cross-sectional study of Bangladeshi women, the plasma ET-1 levels were significantly higher in the subjects with hypertension than in the non-hypertensive subjects, and both the systolic and diastolic blood pressures were positively associated with ET-1 after adjusting for age. To our knowledge, this is the first study in an apparently healthy South Asian population to address the association between the circulating levels of ET-1 and hypertension. The elevated ET-1 levels among the subjects with hypertension and the significant positive association between the ET-1 and the blood pressure in our study is consistent with some of the previous clinical studies.16–19 According to these previous studies, the plasma ET-1 levels were significantly higher in the patients with essential hypertension compared with the healthy controls. Moreover, in line with our study, Seissler et al.26 found a significant positive association between proET-1 and high blood pressure. However, the findings of our study were not consistent with a number of other studies. In an apparently healthy population, Hirai et al.23 found a significant positive association between ET-1 and the systolic and diastolic blood pressures in a univariate analysis; however, in a multiple stepwise regression model, both the systolic and diastolic blood pressures were not associated with ET-1. Similarly, Piatti et al.21 found no significant positive association of ET-1 with the systolic and diastolic blood pressures in a multiple regression analysis. Differences in the background characteristics of the study population may be a potential reason for the inconsistent findings across the studies. The present findings, together with a majority of clinical studies, suggest that hypertension may be associated with elevated ET-1 levels, but more studies from South Asian countries may clarify the relationship between ET and hypertension that we observed in the present study. In the present study, we clearly demonstrated through a tertile analysis that hypertension was significantly increased in subjects as the levels of ET-1 increased (P for trend = 0.001). Among the hypertensive subjects who belonged to the highest tertile of the ET-1 level, 50% of them had a fasting blood glucose level ⩾ 7 mmol l–1, 10% of them were overweight (body mass index ⩾ 25) and 100% of them had Endothelin-1 and hypertension in Bangladesh S Akter et al 211 Table 2 Association between endothelin-1 and others parameters Unadjusted Variables Coefficient (β) s.e. Age-adjusted P-valuea Coefficient (β) P-valuea s.e. Age (years) 0.0170 0.0077 0.029 BMI (kg m–2) Waist circumference (cm) 0.0055 0.0202 0.0225 0.0112 0.808 0.074 0.0090 0.0188 0.0223 0.0112 0.689 0.095 Triglycerides (mg dl–1) HDL cholesterol (mg dl–1) 0.0015 − 0.0213 0.0016 0.0088 0.338 0.017 0.0012 − 0.0193 0.0015 0.0088 0.436 0.030 Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) 0.0144 0.0294 0.0035 0.0078 o0.001 o0.001 0.0132 0.0265 0.0037 0.0083 o0.001 0.002 MAP (mm Hg) Fasting blood glucose (mmol l–1) 0.0232 0.0180 0.0056 0.0540 o0.001 0.001 0.0213 0.0168 0.0061 0.0542 0.001 0.002 − 0.0030 0.0060 0.615 −0.0020 0.0059 0.740 Insulin (μU ml–1) — — — Table 3 Multiple linear regression of plasma levels of endothelin-1 (pg ml–1) and others predictors Characteristics MAP (mm Hg) Fasting blood glucose (mmol l–1) Coefficient of (β) s.e. P-value 0.0251 0.0157 0.0078 0.0617 0.054 0.012 Abbreviation: MAP, mean arterial pressure. Table 4 Multiple logistic regression of hypertension status and others predictors Characteristics Odds ratio s.e. P-value Percentage of hypertensive subjects Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; MAP, mean arterial pressure. aBased on the regression analysis. P for trend < 0.001 80 79.0 60 41.0 40 21.3 20 0 T1 (< 1.99) T2 (1.99 - 2.38) T3 (> 2.38) Tertiles of endothelin-1 (pg/ml) Age (years) 1.058 0.019 0.004 Waist circumference (cm) Triglycerides (mg dl–1) 1.147 1.015 0.037 0.004 o0.001 0.001 17.319 0.829 0.001 Endothelin-1 (pg ml–1) Abbreviations: BMI, body mass index; HDL, high-density lipoprotein; MAP, mean arterial pressure. nn-Whitney test for non-normal continuous variables. Mean ± s.e. for normal variables (all such values). metabolic syndrome (as determined by modified National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III criteria). In addition, in the current analysis we found that the fasting blood glucose level had a positive association with the plasma ET-1 level, as demonstrated through both the univariate and multivariate regression analyses. Moreover, the plasma high-density lipoprotein level had a negative significant association with the plasma ET-1 level only in the univariate regression analysis. Thus, in addition to the blood pressure, the fasting blood glucose level was also a determinant for the plasma ET-1 levels in our subjects. However, when we performed a multiple logistic regression analysis for the hypertensive status and the other predictors, there was no significant association between hypertension and the fasting blood glucose level; conversely, there was a strong association between hypertension and the plasma ET-1 level in the logistic regression analysis. Although the mechanisms involved in the induction of ET-1 in hypertensive subjects remains unclear, several possible explanations have been suggested. The intravenous infusion of ET causes a rapid and transient vasodilation followed by a profound and long-lasting Figure 2 Percentage of subjects with hypertension according to the tertiles of endothelin-1. increase in blood pressure.5,27 In vitro experiments have demonstrated that this increase in blood pressure is related to the profound vasoconstriction of the resistance arteries of the different vascular beds of the circulation.28,29 ET-1 not only exerts direct vasoconstrictor effects but is also able to potentiate other vasoconstrictor substances such as norepinephrine and serotonin,30 which may be involved in the development of hypertension.31 Another mechanism may be the profound renal effects of ET-1.17,23 ET decreases the renal plasma flow and glomerular filtration rate at a level of the range at which no generalized alterations in hemodynamics occur.29 As the kidney plays a central role in the regulation of the chronic hemostasis of the pressure–volume regulation, these effects may have important roles for the development of hypertension. The major strengths of the present study include the use of a large community-based survey with a relatively large sample size and the exclusion of subjects with chronic diseases such as heart diseases and diabetes that may interact with the possible relationship between ET-1 and hypertension. However, the present study also has several limitations that need to be mentioned. First, the existence of an association derived from a cross-sectional study does not necessarily indicate causality. Thus, the present study cannot rule out whether the higher plasma ET-1 level observed in the present study in hypertensive subjects is the result of high blood pressure. A prospective cohort Hypertension Research Endothelin-1 and hypertension in Bangladesh S Akter et al 212 Specificity % 100 80 0 20 60 40 20 0 40 60 80 100 100 Sensitivity % 80 60 40 20 0 1 - Specificity % Figure 3 Receiver operating characteristic for the hypertensive subjects and the tertiles of endothelin-1. study design and serial plasma ET-1 measurements are essential to explore the relationship between the hypertensive status and plasma ET-1 levels. Second, in assessing the association between ET-1 and blood pressure, we were unable to adjust for important lifestyle risk factors, including smoking, alcohol consumption and physical activity. However, alcohol consumption and smoking are very uncommon among Bangladeshi women and thus are unlikely to alter the results after the additional adjustment for these variables. In conclusion, the findings of our study suggest that the plasma ET-1 level was elevated among the hypertensive subjects and was significantly and positively associated with the blood pressure among the rural women in Bangladesh. Prospective studies are needed to confirm the present cross-sectional findings among South Asian subjects. CONFLICT OF INTEREST The authors declare no conflict of interest. ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientific Research (overseas academic) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23406037, 23406016, 23406029, 24406026 and 25305034) and the Japan Society for the Promotion of Science. A current project (WDF11610) on gestational diabetes from the World Diabetes Foundation, Denmark to Health and Disease Research Center of Rural Peoples also supported a part of this work. 1 He J, Whelton PK. Epidemiology and prevention of hypertension. Med Clin North Am 1997; 81: 1077–1097. 2 Whelton PK. Epidemiology of hypertension. Lancet 1994; 344: 101–106. 3 Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: analysis of worldwide data. Lancet 2005; 365: 217–223. 4 Chow CK, Teo KK, Rangarajan S, Islam S, Gupta R, Avezum A, Bahonar A, Chifamba J, Dagenais G, Diaz R, Kazmi K, Lanas F, Wei L, Lopez-Jaramillo P, Fanghong L, Ismail NH, Puoane T, Rosengren A, Szuba A, Temizhan A, Wielgosz A, Yusuf R, Yusufali A, McKee M, Liu L, Mony P, Yusuf S. PURE (Prospective Urban Rural Epidemiology) Study investigators. Prevalence, awareness, treatment, and control of hypertension in rural and urban communities in high-, middle-, and low-income countries. JAMA 2013; 310: 959–968. Hypertension Research 5 Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411–415. 6 Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 1989; 86: 2863–2867. 7 Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal vascular tone. Lancet 1994; 344: 852–854. 8 Khraibi AA, Heublein DM, Knox FG, Burnett JC Jr. Increased plasma level of endothelin-1 in the Okamoto spontaneously hypertensive rat. Mayo Clin Proc 1993; 68: 42–46. 9 Kurihara Y, Kurihara H, Suzuki H, Kodama T, Maemura K, Nagai R, Oda H, Kuwaki T, Cao WH, Kamada N, Jishage K, Ouchi Y, Azuma S, Toyoda Y, Ishikawa T, Kumada M, Yazaki Y. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 1994; 368: 703–710. 10 Komuro I, Kurihara H, Sugiyama T, Yoshizumi M, Takaku F, Yazaki Y. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988; 238: 249–252. 11 Dawes KE, Cambrey AD, Campa JS, Bishop JE, McAnulty RJ, Peacock AJ, Laurent GJ. Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity. Int J Biochem Cell Biol 1996; 28: 229–238. 12 Faraco G, Moraga A, Moore J, Anrather J, Pickel VM, Iadecola C. Circulating endothelin1 alters critical mechanisms regulating cerebral microcirculation. Hypertension 2013; 62: 759–766. 13 Vuurmans TJ, Boer P, Koomans HA. Effects of endothelin-1 and endothelin-1 receptor blockade on cardiac output, aortic pressure, and pulse wave velocity in humans. Hypertension 2003; 41: 1253–1258. 14 Shohet RV, Kisanuki YY, Zhao XS, Siddiquee Z, Franco F, Yanagisawa M. Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy. Proc Natl Acad Sci USA 2004; 101: 2088–2093. 15 Suzuki N, Miyauchi T, Tomobe Y, Matsumoto H, Goto K, Masaki T, Fujino M. Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats. Biochem Biophys Res Commun 1990; 167: 941–947. 16 Saito Y, Nakao K, Mukoyama M, Imura H. Increased plasma endothelin level in patients with essential hypertension. N Engl J Med 1990; 322: 205. 17 Shichiri M, Hirata Y, Ando K, Emori T, Ohta K, Kimoto S, Ogura M, Inoue A, Marumo F. Plasma endothelin levels in hypertension and chronic renal failure. Hypertension 1990; 15: 493–496. 18 Amoroso A, Cossu MF, Mariotti A, Guido F, Ferri GM, De Rosa FG, Sportelli G. Increased plasma levels of endothelin in patients with essential arterial hypertension. Riv Eur Sci Med Farmacol 1996; 18: 33–37. 19 Januszewicz A, Lapiński M, Symonides B, Dabrowska E, Kuch-Wocial A, Trzepla E, Ignatowska-Switalska H, Wocial B, Chodakowska J, Januszewicz W. Elevated endothelin-1 plasma concentration in patients with essential hypertension. J Cardiovasc Risk 1994; 1: 81–85. 20 Kohno M, Yasunari K, Murakawa K, Yokokawa K, Horio T, Fukui T, Takeda T. Plasma immunoreactive endothelin in essential hypertension. Am J Med 1990; 88: 614–618. 21 Piatti PM, Monti LD, Galli L, Fragasso G, Valsecchi G, Conti M, Gernone F, Pontiroli AE. Relationship between endothelin-1 concentration and metabolic alterations typical of the insulin resistance syndrome. Metabolism 2000; 49: 748–752. 22 Davenport AP, Ashby MJ, Easton P, Ella S, Bedford J, Dickerson C, Nunez DJ, Capper SJ, Brown MJ. A sensitive radioimmunoassay measuring endothelin-like immunoreactivity in human plasma: comparison of levels in patients with essential hypertension and normotensive control subjects. Clin Sci (Lond) 1990; 78: 261–264. 23 Hirai Y, Adachi H, Fujiura Y, Hiratsuka A, Enomoto M, Imaizumi T. Plasma endothelin-1 level is related to renal function and smoking status but not to blood pressure: an epidemiological study. J Hypertens 2004; 22: 713–718. 24 Ergul S, Parish DC, Puett D, Ergul A. Racial differences in plasma endothelin-1 concentrations in individuals with essential hypertension. Hypertension 1996; 28: 652–655. 25 Jesmin S, Akter S, Rahman MM, Islam MM, Islam AM, Sultana SN, Mowa CN, Yamaguchi N, Okazaki O, Satoru K, Kimura S, Hiroe M, Mizutani T, Moroi M. Disruption of components of vascular endothelial growth factor angiogenic signalling system in metabolic syndrome. Findings from a study conducted in rural Bangladeshi women. Thromb Haemost 2013; 109: 696–705. 26 Seissler J, Feghelm N, Then C, Meisinger C, Herder C, Koenig W, Peters A, Roden M, Lechner A, Kowall B, Rathmann W. Vasoregulatory peptides pro-endothelin-1 and proadrenomedullin are associated with metabolic syndrome in the population-based KORA F4 study. Eur J Endocrinol 2012; 167: 847–853. 27 Kiowski W, Lüscher TF, Linder L, Bühler FR. Endothelin-1-induced vasoconstriction in humans. Reversal by calcium channel blockade but not by nitrovasodilators or endothelium-derived relaxing factor. Circulation 1991; 83: 469–475. 28 Dohi Y, Lüscher TF. Endothelin in hypertensive resistance arteries. Intraluminal and extraluminal dysfunction. Hypertension 1991; 18: 543–549. 29 Miller WL, Redfield MM, Burnett JC Jr. Integrated cardiac, renal, and endocrine actions of endothelin. J Clin Invest 1989; 83: 317–320. 30 Yang ZH, Richard V, von Segesser L, Bauer E, Stulz P, Turina M, Lüscher TF. Threshold concentrations of endothelin-1 potentiate contractions to norepinephrine and serotonin in human arteries. A new mechanism of vasospasm? Circulation 1990; 82: 188–195. 31 Echizen H, Freed CR. Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension. Life Sci 1984; 34: 1581–1589.