Urotensin-II levels in acute coronary syndromes

International Journal of Cardiology 108 (2006) 31 – 35 www.elsevier.com/locate/ijcard Urotensin-II levels in acute coronary syndromes Dominique Joyala, Thao Huynha,*, Nambi Aiyarb, Brian Guidab, Steven Douglas, Adel Giaida,1 a McGill University Health Center, Montreal General Hospital, Division of Cardiology, E-5-200, 1650 avenue Cedar, Montreal, Quebec, Canada H3G 1A4 b Cardiovascular and Urogenital Centre of Excellence for Drug Discovery, GlaxoSmithKline, King of Prussia, PA, United States Received 22 September 2004; received in revised form 28 March 2005; accepted 1 April 2005 Available online 10 May 2005 Abstract Background: Urotensin-II (U-II) is a vasoactive peptide with diffuse expression in human cardiomyocyte and vascular smooth muscle cells. Recent studies have reported increased plasma levels of U-II in patients with congestive heart failure. Objective: We sought to determine the plasma levels of U-II in patients with acute coronary syndromes (ACS), stable coronary artery disease (CAD) and healthy controls. Methods: We prospectively measured plasma U-II levels in 54 patients with ACS, 51 patients with stable coronary disease and 29 healthy volunteers. Monoclonal antibodies against U-II were generated and plasma U-II levels were determined by radioimmunoassay from extracted venous samples. Results: ACS patients had significantly lower levels than patients with stable CAD and healthy controls (2.53 T 1.62 vs. 3.45 T 2.53 vs. 3.3 T 3.9 ng/ml, p = 0.008, respectively). In both ACS and stable CAD patients, we found a negative relationship between plasma U-II levels and systemic arterial pressures. The correlation coefficients for systolic and mean arterial pressure were 0.272, p = 0.006 and 0.209, p = 0.04, respectively. Conclusions: Plasma U-II levels were significantly decreased in patients with acute coronary syndromes and related negatively to systemic arterial pressures. This finding suggests a down-regulation of U-II expression in patients with acute coronary syndromes. Condensed abstract: Urotensin-II (U-II) is a vasoactive peptide with diffuse staining in human cardiomyocytes and vascular smooth muscle cells. We measured plasma U-II levels in patients with acute coronary syndromes (ACS), stable coronary artery disease (CAD) and healthy controls. We observed lower U-II levels in ACS patients and a negative correlation between U-II levels and systemic arterial pressure. This finding suggests a down-regulation of U-II expression in patients with ACS. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Urotensin; Acute coronary syndrome; Coronary artery disease 1. Background Urotensin-II (U-II) is a cyclic 11-amino-acid vasoactive peptide first isolated from the caudal neurosecretory system of teleost fish. It has a controversial role in the human Abbreviations: U-II, Urotensin-II; ACS, acute coronary syndrome; CAD, coronary artery disease; MAP, mean arterial pressure; RIA, radioimmunoassay. * Corresponding author. Tel.: +1 514 934 1934x43241; fax: +1 514 934 8318. E-mail address: [email protected] (T. Huynh). 1 Dr. Adel Giaid is supported by the Fonds de la Recherche Scientifique du Quebec. 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2005.04.001 cardiovascular system. Initially labeled as the ‘‘most potent mammalian vasoconstrictor to date’’ [1], its importance in human vasculature is being extensively tested. U-II appears to have both vasoconstrictive [2] and vasodilating [3] properties depending on the vascular beds studied. We have recently demonstrated increased expression of U-II in human cardiomyocytes and vascular smooth muscle cells of patients with end-stage heart failure [4]. Plasma levels of U-II are elevated in patients with heart failure [5 –7] and correlate with the endothelial-cell-produced peptides endothelin-1 and adrenomodullin [6]. Although the source of plasma U-II is still debated, its elevation in heart failure patients suggests participation in neurohormonal activation. Furthermore, plasma U-II is increased in diabetes [8], renal 32 D. Joyal et al. / International Journal of Cardiology 108 (2006) 31 – 35 failure [9] and liver cirrhosis [10], advocating for a multisystemic involvement. The role of U-II in acute coronary syndromes (ACS) is currently unknown. Abnormal vasoreactivity is present in acute coronary syndromes and chronic coronary artery disease is associated with endothelial dysfunction manifested by impaired endothelium-derived-relaxing-factor vasodilation in resistance vessels. Vasoactive substances such as endothelin-1 and thromboxane A2 are increased in experimental models of ischemia [11,12]. Endothelin-1, the main vasoconstrictor released by the human endothelium, is increased in the early hours after a myocardial infarction [13] and correlates with clinical events [14]. With the ongoing debate on the vasoactive properties of U-II and its diffuse presence in vascular smooth muscle cells, we sought to determine plasma U-II levels in acute coronary syndrome. We hypothesized that U-II levels are elevated in patients with acute coronary syndrome when compared to patient with stable coronary disease and healthy controls. 2. Methods The study population consisted of patients admitted with ACS, patients with stable CAD and healthy volunteers. ACS was defined as typical ischemic chest pain of more than 20 min duration with additional evidence by one of the following: (a) ST segment deviation of at least 1 mm in at least two electrocardiogram leads, (b) previous myocardial infarction, (c) previous coronary angiogram demonstrating at least 50% narrowing of a major epicardial coronary artery or (d) elevations of cardiac markers (troponin T or CPKMB). Data prospectively obtained at enrollment included age, arterial blood pressure, cardiac enzymes, creatinine, cholesterol and conventional risk factors. All patients with ACS had measurement of their left ventricular ejection fraction during the index hospitalization, either by twodimensional echocardiography or ventriculography. Left ventricular systolic dysfunction was defined as left ventricular ejection fraction  40%. Blood sampling for U-II was performed within 24 h from the onset of the chest pain for all patients with ACS. Stable CAD was defined as previously documented coronary artery disease with no evidence of unstable angina or myocardial infarction within the previous 6 months. Our control group was composed of healthy volunteers without hypertension or hyperlipidemia. Blood pressure measurement was performed in all patients of the three groups at the same time as the blood sampling for U-II. This study was approved by the institutional ethic committee and all patients gave written informed consents. 2.1. Measurements of Urotensin-II levels Venous samples were collected into EDTA containing tubes. Samples were immediately spun and extracted plasma was stored at 70 -C and U-II levels were measured by radioimmunoassay (RIA). Plasma samples were extracted using an acid – acetone method. The recovery of this extraction method, evaluated by adding a known amount of (125I) human U-II to plasma, was between 80% and 90%. 2.2. RIA for U-II measurements The RIA incubation mixture (1 ml/tube) consisted of 100 Al standard human U-II (Phoenix Pharmaceuticals), 100 Al diluted mAb, (1:300,000 final dilution), 100 Al (125I) human U-II (25,000 cpm) and 700 Al standard buffer (50 mM phosphate buffer (pH 7.4) containing 10 AM NaEDTA and 0.1% BSA. Monoclonal antibodies against human U-II were generated from immunized mouse spleen at GlaxoSmithKline as described elsewhere [15]. The mixture was incubated for 16 h at 4 -C. Bound and free ligands were separated by addition of 0.25 ml secondary goat anti-mouse antibody (BioMagnetic Goat Antimouse IgG; Qiagen). Repeated RIA measurements (n = 8) of one selected plasma sample yielded an intra-assay variance of 10.5%. Unlike several pAb from other commercial sources, our antibody interacts with truncated human U-II analogs such as [4– 11] human U-II and [5– 11] human U-II. These fragments show almost identical retention time on rp-HPLC columns and are equipotent agonists at the urotensin receptors compared with mature U-II. Indeed, the antibody generated recognizes these multiple U-II species of pharmacodynamic importance present in human plasma [15] making it more sensitive than that of other commercial sources [16]. 3. Statistical analysis Categorical variables are presented as percentages. Continuous data are presented as means T standard deviation. All p-values were two-sided, with values < 0.05 considered significant. The Kruskal –Wallis test was used to compare the Urotensin-II values between the patients with ACS, stable CAD and healthy volunteers. The baseline clinical characteristic between the ACS and stable CAD patients were compared by Student’s t-test for continuous variables and chi-square testing for categorical variables. The Spearman’s correlation was used to assess the relationship between Urotensin-II and mean arterial systolic blood pressure. Statistical analysis was performed with SPSS 11.5, Chicago, Illinois. 4. Results Fifty-four patients with ACS and 51 patients with stable coronary disease were included. Twenty-nine healthy volunteers composed our control group. Patients baseline characteristics are summarized in Table 1. Mean age, blood D. Joyal et al. / International Journal of Cardiology 108 (2006) 31 – 35 Table 1 Baseline characteristics of patients Age (years) Male (%) Diabetes (%) Mean arterial pressure (mm Hg) Left ventricle’s ejection fraction (%) Troponin T (mmol/L) Low density lipoprotein (mmol/L) Creatinine (mmol/1) Duration of coronary disease (years) ACS (n = 54) Stable CAD (n = 51) P-value 61.7 T 13.7 40 (75%) 17.9% 98.56 T 14.23 64.4 T 10.8 43 (86%) 17.3% 128.23 T 18.10 NS NS NS NS 50.2 T 11.6 49.2 T 12.7 NS 2.7 T 4.6 2.9 T 0.9 NA 2.4 T 0.8 NA NS 110 T 88.5 3.4 T 6.1 113 T 17.7 7.4 T 7.0 NS 0.004 ACS: acute coronary syndromes. CAD: coronary artery diseases. pressure and ejection fraction were similar between ACS and stable CAD patients. Healthy volunteers who composed the control group were younger with a mean age of 45.8 T 12.9 years. The mean blood pressure was 117.9 T 24 mm Hg for systolic and 68.2 T 17.2 mm Hg for diastolic in the controls. The mean plasma U-II level in patients with ACS was significantly lower than in patients with chronic coronary disease and healthy controls (2.53 T 1.62 ng/ml vs. 3.45 T 2.53 ng/ml vs. 3.3 T 3.9 ng/ml, p = 0.008, respectively). There was no significant difference of the mean U-II between chronic stable CAD patients and healthy controls. Fig. 1 shows the boxplots of U-II levels between the three groups of patients. In patients with ACS and systolic dysfunction, we found decreased mean U-II levels compared to patients with ACS and preserved left ventricular systolic function. The mean UII levels in patients with left ventricle’s ejection fraction of > 40% and  40% were 2.78 T 1.73 ng/ml and 1.66 T 0.84 ng/ ml, p = 0.003, respectively. There was no significant correlation between U-II and either serum creatinine or troponin-T levels in patients with ACS. U-II levels in ACS patients with troponin levels of  1 ng/ml and > 1 ng/ml were 2.85 T 1.73 ng/ml and 2.10 T 1.4 ng/ml, p = 0.09, respectively. Mean U-II levels were negatively related to mean arterial blood pressure (Fig. 2). In the patients with ACS and stable CAD, significant negative correlations with both systolic and mean arterial pressure and U-II were observed. The correlation coefficient for systolic and mean arterial pressure with UII were 0.272, p = 0.006 and 0.209, p < 0.04, respectively. 5. Discussion The action of U-II on human vasculature is still a matter of debate. In in vitro studies, U-II emerges as a ubiquitous vasoconstrictor in both human arteries and veins. Human coronary, mammary and radial arteries as well as saphenous and umbilical veins constrict in the presence of human U-II [17]. Russell et al. reported a small increase in tone in left coronary circumflex specimen of one patient when exposed to U-II [18]. When injected into the brachial artery of healthy volunteers, U-II induces a dose-dependent reduction in forearm blood flow [2]. This demonstrates potent in vivo vasoconstrictive properties in humans. However, this response depends on the vascular bed studied. For instance, Urotensin-II (ng/ml) 12 8 4 0 acute coronary syndromes 33 stable coronary artery diseases controls Fig. 1. Urotensin-II levels (ng/ml) in patients with acute coronary syndromes, stable coronary artery disease and control group. 34 D. Joyal et al. / International Journal of Cardiology 108 (2006) 31 – 35 Fig. 2. Correlation between Urotensin-II levels (ng/ml) and mean arterial systolic pressure (mm Hg). in both human pulmonary and abdominal arteries, U-II does not induce vasoconstriction but is in fact a potent vasodilator, of similar magnitude to adrenomodullin [3]. The role of U-II in the human coronary circulation remains unclear and so does the effect of atherosclerosis on U-II’s expression and action. We demonstrated significantly lower U-II levels in patients with acute coronary syndromes when compared to patients with chronic coronary disease and healthy controls. As mentioned above, the plasma levels measured in our study represented the total U-II-like immunoreactivity. The lower U-II level in ACS found in our patients was surprisingly more pronounced in patients with low ejection fraction. This negative correlation with left ventricular function contradicts previous reports of patients with systolic dysfunction and U-II elevation. However none of the previous studies were performed in patients with acute coronary syndromes. Hence, previous findings of U-II elevation in chronic left ventricular dysfunction [5– 7] had not been reproduced in the context of myocardial ischemia. We found no correlation between U-II and troponin T levels, suggesting another mechanism of release of U-II than myocyte necrosis. The human vasculature endothelium expresses impaired vasoreactivity in acute coronary syndromes. It may be hypothesized that the reduced release of this new vasoactive peptide is related to an acutely damaged endothelium. This is in contrast to the potent vasoconstrictor endothelin-1 which remains increased in both instances [11– 14]. We observed a negative correlation between U-II levels and systemic arterial pressures. A significant negative correlation between U-II and mean arterial pressure was present in both ACS and stable CAD patients. Although this finding had not been yet reported in patients with cardiovascular diseases, Heller et al. [10] documented a similar correlation in patients with hepatic cirrhosis. The negative correlation between U-II and systemic arterial pressures and our results of decreased U-II levels in ACS challenge the previously described vasoconstrictive properties of this peptide. Does this imply a preferential vasodilatory action of U-II in human large arteries? This question remains unanswered pending the development of systemic U-II receptor antagonists. To date, both the mechanism and action of U-II on human arteries remain unclear. 6. Conclusions In summary, this is the first report showing significantly lower plasma U-II levels in patients with acute coronary syndromes. This finding suggests a down-regulation of U-II expression in the acute inflammatory process associated with acute coronary syndromes. Whether the decreased U-II levels contributed to the impaired endothelium-dependent relaxation seen with myocardial ischemia remains to be determined. References [1] Ames RS, Sarau HM, Chambers JK, et al. Human Urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999;401:282 – 6. [2] Bohm F, Pernow J. Urotensin II evokes potent vasoconstriction in humans in vivo. Br J Pharmacol 2002;135:25 – 7. [3] Stirrat A, Gallagher M, Douglas SA, et al. Potent vasodilator responses to human urotensin-II in human pulmonary and abdominal arteries. Am J Heart Circ Physiol 2001;280:H925 – 8. [4] Douglas SA, Tayara L, Ohlstein EH, et al. Congestive heart failure and expression of myocardial urotensin II. Lancet 2002;359:1990 – 7. [5] Ng LL, Loke I, O’Brien RJ, et al. Plasma Urotensin in human systolic heart failure. Circulation 2002;106:2877 – 80. D. Joyal et al. / International Journal of Cardiology 108 (2006) 31 – 35 [6] Richards MA, Nicholls MG, Lainchbury JG. Plasma urotensin II in heart failure. Lancet 2002;360:545 – 6. [7] Russell FD, Meyers D, Galbraith AJ. Elevated plasma levels of human urotensin-II immunoreactivity in congestive heart failure. Am J Physiol Heart Circ Physiol 2003;285:H1576 – 81. [8] Totsune K, Takahashi K, Arihara Z, et al. Increased plasma urotensin II in patients with diabetes mellitus. Clin Sci 2003;104:1 – 5. [9] Totsune K, Takahashi K, Arihara Z, et al. Role of urotensin II in patients on dialysis. Lancet 2001;358:810 – 1. [10] Heller J, Schepke M, Neef M, et al. Increased urotensin II plasma levels in patients with cirrhosis and portal hypertension. J Hepatology 2002;37:767 – 72. [11] Brunner F, du Toit EF, Opie LH. Endothelin release during ischemia and reperfusion of isolated perfused rat hearts. J Mol Cell Cardiol 1992;24(11):1291 – 305. [12] Zaugg CE, Hornstein PS, Zhu P, et al. Endothelin-1-induced release of thromboxane A2 increases the vasoconstrictor effect of endothelin-1 in post ischemic reperfused hearts. Circulation 1996;94:742 – 7. [13] Wieczorek I, Haynes WG, Webb DJ, et al. Raised plasma endothelin in unstable angina and non-q wave myocardial infarction: relation to cardiovascular outcomes. Br Heart J 1994;72(5):436 – 41. 35 [14] Omland T, Lie RT, Aakvaag A, et al. Unstable angina/myocardial infarction: plasma endothelin determination as a prognostic indicator of 1-year mortality after acute myocardial infarction. Circulation 1994; 89(4):1573 – 9. [15] Su JL, Ao Z, Aiyar NV, et al. Production and characterization of monoclonal antibodies against human urotensin-II. Hybrid Hibridomics 2003;22:377 – 82. [16] Douglas SA. Human urotensin-II as a novel cardiovascular target: ‘‘heart’’ of the matter or simply a fish ‘‘tail’’? Curr Opin Pharmacol 2003;3:159 – 67. [17] Maquire JJ, Kuc RE, Davenport AP. Orphan-receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin-1. Br J Pharmacol 2000; 131:441 – 6. [18] Russell FD, Molenaar P, O’Brien DM. Cardiostimulant effects of urotensin-II in human heart in-vitro. Br J Pharm 2001;132:5 – 9.