Dr Anthony Davenport - EMIT

Peptides 25 (2004) 1767–1774

Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries Janet J. Maguire∗ , Rhoda E. Kuc, Katherine E. Wiley, Matthias J. Kleinz, Anthony P. Davenport a Clinical

Pharmacology Unit, University of Cambridge, Level 6 Centre for Clinical Investigation, Box 110, Addenbrooke’s Hospital, Cambridge CB22QQ, UK Received 29 October 2003; accepted 9 January 2004 Available online 11 September 2004

Abstract We detected urotensin-II-like immunoreactivity in the endothelium of normal human blood vessels from heart, kidney, placenta, adrenal, thyroid and umbilical cord. Immunoreactivity was also detected in endocardial endothelial and kidney epithelial cells. In atherosclerotic coronary artery, immunoreactivity localized to regions of macrophage infiltration. Urotensin-II constricted human atherosclerotic epicardial coronary arteries with pD2 = 10.58 ± 0.46 (mean ± S.E.M.) and Emax = 11.4 ± 4.2% KCl and small coronary arteries with pD2 = 9.25 ± 0.38 and Emax = 77 ± 16% KCl. Small coronary arteries clearly exhibited a greater maximum response to urotensin-II than epicardial vessels. This enhanced responsiveness may be of importance in heart failure, where circulating concentrations of U-II are increased, or in atherosclerosis where focally up-regulated urotensin-II production may act down stream to produce significant vasospasm, compromising blood flow to the myocardium. We conclude that urotensin-II is a locally released vasoactive mediator that may be an important regulator of blood flow particularly to the myocardium and may have a specific role in human atherosclerosis. © 2004 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Coronary artery; Endothelium; Human pharmacology; Immunocytochemistry; Urotensin-II

1. Introduction A cardiovascular role for the fish peptide urotensin-II (UII) in mammals was initially suggested by its remarkably potent constrictor action on isolated arteries from non-human primates and the dramatic effect on myocardial contractility and systemic pressure observed following infusion of intravenous bolus U-II in the same species that resulted in death [2]. Recently, the plasma concentration of this vasoactive peptide has been reported to be significantly elevated in human diseases such as heart failure [16,18,19], diabetes mellitus [23] and liver cirrhosis [8]. U-II receptors (UT) have been ∗

Corresponding author. E-mail address: [email protected] (J.J. Maguire).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.01.028

identified in human heart and vascular smooth muscle using radioligand binding methods [12] and U-II elicits both constrictor [12,9] and dilator [22] actions in human blood vessels in vitro obtained from some, but not all, patients. Infusion of U-II, in vivo, decreased forearm blood flow in one study [3], suggesting that the predominant effect of U-II may be vasoconstriction, although a second study was unable to detect an effect of U-II [1]. The inconsistency and modest maximum constrictor response observed in the human vasculature both in vitro and in vivo contrasts markedly with effects in non-human primate and is indicative of the manifest species variation exhibited by U-II in mammals [5,11]. Therefore, although these data support the hypothesis that U-II may be an important cardiovascular peptide in man, this remains contentious.

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J.J. Maguire et al. / Peptides 25 (2004) 1767–1774

We have previously demonstrated the widespread distribution of human UT receptors and shown that U-II is a ubiquitous constrictor of human arteries and veins, including large diameter epicardial coronary artery [12]. However, it is not known whether these receptors are exposed to circulating or locally released U-II and, therefore, we have extended our investigation to identify the localization of the endogenous peptide, U-II, in human tissues. To seek additional evidence for a role for U-II in human cardiovascular disease, we have also determined the cellular expression of U-II-like immunoreactivity (U-II-LI) in human atherosclerotic coronary artery and diseased saphenous vein grafts and ascertained whether U-II also constricts atherosclerotic human coronary arteries and down stream small resistance coronary arteries that do not themselves exhibit disease.

2. Methods and materials The localization of U-II immunoreactivity was determined in human tissues. Evidence for increased expression of the peptide in atherosclerosis was obtained by comparison of sections of human normal and diseased coronary artery and failed saphenous vein grafts. The functional responses to UII of human diseased large epicardial coronary arteries and small resistance arteries were compared to those of the potent vasoconstrictor endothelin-1 (ET-1). 2.1. Tissue collection Human tissues were collected with local ethical approval. Human heart left ventricle, right atria, conducting system, aorta, saphenous vein grafts, epicardial and resistance coronary arteries were from the explanted hearts of patients transplanted for cystic fibrosis (n = 3), cardiomyopathies (n = 9), ischemic heart disease (n = 20), valve disease (n = 2), lung disease (normal hearts for whom no recipient available, n = 3) or from donor organs for whom no suitable recipient was available (n = 6). Mammary arteries (n = 3) were from patients receiving coronary artery bypass grafts. Samples of normal human kidney (n = 3) and lung (n = 7) were from patients undergoing nephrectomy for hypernephroma and lobectomy for lung tumors, respectively. Other human tissues investigated were thyroid gland, adrenal gland, placenta and umbilical artery and vein. 2.2. Materials U-II (human), ET-1 (human), somatostatin, substance P and rabbit anti-human U-II antisera were obtained from the Peptide Institute Inc. (Osaka, Japan). Mouse anti-human monoclonal CD68 antisera were from DakoCytomation (Ely, UK). Peptide stock solutions (0.1 mM) were prepared in 0.1% acetic acid (ET-1) or distilled water (U-II, somatostatin, substance P) and stored at −20 ◦ C. U-II antiserum was reconstituted in PBS. All other reagents were from Sigma-Aldrich

Ltd. (Dorset, UK) or VWR International Ltd (Poole, UK). Krebs’ solution comprised (mmol/l); NaCl, 90; NaHCO3 , 45; KCl, 5; MgSO4 ·7H2 O, 0.5; Na2 HPO4 ·2H2 O, 1; CaCl2 , 2.25; fumaric acid, 5; glutamic acid, 5; sodium pyruvate, 5; glucose, 10 (pH 7.4, when gassed with 95% O2 /5% CO2 ). 2.3. Immunocytochemistry Cryostat-cut sections of human tissues (30 ␮m), on polyl-lysine coated slides, were air-dried and fixed by immersion in ice-cold acetone for 10 min. Tissue sections were incubated for 1 h, at 23 ◦ C, in 5% swine serum in phosphate buffered saline (PBS) followed by overnight incubation, at 4 ◦ C, with rabbit-anti-human U-II primary antiserum, at 1:300 dilution in PBS containing 1% swine serum and 0.1% Tween-20. Tissue sections were then washed and specific staining revealed using the peroxidase anti-peroxidase method, with diaminobenzidine as the chromogenic substrate. Following alcoholic dehydration the slides were cleared in xylene and mounted using a coverslip and permanent-mounting medium. Adjacent sections of atherosclerotic coronary artery were incubated with antiserum against the macrophage marker CD68. Control experiments were carried out to check the specificity of the human U-II antibody. These included preabsorbing the antiserum with human U-II peptide (1 ␮mol/l) at 4 ◦ C for 24 h and testing for cross-reactivity with ET-1, substance P and the structurally related peptide somatostatin, in an antibody dilution enzyme-linked immunosorbent assay (ELISA). 2.4. In vitro pharmacology Human heart tissue was transported to the laboratory in oxygenated Krebs’ solution (4 ◦ C). Main stem coronary artery (right or left anterior descending) was removed, cleaned of connective tissue and cut into 4 mm rings. The endothelium was removed by gently rubbing the luminal surface with a blunt seeker (verified by lack of vasodilator response to 1 ␮mol/l acetylcholine in vessels pre-constricted with 10 ␮mol/l phenylephrine). Vessel rings were set up for isometric tension recordings in 5 ml organ baths containing oxygenated (95% O2 /5% CO2 ) Krebs’ solution maintained at 37 ◦ C. Data were collected using the MP100 data acquisition system (Biopac Systems Inc., CA, USA) and chart recorder. Following a 60 min equilibration period, basal tension was optimized by construction of a length–active tension curve using 100 mmol/l KCl as previously described [10] with the response to KCl determined as force developed per millimetre segment of blood vessel (mN mm−1 ). Resistance coronary arteries (1–2 mm segments), isolated from the apex of the heart, were mounted on 40 ␮m steel wires in a Mulvany myograph for isometric tension recordings. The endothelium was removed using a human hair (verified by the absence a of dilator response to 100 nmol/l bradykinin after pre-constriction with 100 nmol/l U46619). Following a 60 min equilibration period, basal tension was optimized


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automatically using the active wall tension to length relationship, with each segment set to 90% of the internal diameter at which optimal force is obtained under these conditions [15]. Maximal force (mN mm−1 ) was measured three times with a potassium rich Krebs’ solution (100 mmol/l) at 15 min intervals. All arteries were allowed to equilibrate for a further 60 min. 2.4.1. Vasoconstrictor studies In endothelium-denuded large and resistance arteries, cumulative concentration response curves were constructed to U-II (1 pmol/l–10 nmol/l) and ET-1 (0.1–300 nmol/l). In large coronary arteries, experiments were terminated by the addition of 100 mmol/l KCl to determine the maximum possible response and agonist responses were expressed as a percentage of this. For small arteries, agonist responses were expressed as a percentage of the mean of the three KCl responses obtained prior to the agonist concentration–response curves. 2.4.2. Vasodilator studies Endothelium-denuded large and small arteries were constricted with 10 nmol/l ET-1 and U-II (1 pmol/l–10 nmol/l) concentration response curves constructed to identify direct vasodilatation of smooth muscle. Adjacent rings were constricted with ET-1 but were not exposed to U-II and acted as time-matched controls. 2.5. Statistical analysis For the in vitro pharmacology experiments, concentration–response curves were fitted to a four parameter logistic equation using the FigP 2.98 Software (Biosoft, Cambridge, UK) to obtain values of EC50 (molar concentration producing 50% of the maximum response) and Emax (maximum response expressed as a percentage of a control response to 100 mmol/l KCl). Potency values were normalized by logarithmic transformation to pD2 values (−log10 EC50 ). Where appropriate, responses to U-II and ET-1 were compared using Student’s two-tailed t-test, with a P value of 0.05, Student’s two-tailed t-test). U-II contracted

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Fig. 2. Examples of the localization of immunoreactive-U-II to human tissues. Staining was present in the endothelium of (A) aorta, (B) coronary artery, (C) mammary artery, (D) saphenous vein and to small vessels within (E) ventricle and (F) atria of the heart, (G) lung, (H) placenta, (I) adrenal gland and (J) vasa vasorum in the adventitia (a) of coronary artery. Immunoreactivity was also identified to (K) endocardial endothelial cells and (L) renal tubules. Scale bars = 100 ␮m.


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Fig. 3. Immunoreactive U-II localized to (A) the atherosclerotic lesion of human coronary artery and (B) the thickened intima of a failed human saphenous vein graft. Adjacent sections of atherosclerotic coronary artery demonstrating (C) localization of infiltrating macrophages detected using CD68 and (D) absence of U-II immunoreactivity following pre-absorption with U-II peptide. Scale bar = 200 ␮m.

atherosclerotic epicardial coronary arteries with a pD2 value of 10.58 ± 0.46 and Emax value of 11.4 ± 4.2% KCl (n = 4). This was significantly more potent than the response to ET-1, pD2 8.06 ± 0.01 (n = 3) (P < 0.005, Student’s twotailed t-test), although the maximum response to ET-1 was significantly greater, 66.8 ± 6.2% KCl (n = 3), than that to U-II (P < 0.001, Student’s two-tailed t-test) (Fig. 4A). The mean internal diameter of the small human coronary arteries was 428.0 ± 43.2 ␮m (n = 14) consistent with the size of arteries that contribute substantially to vascular resistance. The response of these vessels to KCl was 1.15 ± 0.19 mN mm−1 (n = 8) in those segments used to test U-II and 2.21 ± 0.57 mN mm−1 (n = 8) in those used for ET-1 (not significantly different, P > 0.05, Student’s two-tailed t-test). U-II contracted vessels with a pD2 value of 9.25 ± 0.38 and Emax of 77 ± 16% KCl (n = 8) compared to pD2 for ET-1 of 8.61 ± 0.13 and Emax of 137 ± 12% KCl (n = 8). The potency of the two peptides did not differ signif-

icantly (P > 0.05, Student’s two-tailed t-test) whereas the maximum response to ET-1 was approximately double that obtained with U-II (P < 0.01, Student’s two-tailed t-test) (Fig. 4B).

Fig. 4. Concentration–response curves to U-II (䊉) and ET-1 () in endothelium-denuded human (A) atherosclerotic epicardial coronary arteries (n = 4) and (B) small coronary arteries (n = 8). Values are mean ± S.E.M.

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3.2.2. Vasodilatation studies No vasodilator action of U-II was observed in endothelium-denuded large epicardial (n = 4) or small resistance coronary arteries (n = 4) that had been pre-constricted with 10 nmol/l ET-1.

4. Discussion We have previously reported that UT receptors are present on the vascular smooth muscle of human blood vessels and mediate vasoconstriction in endothelium-denuded preparations of human large arteries and veins in vitro [12]. Endothelium-dependent vasodilatation in human resistance vessels in vitro has also been observed [22]. In this study, we have demonstrated that U-II-LI is discretely expressed in the endothelial cells of the human vasculature. These data strongly suggest that, physiologically, U-II is a locally acting vasoactive mediator. Peptide released from endothelial cells may act in a paracrine manner on the underlying smooth muscle to elicit vasoconstriction or in an autocrine manner to release vasodilator substances from endothelial cells. Our functional experiments suggest that smooth muscle UT receptors are present throughout the human coronary artery tree, from large epicardial to small resistance arteries. The lack of any direct vasodilator action of U-II supports our hypothesis that smooth muscle UT receptors mediate vasoconstriction. The maximum response of these vessels to exogenous U-II, expressed as a percentage of a control potassium chloride response, was significantly greater for the smaller compared to larger diameter coronary arteries. This implies that U-II has potential to have a more important role in modulating resting tone in these vessels than in the epicardial arteries and may therefore contribute to the normal regulation of blood flow to the heart muscle. Whether U-II plays an important role in the pathogenesis of human disease is not yet certain. Recent clinical investigations have measured plasma U-II levels in patients with heart failure. In three studies, compared to healthy controls, U-II plasma concentration was elevated in patients with heart failure [16,18,19] with no obvious correlation with the severity of the condition [16,19]. One other study failed to detect any difference in plasma U-II levels in patients with severe and moderate congestive heart failure compared to controls [7]. The variability in control plasma concentrations reported in these studies (from 2 to 60 pmol/l) and the lack of correlation of plasma U-II to disease severity observed [16,19] might suggest that plasma concentration does not reflect local tissue concentration but rather the net effect of spill-over and elimination of U-II. Alternatively, differences in the methodologies used to prepare plasma samples and detect U-II concentrations or specificity of the antibodies employed may be sufficient to explain this discrepancy. However, the finding that U-II plasma levels were significantly elevated from the earliest stages of heart failure may prove useful in diagnosis.

Despite this clinical controversy, in vitro data does support a role for U-II in normal cardiac function and potentially in heart disease. We previously detected low levels of UT receptor expression in human left ventricle [12] and UII is a positive inotrope in human isolated trabeculae, increasing force of contraction without alteration in time to peak force or 50% relaxation [20]. One possible source of U-II acting on the heart is the endocardial endothelial cells, in which we detected U-II-LI. Since we localized U-II-LI to endocardial endothelial cells in sections of left ventricle from patients transplanted for cardiomyopathy and ischemic heart disease it is interesting to speculate, given the proximity of the signal to the target cell, that this peptide may contribute to alterations in cardiac function observed in heart failure. It is unlikely that cardiomyocytes are a major source of U-II in the healthy heart, as we were unable to detect U-II-LI in cardiomyocytes from left ventricle or right atria. This observation confirms initial reports showing a lack of expression of U-II mRNA in human heart by dot blot analysis [4,17] and later studies in native tissue showing low or no expression in human heart [7,14]. Interestingly there is one report of marked up-regulation of U-II mRNA and protein expression in cardiomyocytes and inflammatory cells in patients with end stage heart failure [6] suggesting that the diseased myocardium may become an additional source of the peptide as disease progresses. Up-regulation of U-II peptide expression in the heart has been reported in a rat model of heart failure after myocardial infarction, however the localization of the peptide was restricted to chymase positive mast cells in the infarct area with diffuse staining in the interstitium of peri-infarct regions [24]. In addition to effects on force development and coronary tone, pathophysiological levels of U-II may also contribute to cardiac remodeling. U-II has been shown to induce hypertrophic responses in cultured cardiomyocytes from neonatal rats [24,25] and stimulated collagen synthesis in neonatal cardiac fibroblasts [24]. We observed that U-II potently constricts atherosclerotic epicardial human coronary arteries and have demonstrated the expression of U-II-LI in areas that correspond to regions of macrophage infiltration within the atherosclerotic lesions. Immunoreactivity also localized to the thickened intima of failed saphenous vein grafts. Therefore, in coronary artery or graft disease focally produced U-II may elicit vasoconstriction by a direct effect on the diseased blood vessel. The degree of constriction in the large diseased arteries was modest compared to KCl or ET-1 (Table 1) and by itself may not substantially affect blood flow even in an artery whose lumen is reduced by the presence of atheroma. However, U-II is only one of several vasoactive mediators affected by atherosclerosis and therefore U-II mediated vasoconstriction will contribute to the overall consequence of the disease process. A more significant corollary of potentially macrophage derived U-II might be its direct effect on small resistance arteries further down the coronary tree. These also constrict to low concentrations of U-II, but more


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Table 1 The relative efficacy of urotensin-II and endothelin-1 in human blood vessels indicating the comparably greater maximum response to U-II in small coronary arteries Human blood vessel

Coronary arterya Mammary arterya Radial arterya Saphenous veina Umbilical veina Atherosclerotic coronary artery Small coronary artery a

Urotensin-II

Endothelin-1

Relative efficacy U-II:ET-1

Emax (% KCl)

n

Emax (% KCl)

n

15.4 ± 6.5 16.4 ± 6.2 19.7 ± 6.3 31.5 ± 9.5 16.6 ± 10 11.4 ± 4.2 77.2 ± 16

6/9 5/7 4 5 4 4 8

83.8 ± 6.0 82.1 ± 6.9 57.5 ± 10.1 94.8 ± 3.4 82.6 ± 8.2 66.8 ± 6.2 137 ± 12

9 6 4 3 3 3 8

0.18 0.20 0.34 0.33 0.20 0.17 0.56

Data from reference [12].

importantly, the maximum response to U-II in these vessels was much greater than that in the epicardial arteries and indeed to that observed by us in other human large arteries and veins (Table 1). Therefore, in atherosclerosis locally increased levels of U-II, producing vasoconstriction of small coronary arteries, has the potential to significantly attenuate blood flow to the vulnerable myocardium. The possible lethal consequence of U-II-mediated contraction of the coronary arteries has been graphically demonstrated in monkeys. A bolus infusion of U-II resulted in alterations in the electrocardiogram consistent with myocardial infarction due to acute coronary spasm [2], with death occurring at the highest doses tested. In normal human kidney, in agreement with Shenouda and colleagues [21], we find U-II immunoreactivity localizing to vascular endothelium and to renal tubules and ducts. We have previously detected UT receptors in human kidney cortex [12] and therefore the co-expression of receptor and peptide provides further evidence that U-II may be an important renal peptide with effects on renal blood flow and salt/water balance. Moderate U-II immunoreactivity is reported to be expressed by renal carcinoma cells, blood vessels and inflammatory cells within the tumor mass [21]. These authors suggest a mitogenic role for U-II in renal disease that is supported by the observation that U-II is a growth factor for the porcine renal epithelial cell line, LLCPK1 [13]. In conclusion, we have demonstrated that U-II is a ubiquitous endothelial derived vasoactive mediator in human tissues. Our observation of increased expression of the peptide in atherosclerotic human coronary artery and failed saphenous vein graft together with the marked vasospastic response to U-II in small coronary arteries contributes further evidence of a role for this emerging transmitter system in human cardiovascular disease.

Acknowledgements This work was supported by the British Heart Foundation and the Royal Society. We are grateful to Jean Chadderton and the Theatre and Consultant staff of Papworth Hospital for assistance in tissue collection.

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