ORIGINAL ARTICLE

Heart Vessels (2009) 24:393–398 © Springer 2009DOI 10.1007/s00380-008-1136-8

I. Dimitrijevic (*) · U. Ekelund · M.-L. Edvinsson · L. EdvinssonDivisions of Emergency Medicine and Experimental Vascular Research, Department of Clinical Sciences, Lund University Hospital, BMC A13, SE-221 84 Lund, SwedenTel. +46-46-176-749; Fax +46-46-222-0616e-mail: ivan.dimitrijevic@med.lu.se

Ivan Dimitrijevic · Ulf Ekelund · Marie-Louise Edvinsson Lars Edvinsson

Increased expression of endothelin ETB and angiotensin AT1 receptors in peripheral resistance arteries of patients with suspected acute coronary syndrome

Abstract Patients who experience chest pain, in which isch-emic heart disease has been ruled out, still have an increased risk of future ischemic cardiac events and premature death, possibly due to subclinical endothelial dysfunction. A feature of endothelial dysfunction is an increased expres-sion of arterial vasoconstrictor endothelin (ET) and angio-tensin (AT) receptors. Our aim was to investigate if the arterial expressions of these receptors are changed in patients with suspected but ruled out acute coronary syn-drome (ACS). Small subcutaneous arteries (diameter of 100 μm) were surgically removed in an abdominal biopsy from 12 patients suspicious of ACS (susp ACS), admitted to the medical telemetry unit for chest pain. The vessels were analyzed for their receptor protein expression by quantitative immunohistochemistry using specifi c antibod-ies directed against ETA, ETB, AT1, and AT2 receptors. The control group (controls) consisted of eight healthy volun-teers matched for age and sex with no previous cardiac illness or medication. The susp ACS group had an increased expression of ETB (by 94%) and AT1 (by 34%) receptors in the smooth muscle cells of resistance arteries as com-pared to the control group. There were no signifi cant dif-ferences in AT2 and ETA receptor expression between the groups. The results indicate that the expression of arterial smooth muscle ETB and AT1 receptors are increased in patients with suspected but ruled out ACS. These receptor changes could be important in the regulation of coronary tone and in the development of atherosclerosis, and may be related to increased cardiovascular risk.

Key words Vasculature · Ischemic heart disease · Endothe-lin · Angiotensin · Immunohistochemistry

Introduction

Despite a tremendous improvement in the therapy of isch-emic heart disease over the last decades, our understanding of the disease and its underlying vascular pathophysiology is still incomplete. As an example, there seems to be an increased risk of future ischemic cardiac events and prema-ture death even in chest pain patients where current isch-emic heart disease is ruled out.1 The reason for this is unknown, but it might be related to a subclinical endothelial dysfunction leading to arteriosclerosis. Dysfunctional endo-thelium may also cause inappropriate arterial vasoconstric-tion, in itself or by various stimuli,2,3 causing myocardial ischemia. An important feature of endothelial dysfunction4 is likely an increased expression of vasoconstrictor endothelin (ET), angiotensin (AT), and serotonin (5-hydroxytrypatamine; 5-HT) receptors, and this has been observed in established ischemic vascular disease, stroke,5 hypertension6 and diabetes.7

Patients with chest pain suspicious of unstable angina pectoris or myocardial infarction (i.e. acute coronary syn-drome, ACS) are very common in emergency medicine. Of those who are admitted to in-hospital care for suspected ACS, only around 30% prove to have the syndrome.8 In the present study, we hypothesized that patients with suspected but ruled out ACS have an increased expression of vaso-constrictor receptors in their resistance arteries. The aim of this study was thus to compare the expression of arterial ETA, ETB, AT1, and AT2 receptors in patients admitted for suspected ACS to healthy subjects without known athero-sclerotic disease.

Materials and methods

Patients and tissue collection

After informed consent, we included patients admitted to the telemetry ward at Lund University Hospital solely for nontraumatic chest pain suspicious of ACS. The admitting

Received: October 8, 2007 / Accepted: November 25, 2008

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physician’s suspicion of ACS was not reviewed. At the time of inclusion, all patients had normal or nonischemic ECGs and were negative for repeated blood markers of myocar-dial injury (troponin T < 0.05 μg/l and creatine kinase MB < 6.0 μg/l), and none had a previous myocardial infarction. The control group consisted of healthy volunteers with no history of chest pain and cardiovascular disease. Character-istics of patients and controls are detailed in Table 1.

After local anesthesia (2 ml, 1% lidocaine), subcutane-ous biopsies of 1 cm3 containing small arteries (diameter 100 μm) were removed from the abdomen of all patients and control subjects by the same surgeon. The biopsies were frozen in ice-cold isopentane for immunohistochemis-try. The study was approved by the regional ethics commit-tee at Lund, Sweden (Lu Dnr:308/2004).

Immunohistochemistry

The biopsies were stored at −80°C prior to sectioning into 8-μm-thick slices in a calibrated Microm HM500M cryostat (Microm, Walldorf, Germany). The sections were fi xed and rehydrated in phosphate-buffered saline (PBS). The sec-tions were incubated overnight with rabbit antihuman ETB (16207, IBL) diluted 1:400, goat antihuman ETA (sc-21194, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) diluted 1:50, rabbit antihuman AT1 (sc-1173, Santa Cruz Biotech-nologies) diluted 1:50, rabbit antihuman AT2 (sc-9040, Santa Cruz Biotechnologies) diluted 1:50, mouse antihu-man CD-31 (M0823, DAKO, Glostrup, Denmark) diluted 1:100, mouse antihuman actin (M0851, DAKO) diluted

1:1000. All dilutions were done in PBS with 10% fetal calf serum. The secondary antibodies used were donkey anti-rabbit CyTM3 conjugated (711-165-152, Jackson ImmunoRe-search, West Grove, PA, USA) 1:100, donkey antigoat CyTM3 conjugated (705-165-003, Jackson ImmunoResearch) 1:100 and donkey antimouse Texas Red conjugated (715-076-150, Jackson ImmunoResearch) 1:200 in PBS. As control, only secondary antibodies were used. The samples were examined in a fl uorescence microscope (Bx60F5, Olympus Optical, Tokyo, Japan) and absolute fl uorescence intensity was measured with ImageJ software (http://rsb.info.nih.gov/ij/). The measurements were performed where positive staining for smooth muscle was found, hence in the medial layer of the vessels. Mean fl uorescence values in the area selected were obtained. Each sample was investigated blinded to the observer in three sections and at four differ-ent regions, and the mean values were used.

Statistics

Data are presented as mean ± SD. NPar tests and Mann–Whitney tests were used to process the data. P values below 0.05 were considered to be statistically signifi cant. Statistical analysis were performed using SPSS software (version 15.0; SPSS, Chicago, IL, USA).

Results

Subcutaneous vessels were obtained from 12 patients (age 36–88 years; 4 females) admitted for chest pain with sus-pected ACS (susp ACS) and 8 healthy volunteers (age 30–76 years, 4 females) without known cardiovascular disease (controls). The ages between the two groups were similar (susp ACS; 64 ± 11 years, controls; 62 ± 14, P not signifi cant) and the HbA1c was slightly elevated in the susp ACS group. Despite the signifi cantly more frequent use of beta-blockers in the susp ACS group, the systolic blood pressure was still slightly higher than in the control group (Table 1). No other differences were detected between the groups and none of the patients in the susp ACS group had ACS at the time of discharge.

ETB and AT1 receptor expressions were higher in the smooth muscle cell layer in the arteries from the patients with susp ACS than in the arteries from the control group (Fig. 1A). In endothelial cells, it was primarily ETB receptor immunostaining that was observed. There were signifi cantly higher expressions of ETB (+94% ± 10%) and AT1 (+34% ± 4%) receptors in the susp ACS group, (P < 0.001 for both) compared to the control group (Fig. 2). The levels of ETA and AT2 receptor expression in the smooth muscle cells were similar in both groups (P > 0.05), as demonstrated in Fig. 1B. The enhanced fl uorescence in the susp ACS sample is seen over the smooth muscle cell layer as demonstrated by the colocalization of the receptors and actin with double immunostaining (Fig. 3). We also observed a weak ETB and AT1 receptor expression in the endothelial cells, but there were no differences between the two groups (Fig. 3).

Table 1. Characteristics of patients and controls

Controln = 8

Suspected ACSn = 12

Age (years) 62 ± 14 64 ± 11Smokers (n) 1 3SBP (mmHg) 126 ± 10 142 ± 7*DBP (mmHg) 70 ± 8 76 ± 7Total cholesterol (mg/dl) 4.8 ± 1.9 5.2 ± 1.3LDL (mg/dl) 3.5 ± 0.8 3.4 ± 1.2HDL (mg/dl) 1.1 ± 0.3 1.6 ± 0.5Triglycerides (mg/dl) 1.2 ± 0.5 1.8 ± 1.2Apolipoprotein A (mg/dl) 1.7 ± 0.2 1.5 ± 0.2Apolipoprotein B (mg/dl) 0.8 ± 0.2 0.9 ± 0.3NT-pro-BNP (ng/l) 120 ± 123 242 ± 267C-reactive protein (mg/dl) 1.6 ± 0.9 7.1 ± 17.1Beta blockers (%) 0 50%*Statins (%) 0 0ARB or ACE (%) 0 25%HbA1c (%) 4.3 ± 0.4 4.7 ± 0.4*BMI (kg/m2) 27.5 ± 1.7 30.0 ± 4.9

Background characteristics for patients with suspected ACS without established myocardial infarction and cardiovascular healthy controls. Values are expressed as mean ± SD. Statistical analysis was performed using NPar tests and Mann–Whitney tests. Signifi cance was defi ned as * P < 0.05SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; NT- proBNP, N-terminal pro B-type natriuretic peptide; ARB, angiotensin receptor blockers; ACE, angiotensin-converting enzyme; BMI, body mass index

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Discussion

This is the fi rst study demonstrating that the expression of smooth muscle cell ETB and AT1 receptors are increased (by 94% and 34% respectively) in small resistance arteries in patients with suspected but ruled out ACS, compared to healthy matched individuals. The enhanced receptor expres-sion seems limited to specifi c receptors, since no differences in the expression of smooth muscle ETA and AT2 receptors were found between the two groups. In addition, the differ-ent expression appears to be confi ned to the smooth muscle cells, as there were no differences in the endothelial recep-tor expressions between controls and susp ACS patients. The small arteries used are well suited for the study, since endothelin receptor mediated constriction is more impor-tant in small resistance arteries9–11 than in larger conduc-tance arteries.10,12,13

The cause of the increased ETB receptor expression in the susp ACS group is unclear. There is increasing evidence that the balance between vascular ETA and ETB receptors may be altered in different conditions,14–16 including diabe-tes mellitus,7 hypertension,17 and increased blood fl ow,18 which are associated with increased expression of ETB

receptors in arterial smooth muscle cells. Also, there is evi-dence to indicate an increased expression of coronary ETB receptor mRNA in established ischemic heart disease.19 Contradicting data exist, however, with an observed down-regulation of endothelin receptors in hypertensive rats.20

The increased ETB receptor expression in our patients with suspected ACS could be a result of both the slightly higher systolic blood pressure and the slightly increased blood glucose levels over time, manifested as an elevated HbA1c. Lind et al. observed enhanced contraction to endothelin-1 in hypertension, suggesting upregulation of endothelin receptors in this state.6 We found, however, no changes in the expression of ETA and AT2 receptors; this may implicate that they are of less importance for the dif-ference in prognosis between our two study groups. Even if the role of AT2 receptors is controversial in essential hypertension,21 we found no change in the expression of this receptor.

In a similar way, several mechanisms might explain the increased AT1 receptor expression in our patients with sus-pected ACS. Infl ammatory mediators such as IL-1 and IL-6 up regulate AT1 receptors and enhance AT II-stimulated vessel hypertrophy.22 C-reactive protein (CRP) may increase AT1 receptor expression,23 and there is thus a distinct

Fig. 1A, B. ETB and AT1 (A), and ETA and AT2 (B) receptor expression assessed by immunohistochemistry, in human subcutaneous arteries from patients with suspected acute coronary syndrome without established myocardial infarction (n = 12) and cardiovascular healthy controls (n = 8). Note that the staining intensity for both ETB and AT1 receptors is higher in the arteries from patients with suspected acute coronary syndrome (susp ACS) than in those from healthy controls. We found no differences in ETA and AT2 receptor expression between the studied groups. Bar 40 μm

Fig. 2. AT1 and ETB receptor expression at protein level assessed by immunohistochemistry in human subcutaneous arteries without known cardiovascular disease (Control, n = 8) and from patients suffering from chest pain associated with suspected ACS (Susp ACS, n = 12). The

immunofl uorescence intensity over the smooth muscle cells was quan-tifi ed by using the Image J program. Values are presented as mean values ± SD. Statistical analyses were performed using the Mann–Whitney test, where P < 0.05 is considered signifi cant (asterisk)

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Fig. 3. Immunohistochemical staining and co-localization of the AT1 and ETB receptors in resistance arteries of control patients. Antibodies to AT1 (green, A1, arrow) and smooth muscle actin (red, A2, arrow), A3 (merged A1 and A2). Antibodies to ETB (green, B1, arrow) and

smooth muscle actin (red, B2, arrow), B3 (merged B1 and B2). Anti-bodies to AT1 (green, C1, arrow) and CD-31 (red, C2, arrow), C3 (merged C1 + C2). Antibodies to ETB (green, D1, arrow) and CD-31 (red, D2, arrow), D3 (merged D1 and D2). Scale: A–D × 40

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possibility that the increased AT1 receptor is associated with vascular infl ammation. Although the difference in the mean CRP levels in the susp ACS and control groups did not reach statistical signifi cance (Table 1), the mean CRP levels were different. Because of the small size of our study, we cannot exclude a higher level of vascular infl ammation in the susp ACS group. If so, this could of course contribute to the observed higher AT1 receptor expression. Altered vascular expression of AT receptors has also been shown in conditions such as hypertension,24 congestive heart failure,25 hypoxia,26 hypercholesterolemia,27 and hyperglycemia.28 The higher systolic blood pressure in the susp ACS group may thus partly explain the higher expression of AT1 receptors in this group.

Specifi c upregulation of AT1 but not AT2 receptors has been found in insulin-dependent diabetes mellitus.29 In the present study we observed a slightly higher HbA1c level in the susp ACS group, indicating a possible impaired glucose metabolism. Since the average level in the susp ACS group was below the cutoff for diabetes, it is unclear whether an impaired glucose metabolism could have caused the increased AT1 receptor expression in this group. There are, however, studies to support this relation.30

AT1 receptor activation leads to vasoconstriction, induc-tion of infl ammatory, fi brotic, and thrombotic processes,31,32 and to increased vascular reactive oxygen species (ROS) production,31 which in turn may result in impaired endothe-lium-dependent vasodilatation.33 There is in fact a clear relationship between the AT1 receptors and endothelial dysfunction. For instance, AT1 receptor inhibition has been shown to inhibit ROS production and improve endothelial function in human vessels.34,35 This could implicate that the increased AT1 receptor expression observed in our patients is instrumental in the development of endothelial dys-function caused by oxidative stress. Since endothelial dysfunction is associated with progression of coronary ath-erosclerosis,36 it is thus possible that the high AT1 receptor expression in the susp ACS group is both a result of, and a key player in, processes leading to atherosclerosis in our patients with suspected ACS. Indeed, both Wilhelmsen et al.37 and McMahon et al.1 have presented data that indicate a high cardiovascular mortality among chest pain patients to whom ACS was ruled out. Clinical evidence supports the antiatherogenic effect of angiotensin-converting enzyme inhibitors or AT II receptor blocking agents.38

Study limitations

The present study shows increased expression of ETB and AT1 receptors in resistance arteries of patients with sus-pected ACS compared to controls. However, the two groups were not identical. The systolic blood pressure was higher in patients with chest pain than in the healthy controls. Furthermore, the prevalence of medication with angio-tensin receptor blockers, angiotensin-converting-enzyme inhibitors, and beta-blockers was common in the patients with suspected ACS while nonexistent in healthy controls. The effects of these confounding factors and how ET-1 and

angiotensin II levels in plasma infl uenced the receptor expression of the endothelin and angiotensin II receptors cannot be concluded from this study.

Conclusions

The study indicates that in patients with chest pain and suspected but ruled out ACS, arterial smooth muscle ETB and AT1 receptors are upregulated. There were no differ-ences in the expression of ETA and AT2 receptors between the two study groups. The mechanisms are unclear, but might be related to a higher blood pressure and an impaired glucose metabolism in patients with suspected ACS com-pared to healthy control subjects. We speculate that the increased receptor expression is related to ongoing endo-thelial dysfunction and an increased cardiovascular risk.

Acknowledgments This study was supported by the Swedish Research Council grant no 5958 and the Heart and Lung Foundation (Sweden).

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