www.elsevier.com/locate/ijcard

International Journal of Cardio

Impaired effect of endothelin-1 on coronary artery stiffness in

type 2 diabetes

Zenon S. Kyriakides a,*, Dimitrios Th. Kremastinos b, Athanasios E. Raptis b, Neil Johnston c,

Sotirios A. Raptis b, David J. Webb c, Stamatis Kyrzopoulos d, Eftichia Sbarouni d

a Red Cross Hospital, Athens, Greece, 2nd Cardiology Department, University of Athens, School of Medicine,

University General Hospital ‘‘Attikon’’, Athens, Greeceb 2nd Department of Internal Medicine- Propaedeutic, Research Institute and Diabetes Center, University of Athens, School of Medicine,

University General Hospital ‘‘Attikon’’, Athens, Greecec University of Edinburgh, Western General Hospital, UK

d Onassis Cardiac Center, Athens, Greece

Received 13 June 2005; received in revised form 4 August 2005; accepted 18 September 2005

Available online 2 December 2005

Abstract

Aim: We examined whether there is a differential effect of endothelin-A antagonism on coronary artery compliance in type 2 diabetes

mellitus compared to non-diabetic patients.

Patient and methods: We examined 32 patients, 11 type 2 diabetes mellitus and 21 non-diabetic patients, with atherosclerotic epicardial

arteries free of significant luminal stenoses. Intracoronary BQ-123 (6 Amol), an endothelin-A receptor antagonist, was infused over 20 min.

The artery lumen area in the proximal arterial segment was measured at end diastole and end systole before and after BQ-123 administration

using an intravascular ultrasound catheter. Calculations were made of normalized arterial compliance index, in mm Hg�1�103 and of arterialstiffness index h.Results: Pulse pressure and heart rate did not change after BQ-123. In type 2 diabetes mellitus, normalized compliance index decreased from

1.79T1.36 at baseline to 1.29T0.82 after BQ-123 administration, whereas in non-diabetic patients it increased from 2.10T1.36 to 3.00T2.07( p <0.05 versus baseline) (F =6.39, p =0.02). In type 2 diabetes mellitus, b index increased from 1.97T0.53 to 2.46T0.95, whereas in non-

diabetic patients it decreased from 1.83T0.95 to 1.63T0.84 (F =7.80, p =0.009). Big endothelin-1 at baseline was correlated with the

baseline b index ( p <0.0001, r =0.68).

Conclusions: Big endothelin-1 is correlated with the coronary artery stiffness. The effect of endogenous endothelin-1 on coronary artery

stiffness is impaired in type 2 diabetes mellitus. This may have important therapeutic implications with respect to the introduction of

endothelin receptor antagonists as cardiovascular therapeutic agents.

D 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Endothelin; Artery elasticity; Catheterization; Type 2 diabetes mellitus

1. Introduction

Endogenous production of endothelin-1 contributes to

the maintenance of coronary vascular tone in coronary

0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.ijcard.2005.09.018

* Corresponding author. B Cardiology Department Red Cross Hospital, 1

Erythrou Stavrou str., Athens 115 26, Greece. Tel.: +30 210 6414 705, 6414

587, 8104 554; fax: +30 210 6414 587.

E-mail address: zskyr@otenet.gr (Z.S. Kyriakides).

artery disease and healthy controls [1]. In animal models of

diabetes reduced responsiveness to endothelin-1 is seen in

both the large vessels and the microvasculature [2,3]. In

addition, exogenous as well as endogenous endothelin-1

exerts impaired vasoconstriction, in forearm arteries, in

patients with type 2 diabetes mellitus [4,5]. Compensatory

vessel enlargement occurs to a greater degree in patients

with unstable than with stable coronary syndromes, and is

associated with increased coronary artery distensibility

logy 112 (2006) 207 – 212

Table 1

Clinical features of the patients studied

Diabetic

patients

(n =11)

Non-diabetic

patients

(n =21)

Age, years 60T10 55T11Sex, M/F 10/1 17/4

Smoking, n 4 7

Systemic hypertension, n 6 8

Blood cholesterol, mg% 196T41 209T41Triglycerides, mg% 136T80 170T140

HDL-cholesterol, mg% 37T8 37T9

Patients on anti-diabetic diet, n 4 –

Patients taking anti-diabetic tablets, n 7 –

Blood glucose during catheterization, mg% 129T29 99T21*

HbA1c, % 7.4T1.6 5.5T0.6*

Studied artery, n

Left anterior descending artery 3 5

Right coronary artery 8 16

Medications, n

Z.S. Kyriakides et al. / International Journal of Cardiology 112 (2006) 207–212208

[6,7]. In diabetic patients coronary compliance is decreased

[8]. Recently, we showed that endothelin receptor antago-

nists improve coronary artery compliance in patients with

atherosclerotic vessels [9]. The role of endothelin-1 in the

regulation of vascular tone and coronary artery compliance

and the potential benefits of endothelin antagonists in

patients with type 2 diabetes mellitus are less clear.

Endothelin-1 binds to at least 2 receptors: the endothelin-A

receptor appears to be the major receptor causing vasocon-

striction in arteries, the endothelin-B receptor mediates

release of endothelium-dependent vasodilator substances

and is also present in some resistance and capacitance

arteries, where it contributes to vasoconstriction [10].

The aim of this study was to examine whether there is a

differential effect of endothelin-A antagonism on coronary

artery compliance in type 2 diabetes mellitus compared to

non-diabetic patients.

Beta blockers 3 8

Aspirin 10 20

Calcium channel blockers 6 7

Angiotensin converting enzyme

inhibitors or angiotensin receptor blockers

8 11

Statins 11 11

HbA1c: hemoglobin A1c; *p <0.05 versus diabetic patients.

2. Patients and methods

The Hospital Ethics Committee approved the study and

all patients gave written informed consent.

2.1. Study group

Forty patients with chest pain, on the waiting list for

diagnostic coronary angiography, were selected prospec-

tively on the basis of the following criteria: 1) atheroscle-

rotic epicardial arteries without significant coronary artery

stenoses and not calcified, 2) normal left ventricular ejection

fraction, and 3) coronary arteries eligible for intravascular

ultrasound studies [9].

Non diabetic patients underwent an oral glucose toler-

ance test in order the patients with impaired glucose

tolerance to be excluded.

2.2. Exclusion criteria

Acute or old myocardial infarction, unstable angina,

additional cardiac disease and severe non-cardiac disease.

All medications were discontinued at least 12 h before

the procedure.

Five patients were rejected for not having intravascular

ultrasound studies of sufficient quality, and 3 for having

impaired glucose tolerance tests. As a result, 32 patients

completed the study (Table 1).

2.3. Protocol

After the end of diagnostic coronary arteriography 2000

IU of heparin were administered.

Intravascular ultrasound studies were performed with an

Avanar, 2.9 French catheter of JoMed (Rijswijk, The

Netherlands) and image protocol was the same as indicated

earlier [9].

Maximal and minimal lumen areas at the selected sites

were identified during frame-by-frame playback analysis.

Vascular pulsatility, absolute compliance, normalized com-

pliance, and the stiffness index (h), which is considered to

be independent of the changes in blood pressure, were

obtained as indicated earlier [9]. Inter-observer variability

were very low [9].

Blood was obtained from the femoral artery sheath,

immediately before and after BQ-123 infusion for assay of

endothelin-1, big endothelin-1, renin and aldosterone

plasma levels.

2.4. Drug administration

After baseline recordings, while the catheter was in the

same site of the coronary artery, endothelin-A receptor

antagonist BQ-123 (Clinalfa, CH) was administered intra-

coronarily at a constant rate of 1 ml/min (300 nmol/min)

for 20 min. Before the initiation of BQ-123 infusion,

normal saline 0.9% was infused at a rate of 1 ml/min for 5

min and then the first baseline recordings were performed

(heart rate and arterial blood pressure—from the guiding

catheter—synchronously with intravascular ultrasound

recordings). The 2nd recordings were taken immediately

at the end of the 20 min infusion of BQ-123. The dose of

BQ-123 administered in this study (total dose of 6 Amol)

has been shown in other studies not to cause systemic

hemodynamic effects [10]. In the present study, this dose

was administered directly into the coronary circulation in

order to maximise delivery of the drug to the heart. The

Z.S. Kyriakides et al. / International Journal of Cardiology 112 (2006) 207–212 209

local concentrations achieved are likely to be greater than

the IC50 at the ETA receptor, but still selective for inhibition

of the ETA receptor, given the ¨2500-fold greater

selectivity of BQ-123 for this receptor, over the ETB

receptor [11].

2.5. Endothelin-1, big endothelin-1, aldosterone and renin

estimation

The very low levels of endothelin-1 and big endothelin-1

in plasma preclude the direct measurement of these peptides

by radioimmunoassay. Therefore endothelin-1 and big

endothelin-1 must first be extracted from plasma using a

sample preparation column, concentrated and the subse-

quent extract analyzed by radioimmunoassay. The extrac-

tion technique used is an acetic acid extraction and shown to

give the best recovery of endothelin-1 from plasma at 89%

[12]. Subsequent evaluation within our laboratory has also

shown this technique to give a recovery of big endothelin-1

of 91%.

The radioimmunoassays used were based on the com-

mercially available Bachem kits for the determination of

endothelin-1 and big endothelin-1 in plasma. (Bachem

(UK) Ltd, St. Helens, England). Intra assay (within day)

variation was 6.3% and inter assay (day to day) variation

was 7.2%.

Aldosterone was measured by ‘‘coat a count’’ radioim-

munoassay in serum. (Diagnostic Products Corporation, Los

Angeles, CA). Intra and inter assay variation were 3.5% and

6.5%, respectively.

Plasma Renin Activity in plasma was determined by

radioimmunoassay kit (Perkin-Elmer Life Sciences, Boston,

Table 2

The variables before and after BQ-123 administration

Pulse pressure, mm Hg Diabetic patients

Non-diabetic patients

Diastolic area, mm2 Diabetic patients

Non-diabetic patients

Arterial area change during systole, mm2 Diabetic patients

Non-diabetic patients

Pulsatility, % Diabetic patients

Non-diabetic patients

Absolute compliance index, mm2/mm Hg�1000 Diabetic patients

Non-diabetic patients

Normalized compliance index, mm Hg�1�1000 Diabetic patients

Non-diabetic patients

Index h Diabetic patients

Non-diabetic patients

Endothelin-1, pg/ml Diabetic patients

Non-diabetic patients

Big endothelin-1, pg/ml Diabetic patients

Non-diabetic patients

Aldosterone, pg/ml Diabetic patients

Non-diabetic patients

Renin, ng/ml Diabetic patients

Non-diabetic patients

* p <0.05 versus baseline.

MA). Intra and Inter assay variation were 5.2% and 8.6%,

respectively.

3. Statistical analysis

All data were expressed as mean valueT standarddeviation. Analysis of variance with repeated measures

was used for the statistical evaluation of the results,

followed by Tukey’s honestly significant difference test

for post-hoc comparisons. Linear regression analysis using

the least-square difference was used in order to examine

correlations between the different variables studied. A p

value of <0.05 was considered as statistically significant.

4. Results

Clinical characteristics, apart from the increased blood

glucose and hemoglobin A1c concentrations, which were

higher in type 2 diabetes mellitus, were similar in the two

groups (Table 1). Type 2 diabetes mellitus had systolic

hypertension more often, but this was not statistically

significant.

4.1. Intracoronary ultrasound and blood pressure

measurements

Heart rate at baseline was similar in the two groups

(69T9 beats/min in the type 2 diabetes mellitus and 74T16beats/min in the non-diabetic patients and did not change

after BQ-123 administration. Systolic blood pressure

Baseline After BQ-123 F p value

76T17 70T17 0.47 0.5

65T21 61T17

11.5T5.4 12.4T5.0 0.48 0.5

10.2T3.7 11.6T3.41.29T0.63 1.04T0.75 7.17 0.01

1.37T1.02 1.99T1.38*

13T9 9T7 8.07 0.008

13T8 18T12

18T10 15T8 6.31 0.02

23T18 35T27*

1.79T1.36 1.29T0.82 6.39 0.02

2.10T1.36 3.00T2.07*

1.97T0.53 2.46T0.95 7.80 0.009

1.83T0.95 1.63T0.84

1.87T0.71 1.51T0.75 0.05 0.8

1.94T1.44 1.43T0.96

46T22 39T23 1.28 0.3

76T121 188T32353T64 32T29 0.04 0.8

49T53 31T33

0.37T0.15 0.43T0.21 0.65 0.4

0.63T0.65 0.93T1.15

Fig. 1. Line plots of the index h at baseline and after 20 min of infusion of BQ-123 in the two groups.

Z.S. Kyriakides et al. / International Journal of Cardiology 112 (2006) 207–212210

(161T20 mm Hg in the type 2 diabetes mellitus and

151T25 mm Hg in the non-diabetic patients) and pulse

pressure were similar at baseline in the two groups, and

showed the same response to BQ-123 administration

(Table 2).

Diastolic arterial area was the same in the two groups and

showed the same response to drug administration (Table 2).

However, arterial area change during systole was more

pronounced in non-diabetic patients after BQ-123, whereas

in type 2 diabetes mellitus it did not increase.

4.2. Coronary distensibility

The indexes of coronary artery compliance showed a

differential response in the two groups after BQ-123

administration.

Fig. 2. Line plots of the normalized compliance index at baseline and after 20

In type 2 diabetes mellitus after BQ-123, pulsatility de-

creased by 31%, absolute compliance by 17.%, normal-

ized compliance by 28% and the h index increased by

25%. In non-diabetic patients pulsatility increased by

38%, absolute compliance by 52%, normalized compli-

ance by 46% and h index decreased by 11% (Table 2,

Figs. 1 and 2). The patients’ medication as well as glucose

and hemoglobin A1c plasma levels did not affect the

results.

In the patients studied as a whole group, aldosterone

plasma levels decreased from 51T57 before, to 31T30 pg/

ml after BQ-123 administration ( p =0.02). However, the

two groups responded similarly to the BQ-123 administra-

tion. Endothelin-1, big endothelin-1 and renin plasma levels

did not change after BQ-123 administration and the two

groups responded similarly (Table 2). Big endothelin-1 at

min of infusion of BQ-123 in the two groups. *p <0.05 versus baseline.

Fig. 3. Correlations between index h and baseline big endothelin-1.

Z.S. Kyriakides et al. / International Journal of Cardiology 112 (2006) 207–212 211

baseline was correlated to the baseline h index ( p <0.0001,

r =0.68), (Fig. 3).

5. Discussion

The present study demonstrates, for the first time, that

blockade of endothelin-A receptors results in an improve-

ment in coronary artery compliance in non-diabetic patients,

but not in type 2 diabetes mellitus patients, thereby

suggesting that endothelin-A-dependent activity is impaired

in the coronary arteries in type 2 diabetes mellitus patients.

It has been demonstrated that endothelin exerts a tonic

stiffening effect on the in vitro common carotid artery and that

this effect is mediated via the endothelin-A receptor [13]. In

the present study, big endothelin-1 plasma levels were

correlated to the stiffness of the coronary arteries, in

accordance with the above findings. However, in animal

models of diabetes and in patients with type 2 diabetes

mellitus, a reduced responsiveness to endothelin-1 is seen in

both the large peripheral vessels and themicrovasculature [2–

5]. Recently, a human study demonstrated that the effect of

endogenous endothelin-1 on endothelin-A receptors in the

periphery is enhanced in the resistance vessels of patients with

diabetes, whereas their sensitivity to exogenous endothelin-1

is blunted [14]. These discrepancies between the results of the

different studies could be ascribed to the following:

1) The patient’s quality of glucose control. It is possible that

a worse metabolic milieu may have affected the vaso-

dilatory mechanisms secondary to endothelin-A receptor

blockade, reducing the response to BQ-123;

2) The medications the patients were on and whether these

medications had been stopped before the study. Also, the

average duration of diabetes in the population may

contribute to the difference in the groups’ response;

3) Inter-individual variability, or to other unrecognised

factors; and

4) Gender issues.

In this study, we have extended, for the first time, these

observations concerning the effect of endothelin-1 in the

peripheral arteries of diabetic patients to the human

coronary circulation. We have demonstrated an improve-

ment in coronary artery compliance caused by endothelin-A

receptor antagonists in atherosclerotic arteries [9], which

was absent in type 2 diabetes mellitus patients.

BQ-123 has no inherent contractile action and has no

effect on the contractile response induced by various

agonists, indicating that the response to BQ-123 is due to

endothelin-1 antagonism rather than a non-specific action of

BQ-123 itself on the coronary vasculature [11].

There are several possible explanations to account for the

type 2 diabetes mellitus impaired response to endothelin-A

receptor antagonists as regards coronary artery compliance.

Hyperglycemia has been associated with increased basal

forearm blood flow [15] and hyperperfusion in the retinal

and glomerular microcirculation, which is reversible by

strict glycemic control [16]. Reduced contractility to

endothelin-1 of bovine pericytes in high concentrations of

glucose has been described [2]. It is possible that

hyperglycemia and insulin could cause endothelin-1 recep-

tor down-regulation. It is also possible that hyperglycemia

may result in glycosylation of the endothelin receptor and

impair receptor binding; alternatively, hyperglycemia may

impair intracellular events following binding to the endo-

thelin-A receptor. However, we detected no correlation

between the glucose blood levels and the coronary artery

compliance.

In our study we did not find higher endothelin-1 levels in

the type 2 diabetes mellitus than in non-diabetic patients as

in other studies [17]. It is possible, however, that tissue

rather than plasma endothelin-1 is important in determining

endothelin-A receptor down-regulation.

Finally, non-specific impairment of smooth muscle

constriction in diabetes is unlikely, as similar vasoconstric-

tion has been demonstrated in response to nonspecific

smooth muscle vasoconstrictors in diabetic patients and

control groups [18].

Z.S. Kyriakides et al. / International Journal of Cardiology 112 (2006) 207–212212

A consideration arising from our findings relates to their

possible clinical implications. These considerations may

become of practical significance as a result of the recent

introduction of non-peptidic endothelin receptor antagonists

as cardiovascular therapeutic agents.

5.1. Study limitations

Images of intravascular ultrasound represent a thin

tomographic cross-section of the artery and cannot be used

to evaluate changes simultaneously in adjacent regions or

the whole artery. A potential limitation of this study is that

measurement of coronary artery compliance may not reflect

changes in the microcirculation, which may also be

important. It would be valuable if similar findings could

be confirmed directly in the microcirculation itself. We did

not examine the effects of exogenous endothelin-1 on the

coronary artery compliance and the coronary circulation in

general, as this would not be so simple and would involve

risk for the patients studied.

In conclusion, the coronary artery stiffness effect of

endogenous endothelin-1 is impaired in type 2 diabetes

mellitus patients, and this may have important therapeutic

implications with respect to the introduction of endothelin

receptor antagonists as cardiovascular therapeutic agents.

This abnormality may participate in the pathophysiology of

vascular complications associated with diabetes. Further

studies in diabetic patients with systemic or pulmonary

hypertension are required to elucidate whether endothelin

antagonists will be of benefit in this group of patients.

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