ENDOTHELIN-1 SYTEM ACTIVITY IN ADULTS WITH BORDERLINE HIGH LDL-CHOLESTEROL

Abstract

Background

Modest elevations in plasma low-density lipoprotein (LDL)-cholesterol have been shown to confer a significant increase in cardiovascular risk. Endothelin (ET)-1 is a vasoconstrictor peptide with proatherogenic properties. The experimental aim of this study was to determine whether ET-1 system activity is elevated in adults with borderline high LDL-cholesterol, independent of other cardiometabolic abnormalities.

Methods

Forearm blood flow (FBF; plethysmography) responses to intra-arterial infusion of ET-1, selective ET_A receptor blockade (BQ-123), and non-selective ET_A/B blockade (BQ-123 + BQ-788) were determined in 40 middle-aged and older adults (45–70 years): 20 with optimal/near optimal LDL-cholesterol (<3.4 mmol/L) and 20 with borderline-high LDL-cholesterol (3.4–4.1 mmol/L).

Results

Both groups demonstrated a similar, non-significant (~10%) reduction in FBF to ET-1. BQ-123 and BQ-123+788 elicited a modest, but significant, increase in FBF (~15–20%) in each group. However, there were no group differences in the FBF responses to either selective ET_A or non-selective ET_A/B receptor antagonism.

Conclusion

Borderline-high LDL-C is not associated with increased ET-1 mediated vasoconstrictor tone. Disrupted ET-1 system activity may not contribute to the increased cardiovascular risk burden with borderline-high LDL-cholesterol.

INTRODUCTION

Low-density lipoprotein (LDL) cholesterol is a major atherogenic fraction of total cholesterol [1, 2] and an important predictor of myocardial infarction in middle-aged adults [3]. In fact, the risk of cardiovascular disease is estimated to increase by ~30% for every 30 mg/dL rise in plasma LDL-cholesterol concentration, suggesting increased cardiovascular risk burden at levels below the clinically recognized elevated range of >130 mg/dL [1]. Indeed, the relative risk of heart disease is nearly twofold higher in adults with borderline high LDL-cholesterol (3.4–4.1 mmol/L [30–159 mg/mL]) compared with adults with optimal/near optimal LDL-cholesterol levels (<3.4 mmol/L [130 mg/dL]) [1]. Moreover, nearly 40% of the adults in the United States have LDL-cholesterol levels in the borderline high range. Thus, understanding the mechanisms involved with the increased vascular risk with borderline high LDL-cholesterol has great public health importance.

Endothelin (ET-1)-1 is a powerful vasoconstrictor peptide that in addition to contributing to the regulation of vascular tone is also involved in the development and progression of atherosclerotic vascular disease [4, 5]. Indeed, enhanced ET-1 system activity elicits a proinflammatory vascular phenotype and ET-1 immunoreactivity is prominent in the walls of atherosclerotic human vessels [6, 7]. Moreover, ET-1 promotes fibrous tissue formation and inhibits endothelial nitric oxide synthesis [8, 9]. Increased ET-1 vasoconstrictor tone is common characteristic of numerous cardiovascular conditions and risk factors [10–12], including clinical hypercholesterolemia [13]. Whether this vascular impairment is already apparent in the borderline-high cholesterolemic state with its corresponding increase in vascular risk is unknown.

Accordingly, the experimental aim of this study was to determine whether ET-1 system activity is elevated in adults with borderline high LDL-cholesterol, independent of other cardiometabolic abnormalities. If so, this may contribute mechanistically to endovascular dysfunction and increased cardiovascular risk in this population.

METHODS

Subjects

Forty, sedentary middle-aged adults were studied: 20 with optimal/near optimal LDL-C (<130 mg/dL) and 20 with borderline-high LDL-C (130–159 mg/dL) [14]. All subjects were sedentary, non-obese, non-smokers, normotensive, non-medicated (including vitamins) and free of overt cardiovascular disease as assessed by physical examination, fasting blood chemistries and electrocardiogram and blood pressure at rest and during incremental exercise performed to volitional exhaustion. All women were at least 1 year postmenopausal and had never taken or had discontinued use of hormone replacement therapy at least 1 year before the start of the study. Written informed consent was obtained according to the guidelines of the University of Colorado at Boulder.

Intra-Arterial Infusion Protocol

All of the studies were performed between 7 AM and 10 AM after a 12-hour overnight fast in a temperature-controlled room as previously described [15]. Briefly, a 5-cm, 20-gauge catheter was inserted into the brachial artery of the nondominant arm under local anesthesia (1% lidocaine). Forearm blood flow (FBF) at rest and in response to each pharmacological agent was measured by strain-gauge venous occlusion plethysmography (D.E. Hokanson, Bellevue, Washington). To rule out the possibility of nonspecific group differences to vasoconstrictor agents, vascular responses to norepinephrine were determined. Norepinephrine was infused at a rate of 260 pmol/min for 5 minutes, and FBF was measured during the last 3 minutes. After a 20-minute rest period to allow FBF to return to baseline levels, ET-1 (Clinalfa, AG) was infused at a rate of 5 pmol/min for 20 minutes, and FBF was measured during the last 3 minutes. After a 30-minute rest period to allow resting FBF to return to baseline, BQ-123 (Clinalfa, AG), a selective ET_A receptor antagonist, was infused at a rate of 100 nmol/min for 60 minutes. FBF was measured every 10 minutes throughout the infusion period. After 60 minutes of BQ-123 infusion, FBF was measured every 10 minutes during the combined infusion of BQ-123 and BQ-788. BQ-788, a selective ET_B receptor antagonist, was infused at a rate 50 nmol/min for 60 mins.

Statistical Analysis

Differences in subject baseline characteristics and the magnitude of change in FBF to norepinephrine and ET-1 were determined by between-groups analysis of variance (ANOVA). Group differences in FBF responses to BQ-123 and BQ-123+BQ-788 were determined by repeated-measures ANOVA. There were no significant gender interactions, therefore the data were pooled and presented together. Data expressed as means±SEM. Statistical significance was set at P <0.05.

RESULTS

Selected subject characteristics are presented in the Table. By design, total cholesterol and LDL-C levels were the only variables significantly different (p<0.05) between the groups. The vasoconstrictor response to norepinephrine was not significantly different between the groups optimal/near optimal (from 4.0 ± 0.3 to 3.0 ± 0.3 mL/100 mL tissue/min) and borderline high (from 4.1 ± 0.3 to 3.0 ± 0.2 mL/100 mL tissue/min) LDL-C groups. Resting FBF was reduced by ~10% in response to ET-1 in both the optimal/near optimal (from 4.0 ± 0.2 to 3.6 ± 0.2 mL/100 mL tissue/min) and borderline high (from 3.9 ± 0.2 to 3.6 ± 0.3 mL/100 mL tissue/min) LDL-C groups (Figure). In response to BQ-123, there was a significant increase in FBF (~15%) from baseline in both groups. The addition of BQ-788 did not significantly alter FBF in either group (Figure). Importantly, there were no group differences in FBF responses to either BQ-123 (P=0.77) or BQ-123 + BQ-788 (P=0.51).

Table.

Selected Subject Characteristics.

Variable	Optimal/Near Optimal LDL-C (n=20)	Borderline-High LDL-C (n=20)
Age, yr	59±2	59±2
Sex, M/F	11/9	11/9
Body mass, kg	80.5±3.5	76.9±3.6
BMI, kg/m2	27.2±0.8	26.3±0.9
Body fat, %	34.1±2.3	34.1±1.8
Waist circumference, cm	91.8±2.7	88.0±3.0
Systolic BP, mmHg	126±2	122±2
Diastolic BP, mmHg	78±1	78±2
Total cholesterol, mmol/L	4.7±0.1	5.6±0.1*
LDL-cholesterol, mmol/L	2.7±0.1	3.7±0.1*
HDL-cholesterol, mmol/L	1.4±0.1	1.3±0.1
Triglycerides, mmol/L	1.2±0.1	1.4±0.1
Glucose, mmol/L	5.2±0.1	5.1±0.1
Insulin, pmol/L	38.1±4.9	40.2±4.8

BMI indicates body mass index; BP, blood pressure; LDL, low-density lipoprotein; HDL, high-density lipoprotein; Values are mean±SEM.

^*P < 0.05 vs. optimal/near optimal LDL-C.

Figure.

Forearm blood flow response to ET-1 (top panel), BQ-123, selective ETA receptor antagonism (middle panel) and BQ-123 + BQ-788, ETA and ETB receptor blockade (bottom panel) in the optimal/near optimal LDL-C and borderline high LDL-C groups.

DISCUSSION

The ET-1 system not only plays a central role in the regulation of vascular tone, but is also involved in the etiology of atherosclerotic vascular disease [4, 15]. The primary finding of the present study is that borderline-high LDL-cholesterol is not associated with enhanced ET-1 system activity. Indeed, forearm vascular responses to exogenous ET-1, selective ET_A receptor antagonism and non-selective ET_A/B receptor blockade were almost identical between adults with LDL-cholesterol in the optimal/near optimal and borderline-high levels.

Cardillo and colleagues [13] were the first to demonstrate a relation between hypercholesterolemia and ET-1 system activity. In a seminal study, employing the same isolated forearm model as used herein, Cardillo et al. [13] reported that ET_A receptor-mediated ET-1 vasoconstrictor tone was significantly elevated in middle-aged adults with high LDL-cholesterol concentrations (5.8±0.2 mmol/L [223±9 mg/dL]). The results of the present study compliment and extend these findings by demonstrating that ET-1 vasoconstrictor is not elevated at subclinical levels of LDL-cholesterol. This finding is in contrast to conditions such prehypertension [12] and impaired fasting glucose [16] where ET-1-mediated vasoconstrictor tone is already elevated to comparable levels seen with clinical hypertension [17] and overt diabetes [18]. Interestingly, borderline high LDL-cholesterol is associated with diminished nitric oxide-mediated endothelial vasodilation [19], thus modest elevations in LDL-cholesterol appear to have differential effects on two key elements of endothelial vasomotor regulation. Moreover, given the inhibitory interaction between ET-1 and nitric oxide [20], lack of enhanced ET-1 system activity argues against an ET-1-related mechanism underlying endothelial vasodilator dysfunction with borderline high LDL-cholesterol.

It should be noted that we did not measure circulating plasma concentrations of ET-1. Since the vast majority of ET-1 produced by the endothelium (>80%) is released towards the vascular smooth [21], circulating levels of ET-1 do not appropriately reflect the vascular effects of the peptide [22]. Rather, we employed a pharmacological approach involving the intra-arterial infusion of exogenous ET-1 and ET receptor antagonists providing a more direct and specific biological assessment of ET-1 system activity in vivo [13, 17].

In conclusion, the results of the present study demonstrate that borderline high LDL-cholesterol is not associated with increased ET-1 vasoconstrictor activity. Although elevations in LDL-cholesterol in the borderline high range is linked with both diminished endothelial vasodilator function [19, 23] and increased cardiovascular risk burden [1], disrupted ET-1 system activity does not appear to be an contributing factor.