Protective effect of high density lipoprotein on endothelium-dependent vasodilatation

International Journal of Cardiology 73 (2000) 231–236 www.elsevier.com / locate / ijcard

Protective effect of high density lipoprotein on endothelium-dependent vasodilatation a a, a b b Xiang-Ping Li , Shui-Ping Zhao *, Xiang-Yu Zhang , Ling Liu, Mei Gao , Qi-Chang Zhou a b

Departments of Cardiology, Second Affiliated Hospital, Human Medical University, Changsha, 410011, P.R. China Departments of Ultrasound, Second Affiliated Hospital, Human Medical University, Changsha, 410011, P.R. China Received 2 August 1999; received in revised form 7 December 1999; accepted 28 January 2000

Abstract Low concentrations of high-density lipoprotein cholesterol (HDL-C) have been associated with increased risk of coronary heart disease (CHD) even when the total cholesterol (TC) and triglyceride (TG) levels are not elevated. The mechanism by which HDL confers protection against atherosclerosis remains speculative. Using high-resolution ultrasound, we measured the dilatation changes of brachial arteries during reactive hyperemia and after sublingual glyceryl trinitrate (GTN) in 63 patients with established (CHD) and 45 controls, in which the serum TC level was normal. The results showed that both flow-mediated dilatation (FMD) and GTN-induced dilatation of brachial arteries in patients with CHD were much reduced compared with control group (2.3 162.46% vs. 7.4364.10% and 16.4166.15% vs. 22.4468.63%, respectively, P,0.001 for all). Univariate analysis indicated that FMD of brachial arteries was inversely related to age (r520.226, P,0.05), hypertension (r520.229, P,0.05), baseline diameter (r520.299, P,0.01) and LDL-C (r520.237, P,0.05) and positively related to HDL-C (r50.491, P,0.01). GTN induced vasodilatation was inversely related to age (r520.216, P,0.05) and baseline diameter (20.476, P,0.01). Multiple stepwise regression analyses in two groups taken together showed that HDL-C and age were the independent predictors of the FMD of brachial arteries ( b 50.466, P50.000 and b 520.184, P50.020, respectively). Baseline diameter was significant predictor of GTN-induced vasodilatation ( b 520.390, P50.000). The analysis in the group of CHD patients showed that only HDL-C was significantly relate to the FMD of brachial arteries ( b 50.295, P50.018) and in controls that hypertension and HDL-C were significantly relate to the FMD of brachial arteries ( b 520.395, P50.004 and b 50.344, P50.011, respectively). These finding suggest that endothelium-dependent and endothelium-independent vasodilatation are impaired in the patients with CHD. HDL exerts a protective effect on endothelium-dependent vasodilatation in TC being relatively normal population.  2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Vasodilatation; High-density lipoprotein; Coronary heart disease

1. Introduction The endothelium has been found to be the central regulator of vascular tone, lipid breakdown, thrombogenesis, inflamation, and vessel growth [1–3]. Endothelial dysfunction appears before the earliest anatomic evidence of atherosclerosis and may repre*Corresponding author. Tel.: 186-731-555-0313; fax: 186-731-5533525.

sent an important initial step in its development [4–6]. The direct relation between hypercholesterolemia and increased risk of coronary heart disease (CHD) is widely accepted in the clinical setting. Considerable experimental and clinical data demonstrate that elevated total and low-density lipoprotein cholesterol (LDL-C) levels are associated impaired endothelial function, independent of the presence of coronary artery disease [7–9]. However, serum total cholesterol (TC) concentrations may be normal in

0167-5273 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 00 )00221-7

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some patients with CHD. In China, low concentrations of high-density lipoprotein cholesterol (HDLC) have been associated with increased risk of CHD even when the TC and triglyceride (TG) levels are not elevated [10]. The mechanism by which HDL confers protection against atherosclerosis remains speculative. A role for HDL in reverse cholesterol transport has been postulated to explain the cardioprotective effect. Recent data suggest that HDL have other properties which consist of inhibiting oxidative modification of LDL [11], ameliorating vascular endothelial function [12–14] and preventing thrombosis [15]. Few studies by intracoronary ultrasound examination and / or quantitative coronary angiography show a positive correlation between HDLC and acetylcholine-induced coronary vasoreactivity [12,13]. These studies suggest that HDL exert a beneficial effect on abnormal vascular reactivity. However, the relation between serum HDL-C level and endothelial function in CHD patients with normal serum TC concentration is unclear. To assess the relation between serum HDL-C level and endothelium-dependent dilatation of brachial arteries in human, we investigated the serum lipid levels and the diameter changes of brachial arteries during reactive hyperaemia and after sublingual glyceryl trinitrate (GTN) in 63 patients with established CHD and 45 controls, in whom the serum TC levels were not elevated (#6 mmol / l).

2. Subjects and methods

2.1. Subjects One-hundred and eight subjects were involved in this study (88 males and 20 females). The average age of the subjects was 60.868.9 years (41–78 years). Among them, there were 63 patients with established CHD, and 45 subjects of no clinic evidence of atherisclerotic vascular disease as controls. All of the patients experienced symptoms of CHD (i.e. chest pain). Among them 54 patients had a history of myocardial infarction and nine patients had stenosis of at least one coronary artery .50% confirmed by coronary angiography. Histories, physical examination, electrocardiograms, ambulatory electrocardiograms for 24 h and B-mode ultrasound images showed that controls had no evidence of

atherosclerosis. There was a history of hypertension (.140 / 90 mmHg) or current smoking in some subjects of two groups. None of the subjects studied had a history of diabetes, peripheral vascular disease or other chronic diseases. All subjects had serum TC level #6.0 mmol / l.

2.2. Study protocol All vasoactive medications, including calcium channel blockers, angiotensin-converting enzyme inhibitors, b-adrenergic blocker agents, and long-acting nitrates, were withheld at least 7 days before the study. All tests were carried out in a temperaturecontrolled room with the subjects in the resting, supine state between the hours of 9 and 10 a.m. after having abstained from alcohol, caffeine, and tobacco, as well as food for 12 h. High-resolution external ultrasound (128xP/ 10 mainframe with a 7.0-MHz linear array transducer, Acuson) was used to measure changes in brachial artery diameter in response to reactive hyperemia and in response to 0.5 mg of sublingual GTN. The ultrasound method has been described in detail previously [6]. A single, highly skilled sonographer who was unaware of the study performed all imaging. The right brachial artery diameter and flow velocity were scanned in longitudinal sections 2–15 cm above the elbow, after 15 min of rest in the supine position. Depth and gain settings were set to optimize images of interface between the lumen and vessel wall. Vessel diameter was measured at end diastole, coincident with the R-wave on the electrocardiogram. Three cardiac cycles were analyzed by each observer for each scan and the measurements were averaged. Doppler flow velocity measurement were obtained by means of range gating focused on the artery’s center using an angle of incidence of 558 to interrogate maximum luminal flow. After a baseline diameter was recorded, a blood pressure cuff was inflated around the forearm (below the scanned part of the artery) to 300 mmHg. Then deflating the cuff after 4 min, flow was increase through the brachial artery blood. The artery was scanned continuously 30 s before and 90 s after cuff deflation. After another 15 min of rest to allow the artery to return to its baseline diameter, sublingual GTN tablet (0.5 mg) was then administered, and 4 min later the last scan was done. During the examination, ultrasound transducer was on the fixed place of

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brachial artery. Flow-mediated dilatation (FMD) and GTN-induced vasodilatation were expressed as a percentage change relative to the baseline diameter. Arterial blood flow was measured as Doppler flow velocity multiplied by the cross sectional area (p 3 r 2 ).

2.3. Serum lipids analysis The fasting venous samples were taken in the morning. Serum TC and TG levels were determined enzymatically. The serum HDL-C level was determined enzymatically after LDL and VLDL were selectively removed from the serum by sodium phosphotungstate / magnesium precipitation. LDL-C was calculated with the Fridewald formula: LDL-C (mmol / l)5TC-(HDLC1TG / 2.2).

2.4. Repeatability The repeatability of the technique has been established previously [16].

2.5. Statistical analysis Data were analyzed using SPSS 7.5 for Windows 95. All descriptive data were expressed as mean6standard deviation (S.D.). The significance of the difference between two groups was analyzed by independent samples t-test. Chi-square test was used for comparison of discontinuous variables. univariate and multivariate stepwise regression analyses were used to analyze the correlation between vasoreactivity and each potential risk factor. The multivariate analyses were done after taking the CHD patients and control subjects together and separately. Only variable that were significantly (P,0.05) related to vascular responses in univariate analyses were entered into the multivariate analyses. Statistical significance was accepted at P,0.05.

3. Results

3.1. Patient characteristics As shown in Table 1, the baseline characteristics of the controls group and the patients with CHD were

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Table 1 Basic characteristics of the patients with CHD and the controls a Variable

Control group (n545)

Patients group (n563)

P value

Age (years) Sex (M / F) Smokers (%) Hypertension (%) BMI (kg / m 2 ) SBP (mmHg) DBP (mmHg) TC (mmol / l) TG (mmol / l) LDL-C (mmol / l) HDL-C (mmol / l) FBS (mmol / l)

59.669.2 36 / 9 47.7 32.6 23.362.8 124.8615.1 78.469.3 4.6560.56 1.4760.52 2.5660.59 1.4560.46 4.6260.58

61.568.2 52 / 11 49.2 33.9 23.562.7 123.6612.1 77.768.0 4.5960.70 1.5760.89 2.7460.66 1.1460.25 4.6860.52

NS NS NS NS NS NS NS NS NS NS ,0.001 NS

a

CHD, coronary heart disease; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; FBS, fasting blood sugar; NS, not significant.

similar, except for mean HDL-C level was significantly lower in CHD patients.

3.2. Vasodilator response to reactive hyperaemia and GTN The baseline diameter of brachial artery was similar in the two groups studied. The baseline blood flow of brachial artery and the degree of reactive hyperaemia by cuff inflation and release were not different in the two groups. However, both FMD and GTN-induced dilatation of brachial arteries in the patients with CHD were much reduced compared with control group (Table 2).

3.3. Serum lipids and response to reactive hyperaemia of brachial artery and GTN Univariate analysis in the total group of participants indicated that FMD of brachial arteries was inversely related to age (r5 20.226, P,0.05), hypertension (r5 20.229, P,0.05), baseline diameter (r5 20.299, P,0.01) and LDL-C (r5 20.237, P, 0.05) and positively related to HDL-C (r50.491, P,0.01). GTN-induced vasodilatation was inversely related to age (r5 20.216, P,0.05) and baseline diameter (r5 20.476, P,0.01). However, no significant correlation was observed between FMD of brachial arteries or GTN-induced vasodilatation and

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Table 2 Brachial artery studies in the patients with CHD and the controls a

Baseline diameter (mm) Baseline flow (ml / min) Hyperemia (% increase in flow) FMD (%) GTN-induced dilation (%) a

Controls (n545)

Patients (n563)

P value

3.7460.58 78.9636.2 468.16336.2 7.4364.10 22.4468.63

3.9560.54 66.7631.6 4336329.1 2.3162.46 16.4166.15

NS NS NS ,0.001 ,0.001

CHD, coronary heart disease; FMD, flow-mediated dilatation; NS, not significant.

gender, smoking, BMI, TC, TG, FBS, systolic or diastolic blood pressure (P.0.05 for all). Multiple stepwise regression analyses in the CHD patients and controls taken together showed that HDL-C and age were the independent predictors of the FMD of brachial arteries [standardized regression coefficient ( b )50.466, P50.000 and b 5 20.184, P50.020, respectively] (Fig. 1). Baseline diameter was significant predictor of GTN-induced vasodilatation ( b 5 20.390, P50.000). The analysis in the group of CHD patients showed that only HDL-C was significantly relate to the FMD of brachial arteries ( b 50.295, P50.018) and in controls that hypertension and HDL-C was significantly relate to the FMD of brachial arteries ( b 5 20.395, P50.004 and b 5 0.344, P50.0 11, respectively).

4. Discussion Recent investigations have demonstrated that the vascular endothelium is an active participant in the regulation of arterial tone and blood flow through secreting vasoactive substances [1–3]. One of most important substances released from the endothelium is endothelium-derived relaxing factor, which has been identified chemically as nitric oxide (NO) or a NO-containing compound [17]. In a state of health, endothelium-dependent vasodilatation is evoked by NO which is released by endothelial cells when acetylcholine is infused into arteries or shear forces are exerted by blood flow. GTN, which supplies exogenous NO and acts directly on vascular smooth muscle, induces endothelium-independent vasodilat-

Fig. 1. Relation between serum HDL-C level and flow-mediated dilatation (FMD) of brachial arteries.

X.-P. Li et al. / International Journal of Cardiology 73 (2000) 231 – 236

ation. In the present study, we observed that both FMD and GTN-induced dilatation of brachial arteries in the patients with CHD were much reduced compared with the control group. The result suggests that endothelium-dependent vasodilatation, as well as endothelium-independent vasodilatation is impaired in the patients with CHD. Our result is consistent with the results of previous studies [18,19]. It has been known that HDL has an antiatherogenic effect. HDL-C level has been repeatedly shown to be an independent inverse predictor of CHD risk in epidemiological and observational studies even when the TC and TG levels are not elevated [10,20–22]. The cardioprotective effect of HDL has generally been attributed to the efficiency of reverse cholesterol transport from peripheral tissues to the liver. However, recent lipid-modifying studies demonstrated a considerable reduction in the number of coronary events despite only minimal changes in coronary luminal diameters achieved by elevating HDL-C levels [23], suggesting that other functional mechanisms are also involved. Kuhn et al. [12] demonstrated that HDL-C was positively correlated to acetylcholine-induced coronary vaso-reactivity in both angiographically smooth and disease coronary segments. Using intracoronary ultrasound examination, Zeiher et al. [13] observed coronary vasomotor response to the endothelium-dependent dilator acetylchline and to sympathetic stimulation by cold pressor test correlate with local atherosclerotic wall thickening and found elevated HDL-C serum levels ameliorate abnormal vasoconstriction at any given extent of atherosclerotic wall thickening. The present study is the first to investigate the relation between lipids and endothelium-dependent dilation of brachial arteries in the CHD patients in which the total cholesterol level is not elevated. The results indicated that HDL-C was the most important predictor of the FMD of brachial arteries in all subjects studied. These findings suggest that low HDL-C serum levels may related to endothelial dysfunction and HDL may exert a protective effect on endothelial function. Oxidized LDL (ox-LDL) has been shown to inhibit endothelium-dependent relaxation [24–26]. The mechanisms underlying the inhibitory effect of oxLDL may involve the impairment of production and / or release of NO and the promotion of inactivation of NO after it is released. Both monocyte-derive macro-

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phages, which produce large amounts of superoxide anions, and ox-LDL are highly potent inactivators of NO. In vitro studies have demonstrated that HDL not only prevents oxidative modification of LDL in the extravascular microenvironment, but also inhibits monocyte transmigration in cocultures of human arterial wall cells [27]. Another study [14] has also demonstrated that HDL may reverse the ox-LDLinduced impairment of endothelium-dependent relaxation by removing lysophosphatidyl-choline from oxLDL. In addition, HDL has been shown to stimulate arterial endothelial cell prostacyclin synthesis [28] and acutely to reverse the impairment of endothelium-dependent relaxation induced by ox-LDL, which also could contribute to the reduced constrictor response of atherosclerotic vessels. However, the certain mechanism of HDL ameliorating and protecting endothelial function is not fully understood. In prior studies, both coronary and brachial artery endothelial dysfunction have been observed to correlate with the traditional risk factors, including advanced age, hypercholesterolemia, cigarette smoking, hypertension, and diabetes mellitus. Considerable experimental and clinical data suggest that elevated TC and LDL-C are associated with impaired endothelial function [26,29]. In the present study, we found LDL-C slightly influences FMD of brachial arteries, but no significant correlation was observed between TC and FMD. This discrepancy may be related to differences in study populations. The subjects in the present study were older and had normal serum TC levels. Data from the Framingham study suggests that HDL-C, and not TC, is the most important predictor of CHD risk in subjects aged .50 years. Some investigators also believed that HDL has a stronger influence on endothelial integrity than TC levels in older patients with early CHD [12]. Thus, we conclude that HDL has a more significant effect on endothelial function than TC and LDL-C in older populations with relatively normal serum TC level.

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