Restoration of Endothelial Function by Increasing
High-Density Lipoprotein in Subjects With Isolated Low
High-Density Lipoprotein
Radjesh J. Bisoendial, MD; G. Kees Hovingh, MD; Johannes H.M. Levels, PhD;
Peter G. Lerch, PhD; Irmgard Andresen, MD, PhD; Michael R. Hayden, MB, ChB, PhD;
John J.P. Kastelein, MD, PhD; Erik S.G. Stroes, MD, PhD
Downloaded from http://circ.ahajournals.org/ by guest on July 28, 2017
Background—Loss-of-function mutations in the ATP-binding cassette (ABCA)-1 gene locus are the underlying cause for
familial hypoalphalipoproteinemia, providing a human isolated low-HDL model. In these familial hypoalphalipoproteinemia subjects, we evaluated the impact of isolated low HDL on endothelial function and the vascular effects of an
acute increase in HDL.
Methods and Results—In 9 ABCA1 heterozygotes and 9 control subjects, vascular function was assessed by venous
occlusion plethysmography. Forearm blood flow responses to the endothelium-dependent and -independent vasodilators
serotonin (5HT) and sodium nitroprusside, respectively, and the inhibitor of nitric oxide synthase NG-monomethyl-Larginine (L-NMMA) were measured. Dose-response curves were repeated after systemic infusion of apolipoprotein
A-I/phosphatidylcholine (apoA-I/PC) disks. At baseline, ABCA1 heterozygotes had decreased HDL levels
(0.4⫾0.2 mmol/L; P⬍0.05), and their forearm blood flow responses to both 5HT (maximum, 49.0⫾10.4%) and
L-NMMA (maximum, ⫺22.8⫾22.9%) were blunted compared with control subjects (both Pⱕ0.005). Infusion of
apoA-I/PC disks increased plasma HDL to 1.3⫾0.4 mmol/L in ABCA1 heterozygotes, which resulted in complete
restoration of vasomotor responses to both 5HT and L-NMMA (both Pⱕ0.001). Endothelium-independent vasodilation
remained unaltered throughout the protocol.
Conclusions—In ABCA1 heterozygotes, isolated low HDL is associated with endothelial dysfunction, attested to by
impaired basal and stimulated NO bioactivity. Strikingly, both parameters were completely restored after a single, rapid
infusion of apoA-I/PC. These findings indicate that in addition to its long-term role within reverse cholesterol transport,
HDL per se also exerts direct beneficial effects on the arterial wall. (Circulation. 2003;107:2944-2948.)
Key Words: lipoproteins 䡲 apolipoproteins 䡲 endothelium 䡲 nitric oxide
D
espite a clinically significant reduction in coronary
event rates after the use of statins, both primary and
secondary prevention trials indicate that 60% to 70% of major
cardiovascular events cannot be prevented with current therapeutic strategies. This has prompted the search for novel
drug targets to further improve cardiovascular outcome.
Among these options, strategies aiming at increasing HDL
levels hold great promise.1–3 In this respect, a strong inverse
relationship has been established between HDL levels and the
incidence of coronary artery disease.4 In line with this, the
prevalence of HDL deficiency (⬍35 mg/dL) in men with
premature coronary artery disease reaches up to 40%.5,6
Nevertheless, the implications of increasing HDL levels for
cardiovascular outcome remain to be determined. The fact
that low HDL is often associated with other risk factors, such
as enhanced triglyceride levels and insulin resistance (composing the metabolic syndrome), suggests that those, instead
of low HDL per se, might be primarily responsible for the
observed relationship with cardiovascular events. This has
promoted the search for human models with an isolated
low-HDL phenotype in which the consequences of isolated
low HDL and pharmacological modalities targeting HDL for
endothelial phenotype and cardiovascular outcome can be
studied.
A proper model is familial hypoalphalipoproteinemia,
which was recently found to result from loss-of-function
mutations in the ATP-binding cassette (ABCA)-1 gene.
Heterozygous carriers are characterized by a moderate to
severe decrease of HDL but have normal levels of apolipoprotein B (apoB)– containing lipoproteins, thus enabling
Received February 5, 2003; accepted March 4, 2003.
From the Department of Vascular Medicine, Academic Medical Centre, Amsterdam, Netherlands (R.J.B., G.K.H., J.H.M.L., J.J.P.K., E.S.G.S.); ZLB
Bioplasma AG, Bern, Switzerland (P.G.L., I.A.); and the Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver,
Canada (M.R.H.).
Correspondence to John J.P. Kastelein, MD, PhD, Department of Vascular Medicine, G1.146, Academic Medical Center, Meibergdreef 9, 1105 AZ
Amsterdam, Netherlands. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000070934.69310.1A
2944
Bisoendial et al
TABLE 1. Demographic and Baseline Parameters of the
Study Cohort
ABCA1
Heterozygotes
(n⫽9)
Control
Subjects
(n⫽9)
42.9⫾13.9
46.1⫾13.5
Age, y
Sex, M/F
3/6
3/6
BMI, kg/m2
26.1⫾2.7
25.8⫾3.5
Smoking, n
2/9
2/9
Cardiovascular disease, n
1/9
0/9
Systolic blood pressure, mm Hg
131.6⫾23.4
130.1⫾13.4
Diastolic blood pressure, mm Hg
79.0⫾8.9
78.1⫾5.9
HR, bpm
62.2⫾9.0
60.1⫾10.5
3.2⫾1.0
2.6⫾0.6
Basal FBF, mL 䡠 100 mL FAV⫺1 䡠 min⫺1
Values represent mean⫾SD. BMI indicates body mass index; HR, heart rate.
Downloaded from http://circ.ahajournals.org/ by guest on July 28, 2017
assessment of the role of isolated low HDL in vivo.7 Because
the initial step in the reverse cholesterol transport (RCT)
pathway is impeded in these individuals, cell-derived cholesterol accumulates within the arterial wall, clinically manifested by advanced carotid intima-media thickening and
markedly increased susceptibility to atherosclerosis.8,9
Endothelium-derived nitric oxide (NO) has emerged as a
pivotal antiatherogenic mediator. Diminished NO bioavailability is a hallmark of (early) atherosclerosis, which can be
measured indirectly in patients as impaired endotheliumdependent vasomotor response.10 In this respect, endothelial
vasodilator dysfunction has been demonstrated in subjects
with established coronary risk factors even before the onset of
morphological changes, although most studies have indicated
its reversibility after risk factor exclusion.11–14 More recently,
the predictive value of endothelial vasodilator dysfunction for
future cardiovascular events has been underscored by several
research groups.15–18
In the present study, we evaluated the consequences of
isolated low HDL for vascular function in subjects carrying
heterozygous ABCA1 gene mutations compared with normolipidemic sex- and age-matched control subjects (controls).
Previous data in these individuals, including accelerated
carotid atherogenesis, suggested a priori compromised endothelial function. Subsequently, we determined whether an
acute increase in HDL level (on infusion of apolipoprotein
A-I/phosphatidylcholine [apoA-I/PC] disks) would translate
into an improvement of vascular function.
Methods
Subjects
Nine ABCA1 heterozygotes (6 men and 3 women; mean [⫾SD] age,
42.9⫾13.9 years) and 9 age- and sex-matched normolipidemic
controls (6 men and 3 women; mean [⫾SD] age, 46.1⫾13.5 years)
were enrolled in the study. Affected subjects were selected from
available family members of 4 Dutch kindreds with known mutations
on the ABCA1 gene locus, as described previously.11 At baseline,
HDL cholesterol levels were 0.5⫾0.2 versus 1.2⫾0.3 mmol/L
(P⬍0.05) in the control group. Four subjects were smokers, equally
divided between the 2 groups (Table 1). All subjects had normal
plasma levels of apoB-containing lipoproteins (total cholesterol,
LDL, or triglycerides below the 95th percentile), whereas none of the
subjects had diabetes mellitus, hypertension (defined as systolic
Restoration of Endothelial Function With HDL
2945
blood pressure ⬎140 mm Hg and/or diastolic blood pressure
⬎90 mm Hg), or congestive heart failure. Before entering the study,
subjects discontinued all lipid-lowering medication for a period of
ⱖ6 weeks. All subjects refrained from alcohol, tobacco, and
caffeine-containing drinks for more than 12 hours before the study.
The study protocol was approved by the Institutional Review Board
at the Academic Medical Center, University Hospital of Amsterdam.
All subjects gave written informed consent.
Study Protocol
Assessment of vascular function was performed at baseline and after
apoA-I/PC infusion by use of venous occlusion strain-gauge plethysmography (EC4; Hokanson Inc). All experiments were performed
in a quiet, air-conditioned room (temperature, 22°C to 24°C), with
the subjects in the supine position throughout the study. The
nondominant brachial artery was cannulated with a 20-gauge polyethylene catheter under local anesthesia, and blood was drawn for
baseline measurements. Insertion was followed by a 20-minute
interval for the artery to reestablish baseline conditions. Thereafter,
forearm blood flow (FBF), expressed in milliliters per minute per
100 mL of forearm tissue volume (FAV), was measured simultaneously in both arms. During each measurement, blood pressure
cuffs around both upper arms were inflated (40 mm Hg) by use of a
rapid cuff inflator. Simultaneously, bilateral wrist cuffs were inflated
to above-systolic blood pressure to exclude hand circulation
(200 mm Hg). Intra-arterial blood pressure and heart rate were
monitored continuously. Next, FBF response to cumulative doses of
the endothelium-dependent vasodilator serotonin (5HT, Sigma; 0.6,
1.8, and 6 ng · 100 mL FAV⫺1 · min⫺1), the endothelium-independent
vasodilator sodium nitroprusside (SNP, Spruyt Hillen; 6, 60, 180,
and 600 ng · 100 mL FAV⫺1 · min⫺1), and the competitive inhibitor
of endothelial NO synthase (eNOS) NG-monomethyl-L-arginine (LNMMA, Kordia; 50, 100, 200, and 400 ␮g · 100 mL FAV⫺1 · min⫺1)
was measured. All agents were administered intra-arterially for 6, 4,
and 8 minutes at each dose, respectively, with a constant-rate
infusion pump. Six measurements during the last 2 minutes (steady
state) were averaged to determine mean FBF. The 3 different
infusion blocks proceeded after a 15-minute rest period or until FBF
had returned to baseline. Then, a venous catheter was inserted in the
contralateral arm for systemic administration of apoA-I/PC disks at
a dose of 80 mg/kg body weight over a period of 4 hours (ZLB
Bioplasma AG).19,20 Subsequently, all 3 infusion blocks were
repeated.
Biochemical Analysis
Blood samples were drawn from the subjects after a 12-hour
overnight fast, immediately and 3 hours after apoA-I/PC infusion.
After centrifugation within 1 hour after collection, aliquots were
snap-frozen in liquid nitrogen and stored at ⫺80°C until the assays
were performed. All measurements were performed at the vascular
and clinical laboratory of the Academic Medical Center, University
Hospital of Amsterdam. Liver transaminases were measured with
standard laboratory methods. Plasma triglycerides, total cholesterol,
and LDL and HDL levels were determined with high-performance
gel permeation chromatography.21 In brief, the system contained a
PU-980 ternary pump with an LG-980-02 linear degasser and an
FP-920 fluorescence and UV-975 UV/VIS detector (Jasco). An extra
P-50 pump (Pharmacia Biotech) was used for addition of in-line
cholesterol (or triglyceride) PAP enzymatic reagent (Biomerieux) at
0.1 mL/min. EDTA plasma was diluted 1:1 with Tris-buffered saline,
and 30 ␮L of a sample/buffer mixture was loaded on a Superose 6
HR 10/30 column (Pharmacia Biotech) for lipoprotein separation at
a flow rate of 0.31 mL/min. Plasma levels of apoB and apoA-I were
assayed on stored plasma by rate nephelometry. The serum concentration of malondialdehyde-modified LDL was assayed by a murine
monoclonal antibody 4E6 – based sandwich ELISA (Mercodia AB)
as described previously.22
Statistical Analysis
All results of clinical parameters, including plethysmographic data,
are expressed as mean⫾SD. Descriptive statistics between the 2
2946
Circulation
June 17, 2003
TABLE 2.
Laboratory Parameters of the Study Cohort
ABCA-A1 Heterozygotes
Control Subjects
Before HDL Increase
(n⫽9)
After HDL Increase
(n⫽9)
Before HDL Increase
(n⫽9)
After HDL Increase
(n⫽9)
TC, mmol/L
4.3⫾1.5
5.6⫾1.5
5.4⫾1.2
6.5⫾1.4
HDL, mmol/L
0.4⫾0.2*
1.3⫾0.4†
1.0⫾0.3
2.1⫾0.5†
LDL, mmol/L
3.5⫾1.0
3.5⫾0.8
4.1⫾1.1
3.7⫾1.2
TG, mmol/L
1.6⫾0.9
2.9⫾2.3
1.2⫾0.6
2.0⫾1.1
ApoA-I, g/L
0.7⫾0.3*
2.3⫾0.5†
1.2⫾0.2
2.6⫾0.4†
ApoB, g/L
1.0⫾0.3
0.9⫾0.2
1.0⫾0.3
1.0⫾0.3
oxLDL, U/L
46.1⫾16.1
40.2⫾16.0
44.0⫾25.7
37.8⫾20.1
Values represent mean⫾SD. TC indicates total cholesterol; TG, triglycerides; and oxLDL, oxidized LDL.
*P⬍0.05 vs normocholesterolemic control subjects.
†P⬍0.05 vs baseline.
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groups were compared by means of 2-tailed paired or unpaired
Student’s t test. FBF was averaged over 6 consecutive recordings
during the last 2 minutes of each infusion. Statistical analysis of FBF
measurements for individual subjects between the 2 groups was
performed by 2-way ANOVA for repeated measures. A probability
value of P⬍0.05 was considered significant and a value of P⬍0.01
as highly significant.
Results
Biochemistry
Circulating levels of oxidized LDL were not significantly
different between ABCA1 heterozygotes and controls. Also,
the levels of these parameters remained unaltered after
apoA-I/PC infusion.
Discussion
Effects of ApoA-I/PC Infusion on eNOS Activity
In the present study, we show that both basal and stimulated
NO activity is sharply reduced in ABCA1 heterozygotes
compared with age- and sex-matched controls. Strikingly, an
increase in HDL by a single, rapid infusion of apoA-I/PC
disks results in complete recovery of endothelial vasomotor
response. These novel data indicate that isolated low HDL in
ABCA1 heterozygotes is associated with endothelial dysfunction, which is entirely reversible on HDL increase.
The ABCA1 transporter facilitates apoA-I–mediated transfer of cellular cholesterol and phospholipids from the plasma
membrane to poorly lipidated (apo)lipoproteins. Loss of
ABCA1 function leads to defective lipidation, resulting in
At baseline, the vasoconstrictor response to L-NMMA, reflecting basal NO activity, was blunted in ABCA1 heterozygotes compared with controls (P⫽0.001; Figure 1). After a
single rapid infusion of apoA-I/PC disks, the L-NMMA
constrictor response was increased significantly (P⫽0.001;
Figure 1), reaching levels comparable to those of control
subjects. In contrast, apoA-I/PC infusion had no effect on
L-NMMA response in control subjects (Figure 1).
Intra-arterial infusion of the endothelium-dependent vasodilator 5HT increased FBF in a dose-dependent manner in the
2 groups. At baseline, the FBF response to 5HT was impaired
significantly in ABCA1 heterozygotes compared with controls (P⬍0.0001; Figure 2). After apoA-I/PC infusion, FBF
response to 5HT increased significantly to levels comparable
to those of control responses (P⬍0.001; Figure 2). In line
with L-NMMA data, apoA-I/PC infusion had no effect on
5HT-induced vasodilator responses in control subjects (Figure 2).
At baseline, endothelium-independent vasodilation in response to SNP was not different between ABCA1 heterozygotes and controls, and apoA-I/PC infusion likewise showed
no effect on the SNP vasodilator response in either group
(Figure 3).
Figure 1. L-NMMA induced vasoconstriction before and after
HDL increase. FBF dose-response curves to inhibitor of NOS
L-NMMA before (䡩) and after (䢇) apoA-I/PC infusion in ABCA1
heterozygotes and normocholesterolemic controls (before, ‚;
after, ‘). L-NMMA–induced vasoconstriction at baseline was
blunted in ABCA1 mutation carriers compared with control subjects (*P⫽0.001). After HDL increase by apoA-I/PC infusion, FBF
response to L-NMMA in ABCA1 heterozygotes normalized completely (†P⫽0.001).
Baseline clinical characteristics of the ABCA1 heterozygotes
and control subjects are summarized in Table 1. HDL levels
were significantly lower in ABCA1 heterozygotes than in
controls (P⬍0.05). Other lipid parameters, as well as systolic
and diastolic blood pressures and body mass index, were not
significantly different. After apoA-I/PC infusion, plasma
HDL cholesterol increased from 0.4⫾0.3 to 1.3⫾0.4 mmol/L
and from 1.0⫾0.3 to 2.1⫾0.5 mmol/L in ABCA1 heterozygotes and controls, respectively (Table 2).
Bisoendial et al
Downloaded from http://circ.ahajournals.org/ by guest on July 28, 2017
Figure 2. 5HT-induced vasodilation before and after HDL
increase. FBF dose-response curves to endothelium-dependent
vasodilator serotonin (5-HT) before (䡩) and after (䢇) apoA-I/PC
infusion in ABCA1 heterozygotes and normocholesterolemic
controls (before, ‚; after, ‘). Endothelium-dependent vasodilation to 5-HT at baseline was attenuated in ABCA1 heterozygotes compared with control subjects (*P⬍0.0001). After HDL
increase by apoA-I/PC infusion, FBF response to 5HT in ABCA1
mutation carriers normalized completely (†P⬍0.001).
enhanced clearance of the HDL precursor fraction, with
ensuing low plasma HDL levels.7,8 We recently demonstrated
that heterozygous carriers of ABCA1 gene mutations are
characterized by advanced arterial wall thickening and an
increased risk for early-onset coronary artery disease. The
correlation between their cholesterol efflux rates and arterial
wall thickness suggests a critical role for the impairment of
the apoA-I/RCT pathway in mediating these changes in
vascular morphology.
In this study, we also show an impairment in both basal and
stimulated NO bioavailability, which is completely reversible
on HDL increase. The observed vascular effects shortly after
HDL increase seem less likely to be related to the RCT
pathway, for 2 reasons. First, ABCA1 heterozygotes are
Figure 3. SNP-induced vasodilation before and after HDL
increase. FBF dose-response curves to endotheliumindependent vasodilator SNP before and after apoA-I/PC infusion in ABCA1 heterozygotes and normocholesterolemic controls. At baseline, SNP-induced vasodilator response was not
different between ABCA1 heterozygotes and controls (P⫽0.30)
and remained unaltered by apoA-I/PC infusion.
Restoration of Endothelial Function With HDL
2947
characterized by a significantly impaired RCT pathway in
which the apoA-I concentration is not the rate-limiting
factor.23 Second, vascular effects as a result of modulating the
apoA-I/RCT pathway are not expected to occur that soon,
because cholesterol is mobilized primarily from easily mobilizable stores, eg, liver and spleen.8,24 Hence, these data
suggest that HDL by itself is a potent regulator of the
endothelial NO pathway in vivo.
The interactions between (low) HDL and endothelial NO
bioavailability can be explained by several mechanisms.
First, because the localization of eNOS in cholesterol-rich
microdomains of the plasma membrane (including caveolae)
is critical for its activation, caveolar disruption has a profound
effect on endothelial NO release.25 Recently, caveolar processing was shown to be disturbed in ABCA1-defective
human fibroblasts, resulting in a substantial decrease in the
number of caveolae on the plasma membrane.26 Given that
ABCA1 is also present in endothelial cells, similar processes
are likely to contribute to the impaired eNOS functionality.27,28 Next, low HDL may be associated with increased
vascular oxidative stress, secondarily contributing to impaired endothelial NO bioavailability. Partially because of the
presence of the enzymes paraoxonase and platelet-activating
factor hydrolase, HDL has been found to exert potent antioxidant effects.29,30 In line with this, infusion of apoA-I/PC
disks, as in our protocol, resulted in a decreased susceptibility
to oxidation of LDL in humans. However, in the present
study, we were unable to find support for a pro-oxidant state
in ABCA1 heterozygotes, because oxidized LDL levels were
not significantly higher, nor were they altered on apoA-I/PC
infusion. Third, recent data have elegantly shown a direct
stimulatory effect of HDL on eNOS via a scavenger receptor,
class B, type I– dependent pathway.31–33 In particular, the low
basal NO activity in the ABCA1 heterozygotes may support
a physiological role of HDL as a direct regulator of eNOS
activity in vivo.
Infusion of apoA-I/PC promotes intravascular generation
of nascent HDL, which has a natural course in humans.24
Subsequently, HDL increase may contribute to improvement
of endothelial vasomotor function by several mechanisms.
First, recent data have shown acute stimulatory effects of
apoA-I on intracellular cholesterol trafficking from the Golgi
apparatus to the cell surface in human fibroblasts,34 resulting
in a rapid increase in the number of membrane caveolae.
Theoretically, in these ABCA1 heterozygotes, apoA-I may
have initiated restoration of intracellular events involving
caveolar processing, with subsequent reshuttling of eNOS to
caveolar regions in endothelial cells. Finally, along with the
aforementioned direct eNOS-regulating mechanisms, an increase in apoA-I– containing HDLs may have resulted in
enhanced eNOS activity, as attested to in particular by
augmented basal NO bioavailability.
Clinical Perspectives
In the present study, we show that low HDL by itself can be
independently responsible for adverse cardiovascular effects,
assessed as endothelial dysfunction. Importantly, the reversibility of vascular dysfunction in low-HDL patients on acute
HDL increase constitutes a powerful stimulus for ongoing
2948
Circulation
June 17, 2003
efforts targeting HDL increase as a tool in cardiovascular
prevention. The present findings not only underscore the
beneficial cardiovascular potential with regard to the endothelium but also indicate that endothelial function testing is a
suitable intermediate end point in future trials evaluating the
effects of HDL increase on cardiovascular disease
progression.
Acknowledgments
Dr Kastelein is an established investigator of the Netherlands Heart
Foundation (2000D039). Dr Hovingh is supported by the Netherlands Heart Foundation (2000.115). Information on ABCA1 gene
mutations was obtained through a collaboration with Xenon Genetics
Inc. We thank R.J. Berckmans, K. Bakhtiari, and B. Karstenkov
(Department of Clinical Chemistry and Vascular Medicine, AMC
Hospital, Amsterdam) for laboratory support.
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Circulation. 2003;107:2944-2948; originally published online May 27, 2003;
doi: 10.1161/01.CIR.0000070934.69310.1A
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