© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Hypercholesterolemia Increases Coronary Endothelial Dysfunction, Lipid Content, and Accelerated Atherosclerosis After Heart Transplantation
From the Research Center and Department of Surgery (L.P.P.), Montreal Heart Institute, Montreal, Quebec, Canada, and Institut de Recherches Servier (F.M., C.B., J.-P.B., N.V., J.-P.V., P.M.V.), Suresnes, France.
Correspondence to Paul M. Vanhoutte, MD, PhD, Institut de Recherches Internationales Servier, 6 place des Pléiades, 92415 Courbevoie, France.
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Abstract |
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Abstract—Hyperlipidemia may increase endothelial damage and promote accelerated atherogenesis in graft coronary vasculopathy. To study the effects of hypercholesterolemia on coronary endothelial dysfunction, intimal hyperplasia, and lipid content, a porcine model of heterotopic heart transplantation, allowing nonacute rejection without immunosuppressive drugs, was used. A high cholesterol diet was fed to donor and recipient swine 1 month before and after transplantation. The endothelial function of coronary arteries of native and transplanted hearts from cholesterol-fed animals was studied in organ chambers 30 days after implantation and compared with endothelial function in arteries from animals fed a normal diet. The total serum cholesterol increased 3-fold in donors and recipients. Endothelium-dependent relaxations to serotonin, to the
2-adrenergic agonist UK14,304, and to the direct
G-protein activator sodium fluoride were decreased
significantly in allografted hearts compared with native hearts from
both groups. Relaxations to the calcium ionophore A23187 and bradykinin
were decreased significantly in allografts from animals fed the high
cholesterol diet. The prevalence of intimal hyperplasia was
significantly increased in coronary arteries from
hypercholesterolemic swine. There was a significant
increase in the lipid content of allograft arteries of
hypercholesterolemic recipients.
Hypercholesterolemia causes a general
coronary endothelial dysfunction, increases the
prevalence of intimal hyperplasia, and augments the incorporation of
lipids in the vascular wall after heart transplantation.
Hyperlipidemia accelerates graft coronary
atherosclerosis through its effects on the
endothelium.
Key Words: endothelium • lipids • coronary arteries • transplantation • atherosclerosis
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Introduction |
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The normal endothelium contributes to the local regulation of vasomotor tone and the maintenance of a nonthrombogenic surface, acts as a selective barrier controlling permeability and transport, and regulates the adhesion and extravasation of neutrophils, monocytes, and lymphocytes1 and the proliferation of underlying vascular smooth muscle cells. These properties are due to the ability of endothelial cells to secrete endothelium-derived relaxing factors such as nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin.2 Endothelial dysfunction is a fundamental initial step in the progression of atherosclerosis.
Although heart transplantation remains the treatment of choice for
medically unresponsive terminal heart disease and is associated with a
5-year survival of 
70%, coronary graft vasculopathy
develops in a majority of transplant recipients and is the main cause
of death beyond the first year after transplantation.3
Accelerated atherosclerosis is preceded by reduced
dilatation of the coronary artery to
endothelium-dependent agonists.4 This
endothelial dysfunction is due to an immunologic injury
directed at the endothelial cells and to other factors
that cause endothelial activation and trigger a cascade
of pathological events.5 Endothelial
dysfunction is predictive of the development of graft coronary
disease, which can be detected by intracoronary ultrasound, 1
year after graft implantation4 and of the occurrence of
morbid events and death after transplantation.
In experimental animals and humans, hypercholesterolemia impairs endothelium-dependent relaxations both in the macrocirculation and microcirculation,6 7 8 which can occur in the absence of intimal thickening.9 In the early stage of the atherosclerotic process, endothelial dysfunction is limited to the pertussis toxin–sensitive Gi-protein–dependent pathway leading to nitric oxide formation.7 Oxidized LDLs induce, in vitro, a similar selective Gi-protein pathway leading to endothelial dysfunction and, at high concentrations, inhibit endothelium-dependent responses evoked by receptor-independent stimuli (A23187). Hypercholesterolemia increases production of the superoxide anion, which scavenges the endothelium-derived vasodilator, nitric oxide.10
Hypercholesterolemia occurs in up to 80% of patients after transplantation,11 with significant increases in the ratio of total cholesterol to HDL cholesterol creating an atherogenic milieu, and is incriminated in the development of graft atherosclerosis, early myocardial infarction, and death. Dietary and pharmacological control of dyslipidemia can revert the endothelial dysfunction and slow the progression of senile atherosclerosis, leading to an increased overall survival and freedom from cardiac ischemic events.12 13 Pravastatin reverses the early endothelial dysfunction and decreases the occurrence of graft coronary vasculopathy.14
The purpose of the present study was to determine the effects of diet-induced hypercholesterolemia (1) on coronary endothelial dysfunction due to immune injury after transplantation, (2) on the development of intimal thickening in the vascular wall of coronary arteries, and (3) on the cholesterol content of coronary arteries from allografted hearts in a porcine model of heterotopic heart transplantation. The working hypothesis is that hypercholesterolemia is a compounding injurious factor to the endothelium of coronary arteries that is due to the immune injury from recipient cells and that it accelerates the development of vasculopathy, which is manifested by increased intimal hyperplasia and incorporation of lipids in the vascular wall.
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Methods |
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Animals and Immunologic Studies
Forty-nine Large-White swine (EARL de Fresnelles, Boisemont, France) of either sex, aged 12±0.8 weeks and weighing 20.2±0.9 kg, were used. The experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85–23, revised 1985). All procedures used in the present study were approved by the local institutional committee on animal care. Preoperative blood samples were drawn for determination of blood type and the class I antigen of the Swine Lymphocytes Alloantigen (SLA) system by the microlymphocytotoxicity technique.15 Swine from the same litter were used to ensure a rate of recombination between the class I and class II regions of the major histocompatibility complex of <1% (Institut National de Recherches Agronomiques). The transplantations were performed between animals compatible for blood type and for the SLA class I antigen.15 16
There was no statistically significant difference in sex distribution, age, or weight at the time of transplantation of donors and recipients in the normocholesterolemic group versus the high cholesterol group (data not shown).
Experimental Groups and Diets
Two different diet regimens were used: a standard piglet chow ad
libitum (number 8, Pietrement) and a high cholesterol (2%
cholesterol plus 10% lard) diet. To prevent excessive
weight gain, the daily food intake was limited to an amount equal to
3% of body weight daily in swine fed the high cholesterol
diet.
Six experimental groups were studied (Table 1
). All animals were weighed weekly.
Blood sampling was performed on day 1 of the diet, on the day of
transplantation (4 weeks of the diet), and at euthanasia 30 days after
graft implantation (8 weeks of the diet).
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Regimens and Biochemistry Studies
Hematology
Blood samples were processed for white and red blood cell
counts, hemoglobin content, hematocrit, platelet number, and red
blood cell indices (mean globular volume, mean globular hemoglobin
content, and mean concentration of hemoglobin) by a Minos Vet
analyzer (ABX).
Biochemistry
Blood samples were processed with a Cobas 12 analyzer
(Cobas-Mira, Roche) for liver function tests (lactate dehydrogenase,
creatine kinase, glutamate oxalate transferase, glutamate pyruvate
transferase, 
-glutamyltransferase, alkaline phosphatase, total
bilirubin, albumin, and total protein content), renal function
tests, and electrolytes (urea, creatinine, sodium,
potassium, chloride, and calcium).
Serum Lipids
Blood samples were processed for the following lipid profile
with a Cobas 12 analyzer: total cholesterol, free
cholesterol, triglycerides, phospholipids, and
HDL cholesterol (HDL-C). LDL cholesterol
(LDL-C) was calculated as the difference between total
cholesterol and HDL-C.
Plasmatic Lipoprotein Distribution Studies
Gel electrophoresis of the blood samples was performed for
measurement of the HDL-C, LDL-C, and VLDL fractions of the
cholesterol. Serum samples (40 mL) were left to coagulate
at room temperature. Separation was performed on a
polyacrylamide gel with a discontinuous gradient (Lipofilm
SEBIA). Densitometric analysis of the lipid profiles was
performed with a GS/670 scanner (Bio-Rad). Dehydrated gels were stocked
at room temperature by heating (warm air <80°C).
Surgical Technique
Anesthesia and Cardioplegia
A 4:1 blood/crystalloid solution ratio was used to achieve
cardioplegia of the donor heart. After systemic heparinization (3
mg/kg), a left retroperitoneal heterotopic transplant was performed
with an end-to-side anastomosis between the donor ascending aorta and
recipient abdominal aorta and between the donor main pulmonary
artery and the recipient inferior vena
cava.15 16 There was a statistically significant longer
ischemic time in the normocholesterolemic group
compared with the high cholesterol group (64±6 versus
38±3 minutes, respectively; P<0.05; Table 2).
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Postoperative Care
After standard ventilatory weaning, the animals were left to
recover in temperature-controlled quarters. The preoperative diet was
resumed, and water was fed ad libitum. No immunosuppressive drugs were
used. The recipients were euthanized 30 days after transplantation.
Explantation Protocol and Experimental Groups
In allografted hearts from normocholesterolemic
and hypercholesterolemic swine, after pentobarbital
anesthesia and ventilation, the allograft was excised
rapidly. Native hearts were excised through a median sternotomy.
Control hearts from nonoperated swine fed a 2%
cholesterol+10% lard diet for 60 days and receiving the
same sedation were also used. Hearts were excluded if the
coronary arteries were thrombosed (n=4 allografted hearts 30
days after transplantation in the standard chow group). Experimental
groups are summarized in Table 1
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Vascular Reactivity
The native, allografted, and control hearts were placed in a
modified Krebs’ bicarbonate solution (composition in mmol/L: NaCl
118.3, KCl 4.7, MgSO4 1.2,
KH2PO4 1.2, glucose 11.1,
CaCl2 2.5, NaHCO3 25, and
calcium EDTA 0.026; control solution). Oxygenation was
ensured by using a 95% O2/5%
CO2 gas mixture. The epicardial coronary
arteries of the native and allografted hearts were dissected free from
the epicardium, myocardium, and adventitial tissue and
divided in rings (4 mm wide, 20 rings from the allograft and 20
rings from the native heart). Rings from the left anterior descending,
left circumflex, and right coronary arteries were used randomly
but were matched between native and allograft preparations in all
experiments. The vascular reactivity of native, transplanted, and
control coronary arteries was studied in organ chambers filled
with control solution (20 mL) at 37°C. The rings were suspended
between 2 metal stirrups, 1 of which was connected to an isometric
force transducer. Data were collected with data acquisition software
(IOS3, Emka Inc). All studies were performed in the presence of
indomethacin (10-5 mol/L, to
exclude production of endogenous prostanoids) and
propranolol (10-7 mol/L, to prevent
the activation of ß-adrenergic receptors).
Each preparation was stretched to the optimal point of its active
length-tension curve (4 g), as determined by measuring the
contraction to potassium chloride (30 mmol/L) at different levels
of stretch, and then allowed to stabilize for 90 minutes. A maximal
contraction was determined with potassium chloride (60 mmol/L).
Rings were excluded if they failed to contract to potassium chloride
(exclusion rate <5%).
After a wash and 30 minutes of stabilization,
endothelium-dependent relaxations were studied in
preparations contracted with prostaglandin
F2 (range 2x10-6 to
2x10-5 mol/L) to achieve a contraction
averaging 50% of the maximal contraction to KCl (60 mmol/L).
Responses to 5-hydroxytryptamine (5HT)
creatinine sulfate (serotonin,
10-10 to 10-5 mol/L, in
the presence of 10-6 mol/L ketanserin, incubated
40 minutes before the addition of serotonin to block
serotonin 5HT2 receptors), calcium
ionophore A23187 (10-9 to
10-5 mol/L), sodium fluoride (NaF, 0.5
to 9.5 mmol/L in the presence of aluminum 5 µmol/L chloride
added 5 minutes before the addition of NaF), UK14,304
(10-9 to 10-5 mol/L, an
2-adrenergic agonist), and bradykinin
(10-10 to 10-5 mol/L)
were compared between control, native, and allografted coronary
rings 30 days after transplantation from swine fed either the standard
chow or the lipid-rich regimen. No rings were exposed to more than one
agonist in the course of the experiments. At the end of the
experiments, endothelium-independent relaxations were
studied with the use of 10-5 mol/L
3-morpholinosydnonimine (Sin-1), a nitric oxide donor.
Histology
Myocardium
Surgical myocardial biopsies were taken from the septum and
right and left ventricular free walls at the time of
explantation in all allografts and native hearts and fixed in
formaldehyde (10%). Hematoxylin-eosin–safranin O staining was
performed, and the biopsies were evaluated for rejection grade, extent
of necrosis, and ischemic changes (Table 2).
Coronary Artery Rings
After each organ chamber experiment, coronary rings were
fixed in 10% formaldehyde for 20 minutes at their optimal tension. All
formalin-fixed tissue sections were embedded in paraffin, and 5-µm
sections were stained with orcein. Each section was examined for the
presence, extent, and distribution of intimal thickening, luminal
narrowing, inflammatory infiltrates, and disruption of the internal
elastic lamina by light microscopy. Grading of intimal thickening was
performed by using a semiquantitative scale ranging from 0 to
4+.15 16 Intimal hyperplasia was considered to be
present when there was abnormal smooth muscle cell accumulation
within the intima (grades 1+ to 4+). All histological
studies were read in a blinded fashion by an independent observer.
Cholesterol and Phospholipid Determination in
Coronary Arteries
Cholesterol and phospholipids were determined in
epicardial coronary arteries 30 days after transplantation.
Segments from the left circumflex, left anterior descending, and right
coronary arteries were excised from the native and from the
transplanted hearts of 3 normocholesterolemic and 4
hypercholesterolemic animals. The adventitial fatty
tissue was removed, and the dry weight was determined for each segment
after an overnight desiccation at 40°C. Tissues were powdered in
liquid nitrogen by using a Tenbroeck homogenizer and
resuspended in 0.9% NaCl. The lipids were extracted according to the
method of Bligh and Dyer.17 Free and total
cholesterol mass determinations were conducted by gas
liquid chromatography using cholesteryl methyl ester as
an internal standard. Samples were saponified by using the procedure of
Klansek et al18 before total cholesterol
assay. Phospholipid content was quantified by the method of Sokoloff
and Rothblat.19
Drugs
All solutions were prepared daily. Aluminum chloride,
bradykinin, the calcium ionophore A23187, serotonin,
indomethacin, ketanserin, propranolol,
prostaglandin F2, NaF, and
UK14,304 were purchased from Sigma Chemical Co. Sin-1 was synthesized
at the Servier Research Institute.
Statistical Analysis
Relaxations and contractions are expressed as a percentage of
the maximal contraction to prostaglandin
F2 for each group and expressed as ±SEM;
unless otherwise specified, n refers to the number of animals studied.
ANOVA studies were performed to compare concentration-response curves
with a Greenhouse-Geisser correction of sphericity (because of
repeated measures). The Newman-Keuls test was used as the post hoc
test. The Mantel-Haenszel 
2 test was used for
the comparison of the incidence of intimal hyperplasia between native
and allografted coronary arteries at the time of explantation.
A 1-way analysis with repeated measures was conducted to
compare the cholesterol composition between the 3
coronary territories and to evaluate the effect of heart
transplantation on coronary phospholipid content. The influence
of diet and transplantation on the coronary
cholesterol content was analyzed by a 2-way ANOVA.
When differences were significant, a Newman-Keuls test was applied. All
statistical evaluations were performed by setting the Bonferroni
probability of a type I (ß) error at P<0.05.
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Results |
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Hematology
There were no statistically significant differences for white and red blood cell counts, hemoglobin, hematocrit, white blood cell count, platelet count, or red blood cell indices between baseline and end-of-diet values and no significant difference between animals fed the standard chow and those fed the high cholesterol diet (data not shown).
Biochemistry
There was a significant increase in serum creatinine
in control animals and transplant recipients fed the
cholesterol diet for 8 weeks (87±5 and 87±24 versus
144±20 and 144±28 mmol/L, respectively; P<0.001 versus
day 1). There were no statistically significant differences in all
other biochemical values at the time of explantation between animals
fed the standard chow versus the ones fed the high
cholesterol diet (data not shown).
Serum Lipids
There were no statistically significant differences between
baseline distributions in either group and no changes of distribution
after diet feeding (data not shown). There were no statistically
significant differences between baseline values (day 1 of the diet)
between the swine fed the standard chow and those fed the
cholesterol diet. There was a statistically significant
increase in the total cholesterol and LDL-C in controls and
transplant recipients fed the high cholesterol diet
compared with those fed the standard chow. There were no statistically
significant differences in the other measurements and in the ratio of
HDL-C to total cholesterol. There were no significant
changes in plasma lipids in standard chow–fed swine before and after
transplantation (Figure 1). There was a
progressive increase in the total cholesterol after 60 days
of cholesterol feeding to 3-fold the baseline value (Figure 2). There was no effect of
cholesterol feeding on plasma triglycerides.
There was a significant rise in the LDL and HDL fraction as well as a
30% rise of the ratio of TC to HDL. There was potentiation of diet by
transplantation, resulting in a doubling of the ratio of total
cholesterol to HDL (Figure 3).
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Myocardium
There was no statistically significant difference between the
grade of myocardial rejection between the
normocholesterolemic group and the high
cholesterol group, which averaged 3B according to the
International Society for Heart and Lung Transplantation
classification. There were no differences in the ischemic
changes (subendocardial fibrosis) between the allografts of both groups
and no evidence of transmural necrosis in either group. The
myocardium from native hearts was normal in all
instances.
Vascular Reactivity Studies
There were no statistically significant differences between
native groups and allografts for contraction to potassium chloride and
prostaglandin F2 (please
see Table I, published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/728/DC1). There were
statistically lower contractions to KCl in the allografts compared with
native arteries in both diet groups.
Endothelium-Dependent Relaxations
Effect of Diet Per Se
There was a statistically significant decrease of
endothelium-dependent relaxations to
serotonin, bradykinin, and the calcium ionophore A23187 in
control cholesterol arteries compared with control standard
arteries (Figures 4a, 5a, 6a). There were no statistically
significant differences in endothelium-dependent
relaxations to UK14,304 or NaF between the 2 groups (Figures 7a and 8a).
There were no statistically significant differences in
endothelium-dependent relaxations to
serotonin, UK14,304, NaF, and bradykinin between native
arteries from the standard versus the cholesterol group
(Figures 4, 5b, 7, and 8).
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) and allograft cholesterol (
);
b, native standard (•) and native cholesterol (-); and
c, control standard (
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Effect of Transplantation
There was a statistically significant decrease in
endothelium-dependent relaxations to
serotonin, UK14,304, bradykinin, and A23187 from
allografted coronary arteries 30 days after transplantation
compared with their respective native coronary arteries in the
standard chow and cholesterol groups but no differences
between the 2 groups. There was a significant decrease in
endothelium-dependent relaxations to A23187 between
allograft arteries from the cholesterol versus the native
standard group (Figure 6b).
Effect of Diet and Transplantation
There were no statistically significant difference in
endothelium-dependent relaxations to
serotonin, UK14,304, and NaF between allograft arteries in
the standard versus the cholesterol group (Figures 4a, 7a, and 8a). There was a significant decrease in
endothelium-dependent relaxations to bradykinin and
A23187 in allograft arteries from the cholesterol versus
the standard group (Figures 5a and 6a).
Endothelium-Independent Relaxations
There were no significant differences in
endothelium-independent relaxations to all agonists
because all coronary rings relaxed completely to the bolus of
Sin-1 (data not shown).
Histology
Assessment of Intimal Hyperplasia
Effect of Diet per se
There were no statistically significant differences in the
prevalence of subintimal thickening in coronary arteries from
the controls fed cholesterol (without transplantation),
arteries from the controls fed the standard chow, and arteries from
native hearts of animals fed the standard chow and from native hearts
of animals fed the cholesterol diet having undergone
transplantation (Table 3). Normal swine
carry intimal cushions that are preatherosclerotic lesions.
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Effect of Transplantation
There was a statistically significant greater prevalence of
subintimal thickening in coronary arteries from the allografts
of animals fed the standard chow compared with native arteries from the
same group (P<0.05). There was a statistically significant
greater prevalence of subintimal thickening in coronary
arteries from the allografts of animals fed the cholesterol
diet compared with their respective native arteries
(P<0.05, Table 3).
Effect of Diet and Transplantation
There was a statistically significant greater prevalence of
subintimal thickening in coronary arteries from the allografts
of animals fed the cholesterol diet (P<0.05)
compared with those of animals fed the standard chow (Table 3).
There was no statistically significant difference in the severity of
coronary intimal lesions in the allografts from animals fed the
cholesterol-rich diet compared with those from the
normocholesterolemic animals (Table 3).
Cholesterol Content of Coronary Arteries
Comparison of Lipid Composition Between Coronary
Territories
The left anterior descending, the left circumflex, and the
right coronary artery segments from native
normocholesterolemic hearts (please see Figure I;
Figures I to IV are published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/728/DC1) as well as
coronary arteries from normocholesterolemic
allografts and native and transplanted hearts from
hypercholesterolemic animals (data not shown) had a
similar composition in total, free, and esterified
cholesterol. Further analyses were conducted
independently of the coronary territory.
Effect of Diet per se
After 60 days of a cholesterol-rich diet, the total
cholesterol content increased by 46% in coronary
arteries from native hearts compared with arteries from hearts of
normocholesterolemic animals (please see Figure III,
published online) because of an increase in free
cholesterol, whereas esterified cholesterol was
similar in both groups.
Effect of Transplantation
The total and free cholesterol concentrations in
coronary arteries 30 days after transplantation in
normocholesterolemic swine were increased by 56% and
60%, respectively, compared with concentrations in native arteries.
The esterified cholesterol remained unchanged (please see
Figure II, published online), whereas the total phospholipid
concentration increased by 53% after transplantation. The ratio of
free cholesterol to phospholipids was maintained in both
transplanted and native hearts (Table 4).
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Effect of Diet and Transplantation
After 60 days of high cholesterol feeding (30 of which
occurred after heart transplantation), coronary free
cholesterol increased by an average of 50% compared with
control values. Esterified cholesterol content remained
unchanged. In allografted arteries from
hypercholesterolemic animals, there was a greater
accumulation of total cholesterol compared with that
induced by hypercholesterolemia or by
transplantation alone (please see Figure IV, published online). The
effects of transplantation (Figure II, online) and
hypercholesterolemia (Figure III, online) on
coronary total and esterified cholesterol were
additive in allografts from hypercholesterolemic
animals compared with native arteries from
normocholesterolemic animals, whereas the free
cholesterol content remained similar (Figure IV,
online).
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Discussion |
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Hypercholesterolemia
The major findings of the present study are that hypercholesterolemia does not increase the extent of the selective early endothelial dysfunction due to rejection involving the Gi-protein–mediated pathway in this model but generalizes the dysfunction to involve relaxations to bradykinin and the calcium ionophore A23187 in allografts. Hypercholesterolemia affects only relaxations to the calcium ionophore in control and native coronary arteries. Hypercholesterolemia increases the prevalence but not the severity of coronary intimal thickening in the allografts from animals fed the high cholesterol diets compared with allografts from normocholesterolemic animals but has no effect on control or native hearts. There is a significant increase in the lipid content of native hearts from high cholesterol–fed swine compared with swine fed the standard chow. Transplantation, in the absence of hypercholesterolemia, causes a significant increase in cholesterol accumulation. Hypercholesterolemia and transplantation have additive effects on total and esterified cholesterol accumulation.
Vascular Reactivity
Effect of Diet
Hypercholesterolemia has been shown to
cause impairment of relaxation to agonist-stimulating receptors coupled
to endothelial pertussis toxin–sensitive
Gi proteins that are dysfunctional and have a
reduced expression in human coronary arteries.20
Later, relaxations to bradykinin and ADP become impaired because of the
dysfunction of other G proteins (eg, Gq
proteins).7 At the advanced stage of
atherosclerosis, the response to A23187, reflecting the
capacity of the final pathway to release nitric oxide, is reduced. In
the present study, hypercholesterolemia did
not increase the degree of impairment of
endothelium-dependent relaxations to agonists coupled
to Gi-protein or direct G-protein stimulation
(eg, serotonin, 2-adrenergic
agonist UK14,304, and NaF), as evidenced by the absence of significant
difference in relaxations to these agonists in the coronary
arteries from control, native, and allograft arteries of
hypercholesterolemic animals compared with animals fed
the standard chow. This lack of effect of
hypercholesterolemia on responses to
receptor-mediated agonists that are mediated by
Gi proteins in control and native hearts may be
related to the level and duration of
hypercholesterolemia.
Hypercholesterolemia generalized the
endothelial dysfunction to bradykinin and the calcium
ionophore A23187, agonists not using the
Gi-protein pathway (except in the native arteries
for bradykinin). This effect of
hypercholesterolemia on relaxation to
bradykinin in the hypercholesterolemic group has not
been observed before, whereas the effect on A23187 has been described
with exposure to high levels of oxidized LDL.21 Induction
of Gq-protein dysfunction or an alteration of the
endothelium-derived hyperpolarizing factor pathway
could explain these observations.
Effect of Transplantation
Cellular rejection of the coronary
endothelium after heart transplantation, without
initial ischemia/reperfusion injury and in the absence of
immunosuppressive drugs, in swine fed a standard chow causes
coronary endothelial dysfunction without
destruction of the endothelial cell
lining.16 This impairment preferentially involves the
pertussis toxin–sensitive G-protein–dependent pathway as described in
human cardiac transplant recipients, in which vasodilatations to
bradykinin are maintained, whereas there is paradoxical
vasoconstriction to acetylcholine22 early after graft
implantation. Damage sustained during cardiac preservation and
reperfusion may cause endothelial dysfunction
incriminated in the development of graft coronary
vasculopathy.23 In the present model, no
endothelial dysfunction is induced at the time of
transplantation,15 16 which permits the evaluation of the
effect of rejection and of hypercholesterolemia
on the endothelial function and tone of the underlying
vascular smooth muscle.
Effect of Diet Plus Transplantation
Rejection and high cholesterol feeding did not have
additive effects in decreasing vasorelaxation within the time frame of
the present study, probably because endothelial
alterations induced by immune injury are already marked.16
The contribution of ischemia and reperfusion injury, which
could trigger an early allogeneic reaction, cannot be
assessed,24 but there was no histological
difference in ischemic damage between the 2 groups of
transplanted hearts.
Intimal Hyperplasia
Hypercholesterolemia and, specifically,
elevated serum levels of oxidized LDL correlate with the development of
graft coronary vasculopathy, and low HDL levels are associated
with subsequent sudden death and acute myocardial infarction in heart
transplant recipients.25
Hypercholesterolemia increases
endothelial production of superoxide anion
destroying nitric oxide, promoting oxidation of LDL and lipid
accumulation within the vessel wall26 and activating
oxidant-sensitive transcription nuclear factors. This state of
oxidative stress may be increased by low-grade subclinical injury from
rejection and compounded by immunosuppressive drugs used in clinical
heart transplantation, contributing to the development of graft
coronary vasculopathy.27
The present study confirms that hypercholesterolemia increases the prevalence of early intimal lesions in the transplanted hearts; this has been documented 4 weeks after implantation in cholesterol-fed animals.28 29 Acceleration of the proliferative vascular damage by a factor of 3 to 4 is seen with cholesterol feeding, but frequency or severity of lesions is unchanged in chronically rejecting rat aortic allografts in the absence of hypertriglyceridemia30 and in apoE-deficient recipients of allotransplants.31 Cholesterol feeding has a limited effect on normal arteries, as shown in coronary arteries of control and native hypercholesterolemic animals.32 High cholesterol feeding simply accelerates the process without increasing the severity of lesions and may increase the proportion of fatty proliferative lesions in the early stages.
Coronary Artery Lipid Content
Hypercholesterolemia and heart
transplantation independently increase the cholesterol
content of swine coronary arteries. A 60% rise in free
cholesterol is observed in rabbit native arteries after a
2-month cholesterol-rich diet with no accumulation of
cholesteryl esters. In the absence of
hypercholesterolemia, transplantation alone
induces free cholesterol accumulation, which could be due
to impairment of the barrier function of the
endothelium and the secondary increase in vascular
permeability observed during acute rejection in rat heart
transplantation. Lipid overload of allograft coronary arteries
occurs with only modest blood-to-artery lipoprotein gradients and more
rapidly than in native
atherosclerosis33 and could play an
early and ongoing role in the development of transplant
arteriopathy.33 Endothelial injury may
initiate lipid deposition and atherogenesis by enhancing
transendothelial pinocytic transport of lipoproteins by
regenerated endothelium. Moreover, phospholipid content
is also increased in coronary arteries from
normocholesterolemic allografts, resulting in a free
cholesterol–to–phospholipid cholesterol mass
ratio of 0.2. The maintenance of this ratio corresponds to the
initial adaptive response observed in vitro in
cholesterol-loaded macrophages. Under these
circumstances, phospholipid synthesis is induced when cellular free
cholesterol increases to maintain cell viability and
function and a ratio <0.4.32
An additive effect of hypercholesterolemia and transplantation was observed on total coronary cholesterol. The accumulation of esterified cholesterol in coronary arteries of hypercholesterolemic allografts was modest compared with the massive accumulation observed in advanced lesions. Nevertheless, the deposition of cholesteryl ester in hypercholesterolemic allografts is a hallmark of foam cell formation, which occurs through trapping of lipoproteins in the extracellular matrix or the activation of the cellular enzyme acyl coenzyme A cholesteryl ester transferase participating in the maintenance of cellular cholesterol homeostasis by esterifying the free cholesterol released from the lysosomal hydrolysis of lipoprotein-derived cholesteryl esters. Stimulation of acyl coenzyme A cholesteryl ester transferase is directly related to the availability of excess cellular free cholesterol at a threshold that may be attained with hypercholesterolemic allografts but not with hypercholesterolemia or transplantation alone; this stimulation and its results may explain the absence of esterified cholesterol formation in the latter 2 situations.34 Transplantation alters the lipid composition of the arterial wall in the absence of hypercholesterolemia and immunosuppressive treatments secondary to an alteration of the barrier function of the endothelium. Hypercholesterolemia increases the accumulation of coronary cholesterol induced by transplantation. The accumulation of total cholesterol and cholesteryl esters in the coronaries from hypercholesterolemic allografts suggests an initiation of the foam cell formation process not assessed in the present study.
Lipid accumulation has been observed in transplanted human coronary arteries colocalized with proteoglycans and apolipoprotein deposits. In human pathological studies, increases in free esterified cholesterol and phospholipid wall content correlated with the degree of luminal stenosis. These increases did not differ among the 3 coronary territories. No link was observed between the lipid wall content, the duration of implantation, and the cause of death.33
The accumulation of lipids without endothelial inflammation or intimal thickening in the absence of rejection at biopsy suggests that lipid accumulation could be an early marker of endothelial injury that is due to an increased permeability of the endothelium secondary to immunologic injury.31 Focal denudation and functional alterations from repeated waves of alloimmune response could cause pathological endothelial activation, alteration of the permeability to lipids, and elevation of the transport of lipoprotein across the arterial wall leading to an accumulation of lipids.33 This may explain the greater accumulation of lipids in transplanted hearts compared with native hearts in the animals fed a standard diet and those fed a cholesterol-rich diet.
Lipid-lowering strategies achieving decreases in total and LDL cholesterol and increases in the HDL-to-LDL ratio significantly improve endothelial function, slow the progression of native coronary atherosclerotic heart disease, and decrease the rate of myocardial infarction and death.12 13 Lipid lowering by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin lowers the incidence of rejection causing hemodynamic compromise, increases 1-year survival, and decreases the incidence of coronary vasculopathy detected by intravascular ultrasound in transplant recipients.14 Pravastatin treatment also improves the coronary endothelial dysfunction in patients compared with control transplant recipients and could decrease the incorporation of cholesterol and slow the development of transplant coronary vasculopathy.14 Hypercholesterolemia exerts an adverse effect on endothelial function, accelerates intimal hyperplasia, and increases the accumulation of lipids in the vessel wall. Preservation of the release of protective endothelium-derived factors through minimization of immune injury and control of hyperlipidemia would favor maintenance of the homeostasis of the vascular wall through antithrombotic mechanisms and antiproliferative effects on smooth muscle cells.
Received February 26, 1999; accepted October 7, 1999.
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