© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Quantification of Cholesteryl Esters in Human and Rabbit Atherosclerotic Plaques by Magic-Angle Spinning 13C-NMR
From the Department of Biophysics (S.P., W.G., J.A.H.), Boston University School of Medicine, Boston, Mass; the Departments of Medicine and Biochemistry (J.D.M.), Baylor School of Medicine, Houston, Tex; and the Department of Medicine (M.T.J.), Cardiovascular Division, Beth Israel Medical Center, Boston, Mass.
Correspondence to James A. Hamilton, Department of Biophysics, Boston University School of Medicine, Boston, MA 02118. E-mail Hamilton{at}med-biophd.bu.edu
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Abstract—Accumulation of cholesteryl esters (CEs) is a key event in the formation of atherosclerotic plaques. More recent work suggests a role for CEs in plaque rupture leading to thrombosis, which can result in an acute event such as myocardial infarction or stroke. In this study, we present nuclear magnetic resonance (NMR) protocols for quantification of CEs in plaques in situ. Total CEs quantified by 13C magic-angle spinning (MAS) NMR in excised plaques from human carotid arteries and rabbit aortic arteries were in good agreement with the amounts determined by subsequent standard chemical assays. The latter analysis is disadvantageous because it requires that plaque lipids be extracted from the tissue, resulting in the loss of all phase information of CEs as well as other major plaque components. With our MAS-NMR protocol, the plaque components are preserved in their native phases. Combining MAS and off-MAS NMR, we were able to quantitatively distinguish isotropic (liquid) CEs from anisotropic (liquid-crystalline) CEs in plaque tissues. In a recent study, we applied a different 13C MAS-NMR protocol to quantify crystalline cholesterol monohydrate in plaques. Together, these 2 studies describe a new, noninvasive MAS-NMR strategy for the identification and quantification of the major lipid components in plaques in situ. This approach will be useful for investigation of the relationship between plaque rupture and specific lipids in their biologically relevant phases.
Key Words: NMR • cholesteryl esters • atherosclerotic plaques • plaque rupture • lipid phase
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Introduction |
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The formation of a lipid-rich lesion in the arterial wall is a key event in the initiation and progression of atherosclerosis. The lipid content and composition are important factors in predicting the stability of an atheromatous plaque.1 2 Cholesteryl esters (CEs) constitute a major fraction of the lipid-rich core in the atherosclerotic plaque, and their abundance is highly associated with rupture of plaques and formation of thrombi.3 Aortic plaques with a lipid core occupying >40% of the total plaque volume are at the highest risk of rupture.4 It is hypothesized that the accumulation of noncrystalline CEs may soften the lipid core, making plaques with a thin, fibrous cap more prone to rupture.5 A more specific suggestion is that the ratio of CEs to crystalline cholesterol monohydrate is a determinant of plaque "softness."6 However, only very recently has an accurate quantification of crystalline cholesterol monohydrate in a plaque in situ been achieved.7
In addition to the chemical abundance of CEs, the physical state of CEs and the proximity of CEs to other plaque components may be important for plaque stability. CE is a nonpolar lipid that does not mix well with polar lipids (eg, phospholipids) but can serve as a weak solvent for weakly polar lipids such as triglycerides and cholesterol.3 Within plaques, CE is phase-separated from phospholipid/cholesterol bilayers and crystalline cholesterol. In some plaques, the CE pool is enriched with saturated long-chain fatty acids, resulting in a phase-transition between liquid and liquid-crystalline phases near body temperature.8 Variations in phase-transition temperatures of CEs may play important roles in disease progression.9 10 In addition, the proximity of CEs to the fibrous cap and their spatial distribution in the plaque are also likely to be important factors that determine plaque stability.11
Several different methods, including x-ray diffraction, optical microscopy, solution nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, and Fourier transform infrared have been applied to study the physical properties of the lipids in plaques in situ.3 7 Complete quantitative analysis of plaque lipids by chemical assays requires extraction of plaques with organic solvents; thus, the information regarding the lipid phases originally present in the plaques is lost. Raman spectroscopic analysis can also be used to quantify the major lipid components in homogenized and intact plaques,12 but to date this technique has not distinguished the different phases of lipids. The point-to-point spatial variations in the structure of plaque samples reduce the accuracy of the Raman assay and its applicability for noninvasive quantitative analysis.13 14 There is currently no method available for the accurate quantification of liquid and liquid-crystalline phases of CEs in a plaque. The best method available, polarized light microscopy, can estimate the relative amounts of CE phases in a thin section (10 µm) dissected from a plaque. However, the quantification of CEs in even a moderately sized plaque would be very time-consuming and not feasible for a large number of plaque samples.
Compared with these methods, NMR spectroscopy is advantageous because it can detect and quantify each lipid species in different physical states in an intact plaque (ex vivo). Whereas standard "solution" NMR spectroscopy detects mainly the liquid phase, solid-state NMR with magic-angle sample spinning (MAS) can eliminate the signal overlap and line broadening caused by chemical shift anisotropy in nonliquid samples to yield high-resolution spectra suitable for quantitative measurements.15 Hence, MAS-NMR is particularly useful for studying heterogeneous and anisotropic lipid mixtures in plaques and other biological samples.16 17 The improved signal sensitivity of MAS-NMR, compared with solution NMR, also makes it an ideal method to study plaque CEs in a liquid phase. However, because the isotropic chemical shifts of liquid CEs and liquid-crystalline CEs detected by MAS-NMR are essentially the same,18 these 2 phases cannot be identified by MAS-NMR alone. Liquid CEs can be distinguished from liquid-crystalline CEs in NMR spectra either without sample spinning or with sample spinning at an off-magic angle (off-MAS), under which conditions the NMR resonances for the anisotropic liquid-crystalline CEs become much broader18 19 20 than the isotropic liquid phase CEs.
In this study, we applied off-MAS and MAS 13C NMR to detect and quantify the noncrystalline CEs (liquid and liquid-crystalline phase) in excised human and animal atherosclerotic plaques. The accuracy and reliability of this new application are validated by comparison with the results of the chemical analysis of CEs. Together with our previous quantification of solid-phase cholesterol (crystalline cholesterol monohydrate) by a related MAS-NMR experiment,7 this study allows quantification in situ of the phases of lipids that are hypothesized to be key to plaque stability.
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Methods |
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Materials
CEs (>99% pure) were purchased from NuChek. The purity of the CEs was checked by differential scanning calorimetry (DSC) and thin-layer chromatography before and after NMR experiments. No significant impurities were found.
Human carotid plaque tissues were obtained by endarterectomy, fixed in 10% formalin/Tris-buffered saline immediately after excision (except as noted below), and shipped at ambient temperatures from Baylor College of Medicine (Houston, Tex) to Boston University School of Medicine (Boston, Mass).
Animal tissues were obtained from 4-year-old Watanabe heritable hyperlipidemic (WHHL) rabbits,21 which were a gift from Dr Thomas Parker of Cornell University, Ithaca, NY. No dietary or surgical interventions were performed on these rabbits. The rabbit plaque tissue samples were fixed in 10% formalin/saline solution and stored at -80°C.
Sample Preparation
A mixture of CEs with acyl chains composed of 20%
palmitate (C16: 0), 30% oleate (C18:1), 35% linoleate (C18:2), 5%
palmitoleate (C16:1), 5% linolenate (C18:3), and 5% myristate
(C14:0) was used to model the mixture of CEs in
plaque.10 22
This model sample exists in a liquid-crystalline phase between 38°C
and 44°C and in a liquid phase at temperatures >44°C. In real
plaque samples, the CE pool usually contains small fractions of
impurities, including other lipids, such as triglycerides
and cholesterol. These solute molecules generally lower the
melting temperature of the CE pool to below or near body
temperature.9 To
avoid complications, we chose to use the model CE mixture without
adding these minor components. Hence, the melting temperature of our
model system is higher than are CE mixtures from typical plaque
samples. Because the molecular motions (viscosity) near the melting
point may be anomalous, we performed our MAS-NMR experiments at
temperatures 5°C higher than the corresponding phase-transition
temperatures, where the motions are representative of a
pure liquid
phase.20
The CE components were added together in the crystalline or oil phase and then heated to 5°C above the highest melting CE (cholesterol palmitate) and cooled to ambient temperatures. After 5 cycles of heating and cooling, the CE mixture was considered to be homogenous, based on reproducible DSC thermograms. Such a protocol was required because the samples were mixed without solvents.
Plaque samples were cut into small segments (
2.5- to 5-mm
long) to fit into 7-mm ZrO2 sample rotors used
for MAS-NMR measurements. Because rabbit tissues were stored at low
temperature, which results in the crystallization of CEs, these plaque
samples were heated to 60°C and then cooled to the desired
temperature for NMR experiments, generally 37°C. Previous studies had
shown that this preheating will melt the crystalline CE phase, which,
after cooling, will remain in either the isotropic or
liquid-crystalline phase as it was in
vivo.3 Thus, the
original phases of plaque CEs are restored after preheating of the
frozen plaque
tissues.23
To validate that our temperature protocol (freezing, then
heating to 60°C and recooling) did not affect spectroscopic
properties, we performed the following experiments. Two human carotid
plaques (additional to those reported in
the
Table
) were shipped at ambient temperature in
PBS/glycerol buffer (50%) without fixation, and
13C MAS-NMR spectra were obtained at 37°C
before they were frozen. The plaques were then kept at -80°C for 18
hours and thawed at room temperature. After being placed in the NMR
probe, each plaque was heated to 60°C for 30 minutes and cooled to
37°C. The spectra obtained after this protocol were the same as the
initial spectra, indicating that our temperature protocol did not
affect the quantification of lipids. Previously, we have also shown
that fixation in formalin does not affect the NMR spectra of
plaques.7
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NMR Measurements
MAS-NMR experiments were performed on a Bruker
AMX-300 NMR spectrometer equipped with solid-state NMR accessories. For
experiments with model CE mixtures, samples were sealed in a small
Pyrex glass sphere to avoid leakage of the liquid CE mixture. The glass
sphere was then placed in a 7-mm ZnO2 rotor and
balanced with KCl powder in the rotor to avoid vibration during sample
spinning. Off-MAS-NMR experiments were performed under conditions
identical to those of MAS NMR, except that the sample rotation axis was
changed to 7° off the magic angle, as described
previously.18 We
have observed that the off-MAS experiment is more advantageous than the
nonspinning experiment for detecting the liquid phase because for the
same sample size used, rapid sample spinning achieves better field
homogeneity and thus, higher signal sensitivity. When MAS and
off-MAS-NMR experiments are used together, the CE components in liquid
and liquid-crystalline phases can be identified individually for lipids
in model mixtures and plaques in
situ.16 18 22 24
All spectra were obtained with a 5-µs 90° pulse width, 12K scans, and a spinning rate of 4 kHz. The 13C chemical shifts were referenced to the carbonyl carbon resonance of an external glycine sample (176.06 ppm from tetramethylsilane). Peak assignments were based on previous studies.19 25 26 Because the T1 values for C5 and C6 carbons in noncrystalline CEs are <1 second,25 a 5-second pulse interval time was used to allow full magnetization relaxation, which is important for quantification studies at NMR intensity. The sample temperature was controlled within 1°C by the Bruker B-VT-1000 variable temperature unit. The actual probe temperature was calibrated with the known phase-transition temperatures of pure CEs.18 The mild baseline "roll" in some spectra results from the plastic insert in the MAS-NMR rotor. After completion of the NMR experiments, the plaques were homogenized and their lipid composition was determined by standard chemical assays.27
DSC Measurements
Measurements were made with a Perkin-Elmer DSC-7
instrument over the temperature range of 0°C to 95°C at heating and
cooling rates of 0.5°C/min. DSC confirmed that the CE mixture existed
as an isotropic liquid phase above 44°C and as a liquid-crystalline
phase between 34°C and 44°C after being cooled from the liquid
phase. For pure cholesterol oleate or the CE mixture, we
used 2 to 10 mg of sample. Larger sample sizes (wet weight 10 to 120
mg) were used for plaque tissues. The phase-transition temperature was
determined from the position of the transition peak on the DSC
thermograms.
Polarizing Light Microscopy
Microscopy experiments were performed on a
Leitz-Dialux microscope fitted with a polarizer and heating stage and a
Nikon Microflex UFX camera system. For observing the phase transition
of plaque lipids, a heating rate of 1°C/min was
used.
Statistical Analysis
Standard statistical analysis with the
ORIGIN program was used to compare NMR and
chemical analysis results. The F factor was
calculated as the ratio of the variance of chemical analysis
results to the variance of the NMR results:
F=S12 /S22 ,
where S1 represents the
chemical analysis variance and
S2 is the NMR analysis
variance. The F factor of 1.10 indicates close
agreement between these 2 groups of results. Errors are attributable
mainly to baseline error and the signal-to-noise ratios of peaks that
are integrated. The variability of the NMR integration was determined
from 
3 rounds of integration of the C5 and C6 peaks for each
sample.
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Results |
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Identification of CE Phase Transitions in Rabbit Plaques by DSC and Polarizing Light Microscopy
Storage of plaque at low temperature (
-80°C)
results in crystallization of its CEs, thereby altering the phases that
were present in vivo. The existence of crystallized CEs can be
detected by DSC. The first heating trace for a rabbit aortic plaque
after being stored at -80°C (please see Figure I at
http://atvb.ahajournals.org) showed a broad endothermic transition
(26°C to 39°C) representing the melting of crystalline
CEs; no additional phase transitions were detected between 40°C and
95°C. The crystalline CE phase was not detected in subsequent
cooling/heating cycles so long as samples were not frozen again, as
expected. The broad transition reflects poor cooperativity of melting,
probably because CEs with different acyl chains in plaques are
inhomogeneously distributed and the CEs are in different
lipid pools with different phase-transition
temperatures.3
Another possibility is that after rapid freezing, the CE formed a
supercooled solid amorphous phase with imperfect crystalline
structures.22 The
melting of such a solid mixture exhibits a broad transition-temperature
range. Because of limitations in instrument sensitivity, we did not
detect transitions between liquid-crystalline and liquid phases, which
are much less endothermic. Previous studies have shown that after this
preheating of a plaque sample, the original phases of CEs at
physiological temperature are
restored.3
Polarizing light microscopy is a more sensitive method for
observing liquid to liquid-crystalline phase transitions of CEs and
allows a semiquantitative measurement of the phases on each thin
section of tissue examined.
Figure 1A shows a polarized light microphotograph (x60) of
a lipid-rich plaque section (10-µm slice) at 38°C. All of the CEs
were melted in a liquid phase, because heating to 60°C did not reveal
further phase transitions (loss of birefringence). The weakly
birefringent regions (less bright) were observed between 38°C and
60°C may be from phospholipid/cholesterol bilayers or
tissue matrix proteins. In contrast, there was a remarkable change in
birefringence (bright regions) on cooling from 38°C
(Figure 1A) to 25°C
(Figure 1B), corresponding to the formation of
liquid-crystalline CEs. In
Figure 1A, a strongly birefringent component was also
detected near the site of plaque fracture (near the fibrous cap). This
may be crystalline cholesterol, because the adjacent sample
in this plaque was found by cross-polarization MAS
spectroscopy7 to be
rich in cholesterol monohydrate (not shown). The estimation
of the total CE in a specific phase in an entire plaque by polarizing
light microscopy is not feasible.
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13C NMR
Quantification of Pure CEs and a CE Mixture
MAS-NMR can detect and in principle quantify both
liquid and liquid-crystalline CEs in plaques with different
pathological
characteristics.22 24
To quantify CEs in plaques, it is necessary to construct a calibration
curve based on pure CE treated under the same experimental conditions
used to study plaques.
Figure 2A shows the high-resolution, natural abundance
proton-decoupled 13C MAS-NMR spectrum of a
model CE mixture in the liquid phase. The resonances of the C5 and C6
carbons in the sterol ring are well separated from the resonances of
other components in plaques, including resonances of
cholesterol.16 25
The chemical shifts of the C5 and C6 resonances of CE are independent
of acyl chain
composition22 and
potentially suitable for quantification of total CEs in plaques. The
inserts in
Figure 2A show increasing intensities of the C5 and C6 peaks
with increasing amounts of CEs.
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-CH3), and steroid
ring resonances (C, followed by the carbon numbers). All spectra were
obtained at 55°C with a spinning rate of 4 kHz; 12 880 spectral
accumulations were processed with 5-Hz line broadening. B,
13C NMR calibration results based on the
integration of C5 and C6 resonances of cholesterol oleate
and a CE mixture. The average integrated intensities of the C5 and C6
peaks were plotted against the corresponding sample weights: (•) MAS
and (
) off-MAS results for the CE mixture; MAS(
) and
off-MAS(
) for pure cholesterol oleate. The solid line
is the linear regression fit of the data and intersects the
x axis at 2.2 mg. The dashed line is the linear
regression fit of the open circles and intersects the
x axis at 4 mg. It should be emphasized that such
calibration curves must be obtained for each individual spectrometer
and the chosen experimental
conditions.
Similar spectral features were detected for this model
mixture in the liquid phase with off-MAS-NMR. The integrated
intensities of the C5 and C6 peaks were plotted against the
corresponding sample quantities
Figure 2B
; the filled circles represent the results
with MAS, and the open circles, results with off-MAS. The solid line
representing the linear regression fit of the MAS-NMR data
crosses the x axis at 2.2 mg, implying a detection
threshold of 2.0 mg by this method. The dashed line is the linear
regression fit of the off-MAS-NMR data, and it crosses the
x axis at 4.0 mg, implying a detection limit of 4.0
mg for the off-MAS experiment. The intensity from the off-MAS spectrum
for a given amount of CEs (open circle) is 70% of the intensity of
that for MAS, which indicates an instrumental sensitivity difference
between the MAS and off-MAS measurements, because the model CE mixtures
are completely isotropic at 55°C. This lower sensitivity results in a
higher threshold of detection for the off-MAS experiment. Therefore,
different calibration curves are required for off-MAS and MAS
quantification of liquid CEs. Additional data showing the linear
relationship between off-MAS-NMR peak intensity and the sample quantity
of cholesterol oleate are shown in
Figure 2B (filled circle). The intensities in off-MAS and
on-MAS measurements of pure cholesterol oleate at 86°C
fell on the same curves as those for the model CE mixture at 55°C
(Figure 2B). This indicates that liquid CEs can be quantified
by using the same calibration curve, independent of their acyl chain
composition.
To test further the feasibility of using NMR to quantify CEs in different phases (liquid and liquid-crystalline states), we prepared a heterogeneous mixture of these 2 phases by placing cholesterol oleate (C18:1; 6.0 mg) and cholesterol myristate (C14:0; 13.2 mg) in the same sample rotor without direct contact between the 2 species. This was achieved by sealing each sample inside a separate, small Pyrex glass sphere and then placing both sealed glass spheres into the same 7-mm rotor for NMR measurements. In this way, the thermotropic phase behavior of each individual component is preserved (details of the phase transitions of each of these CE are found in Reference 2828 ).
Figure 3 shows the off-MAS-NMR spectra at 80°C and 86°C.
At 80°C, cholesteryl oleate is completely liquid and
cholesterol myristate, completely
liquid-crystalline. By using the calibration curve of
Figure 2B for off-MAS experiments, the integrated
intensities of C5 and C6 peaks at 80°C correspond to 6 mg total
CE. This is the amount of cholesterol oleate present in
the sample mixture, indicating that only signals of the liquid phase
were detected, as expected. At 86°C, cholesterol
myristate also melted into the isotropic phase. The integrated
intensities of C5 and C6 peaks at this temperature correspond to
19.0 mg total CE, equivalent to the total mass of
cholesterol oleate and cholesterol
myristate. This finding indicates that the off-MAS spectrum at
86°C reflects the combined signals from the cholesterol
oleate and cholesterol myristate.
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In contrast to the off-MAS spectrum, the MAS spectrum (not
shown) detected the same total intensity of CE signals at both
temperatures, 80°C and 86°C (19 mg, estimated from the
calibration curve for MAS NMR experiments in
Figure 2B). By comparing the MAS and off-MAS NMR results at
80°C, we estimated that 13.0 mg of the total CE was
liquid-crystalline at this temperature. This corresponds to the amount
of cholesterol myristate present in this model
system. Hence, we have demonstrated that the combination of MAS and
off-MAS NMR can be used to quantify CEs in different phases. This
method has potential application to studies of atherosclerotic plaques
and other biological samples.
Quantification of CEs in Rabbit and Human
Atherosclerotic Lesions
Selected 13C MAS-NMR spectra
at 37°C of atherosclerotic aorta tissues from 3 different WHHL
rabbits are shown in
Figure 4. The NMR spectra contain well-defined signals of
CEs, as in the model CE mixture
(Figure 2A). The quantity of CEs in these and some additional
plaques was determined by using the calibration method described above.
After the NMR experiments, the plaque tissues were
homogenized and the lipids extracted for chemical
analysis. The results of chemical analysis and MAS-NMR
quantification
(the
Table) are in good agreement. MAS-NMR spectra of
human carotid plaques (not shown) obtained from
endarterectomy samples were generally similar to
those of the rabbit aortic plaques, as published
previously.16 The
MAS-NMR and chemical analysis of the CE content of selected
individual human plaques are in good agreement
(the
Table).
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The liquid and liquid-crystalline phases of CEs in plaques
were identified as described
before16 and
quantified by the approach described above for the model CE mixture.
The results thus obtained for selected rabbit plaques
(the
Table) show variable amounts of CEs in the
liquid-crystalline phase at 37°C, ranging from 6% to 41% (for
additional MAS and off-MAS spectra of these plaques, please see Figure
II at http://atvb.ahajournals.org). This indicates that most of the CEs
in the plaques used for this study were liquid at or near body
temperature. These results agree with the previous NMR studies that
show rabbit aorta plaque CEs were either in a liquid
phase25 or that
liquid and liquid-crystalline phases coexisted near body
temperature.29
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Discussion |
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In recent years, the presence of soft, lipid-rich cores in plaques and the occurrence of plaque rupture in moderately stenosed vessels have become well recognized and documented.5 11 The "softness" of an atherosclerotic core is associated with the accumulation of noncrystalline (mainly liquid) CEs. CE usually comprises >50% of the lipid content of rabbit aortic30 and human aortic31 and carotid32 atherosclerotic plaques by weight. In heterogeneous plaque tissues, CE exists in both intracellular and extracellular domains. Within cells, these domains can be formed by direct uptake of lipoproteins or by intracellular esterification of cholesterol. CEs in each domain may have slightly different acyl chain compositions and thus, different melting temperatures. Although most CE deposits in lesions exist in an isotropic phase under physiological conditions, the liquid to liquid-crystalline transition may be near body temperature.3 5 28 Fluctuation of the CE component between liquid and liquid-crystalline phases near body temperature could contribute to destabilizing a plaque when subjected to external mechanical or temperature stresses.11 33 34 Therefore, identifying and quantifying the different phases of CEs in atherosclerotic plaques is important for understanding some of the underlying pathological mechanisms.
This study demonstrates a nondestructive method for the
quantification of total noncrystalline CEs in plaques by MAS-NMR and of
the liquid and liquid-crystalline phases separately by a combination of
MAS and off-MAS NMR. After these NMR studies, additional studies could
be performed (such as quantification of crystalline
cholesterol monohydrate by MAS-NMR or
histological analysis of selected plaque
regions) before the tissue is homogenized for chemical
analysis. Quantification of total CEs in both rabbit and human
plaques was accurate and reliable, as assessed by subsequent chemical
analysis. In 5 rabbit aortic plaques, we found a range of CE
(6.9 to 17.2 mg) for tissue wet weights of 33.3 to 93.3 mg. The
liquid-crystalline CEs generally amounted to <20% of the total CEs
but 1 sample was 40% at 37°C (plaque E,
the
Table). The CE content in mature human carotid
plaques obtained from patients undergoing
endarterectomy ranged widely, from 3 to 27 mg, with
sample sizes varying from 50 to 100 mg of tissue dry weight. We also
showed a wide range of CE content in human carotid plaque samples
(analyzed by chemical methods) together with a large variation
in crystalline cholesterol
content.7 Because
most of the "soft" plaques are CE rich, the ratio of CE to free
cholesterol in plaques could be 1 of the key factors for
determining the stability of the fibrous
cap.6
In future work, MAS-NMR detection and quantification of CE phases and cross-polarization MAS-NMR quantification of crystalline cholesterol will be combined with MR imaging. The nondestructive detection and quantification of liquid and liquid-crystalline CEs in atherosclerotic plaques may provide important information for studying the role of progression and regression of the ‘soft" lipid core in plaque rupture and stability. In CE-rich plaques, our NMR methods can be applied to the study of CEs in different regions in a plaque, such as shoulders and caps.5 28
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Acknowledgments |
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This work was supported by AHA135-199-67 (W.G.), NIH HL41904 (J.A.H.), Welch Grant Q-1325 (J.D.M.), and NIH training grant fellowship (T32 HL07291-17) (S.P.). The authors would like to thank Jennifer Weissberg for assistance in obtaining rabbit aorta samples.
Received June 29, 2000; accepted September 15, 2000.
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