© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Rapid Regression of Atherosclerosis Induced by Liver-Directed Gene Transfer of ApoE in ApoE-Deficient Mice
From the Department of Medicine (K.T., R.T., D.J.R.), University of Pennsylvania School of Medicine, and the Wistar Institute (S.H.C., E.P.), Philadelphia, Pa.
Correspondence to Daniel J. Rader, MD, University of Pennsylvania Medical Center, 409 Stellar Chance Labs, 422 Curie Blvd, Philadelphia, PA 19104. E-mail rader{at}mail.med.upenn.edu
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Abstract |
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Abstract—Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and tissue macrophages. ApoE-deficient mice have severe hyperlipidemia and develop accelerated atherosclerosis on a chow diet. Both liver-derived and macrophage-derived apoEs have been shown to reduce plasma lipoprotein levels and slow the progression of atherosclerosis in apoE-deficient mice, but regression of atherosclerosis has not been demonstrated in this model. We utilized second-generation adenoviruses to achieve hepatic expression of human apoE in chow-fed, apoE-deficient mice with established atherosclerotic lesions of different stages. As expected, hepatic expression of human apoE3 significantly reduced plasma cholesterol levels. Liver-derived apoE also accumulated substantially within preexisting atherosclerotic lesions, indicating that plasma apoE gained access to the arterial intima. Hepatic expression of human apoE3 for 6 weeks resulted in significant quantitative regression of both early fatty streak lesions as well as advanced, complex lesions in both the aortic root and the aortic arch. In addition, hepatic expression of apoE induced substantial morphological changes in lesions, including decreased foam cells and increased smooth muscle cells and extracellular matrix content. In parallel, human apoE4 and apoE2 were also expressed in the liver by using recombinant adenoviruses. ApoE4 reduced cholesterol levels to the same extent as did apoE3 and also prevented progression but did not induce significant regression of preexisting lesions. ApoE2 reduced cholesterol levels to a lesser degree than did apoE3 and apoE4 and lesion progression was reduced, but regression was not induced. In summary, (1) regression of preexisting atherosclerotic lesions in apoE-deficient mice can be rapidly induced by hepatic expression of apoE, despite the absence of macrophage-derived apoE; (2) the morphological changes seen in this model of regression resemble those in other animal models, induced over longer periods of time; (3) liver-derived apoE gained access to and was retained by intimal atherosclerotic lesions; and (4) apoE4 was less effective in inducing regression, despite its effects on plasma lipoproteins that were similar to those of apoE3. The rapid regression of preexisiting atherosclerotic lesions induced by apoE gene transfer in apoE-deficient mice could provide a convenient murine model for investigation of the molecular events associated with atherosclerosis regression.
Key Words: atherosclerosis • regression • gene transfer • adenoviral vectors • apolipoproteins
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Introduction |
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Regression of atherosclerosis has been demonstrated in several animal models,1 2 3 4 5 6 largely through dietary manipulation of cholesterol levels. Regression is characterized by morphological changes in lesions, such as increased fibrotic components and decreased lipid and inflammatory components. These changes are consistent with the concept of lesion stabilization,7 8 which is thought to be responsible for the reduction in acute coronary events associated with cholesterol reduction in humans. However, the cellular and molecular mechanisms of atherosclerosis regression and stabilization are not yet well understood.
ApoE is a multifunctional protein that is synthesized in a variety of tissues, including the liver and macrophages.9 ApoE-deficient mice have hypercholesterolemia and develop extensive atherosclerosis on a chow diet.10 11 12 13 Expression of human apoE in either the liver14 15 or macrophages16 17 of apoE-deficient mice reduces cholesterol levels and slows progression of atherosclerosis. In addition, macrophage-derived apoE has been shown to slow progression of atherosclerosis, independent of plasma lipoprotein levels.18 19 However, neither liver- nor macrophage-derived apoE has been shown to induce regression of preexisting atherosclerotic lesions in apoE-deficient mice. Furthermore, it is not known whether regression of established atherosclerotic lesions is dependent on macrophage production of apoE. Finally, the ability of liver-derived plasma apoE to gain access to the arterial intima has been suggested by the identification of lipid-poor forms of apoE in plasma20 21 but has not been directly investigated.
ApoE occurs in 3 major, common isoforms in humans.9 ApoE3 is the most common isoform. ApoE2 has a single substitution of 158 Cys for Arg. It has impaired binding to the LDL receptor in vitro22 23 and slower turnover in humans in vivo compared with apoE3.24 Homozygosity for apoE2 is associated with familial dysbetalipoproteinemia due to impaired clearance of remnant lipoproteins.25 ApoE4 has a single substitution of 112 Arg for Cys. It has normal binding to the LDL receptor in vitro23 and faster turnover in humans in vivo compared with apoE3.26 Heterozygosity for apoE4 is associated with an increased risk of atherosclerotic cardiovascular disease.27 28 ApoE4 has certain cellular effects in vitro that differ substantially from those of apoE3.29 30 31 32 We previously demonstrated33 that hepatic expression of the 3 human apoE isoforms in apoE-deficient mice had effects on lipoproteins in vivo that were predicted on the basis of receptor-binding and human kinetic studies.
In the current experiments, we utilized recombinant, second-generation adenoviruses to achieve liver-directed gene transfer of apoE in apoE-deficient mice to test the following hypotheses: (1) that hepatic expression of apoE can induce rapid regression of and morphological changes in established atherosclerotic lesions, even in the absence of macrophage-derived apoE; (2) that liver-derived apoE can gain access to the arterial wall via the plasma compartment; and (3) that apoE isoforms differ in their ability to induce regression of preexisting atherosclerotic lesions in apoE-deficient mice.
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Methods |
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Construction of Recombinant, Second-Generation Adenoviruses
We utilized second-generation adenoviruses, which result in longer transgene expression in the mouse liver34 35 and with which we previously demonstrated prolonged expression of human apoE isoforms in apoE-deficient mice.33 Recombinant adenoviruses were constructed as previously described.33 In brief, human apoE2 and apoE4 cDNAs were generated from human apoE3 cDNA by utilizing the Altered sites II in vitro mutagenesis system kit (Promega Corp). The sequence of the cDNAs was confirmed by the dideoxy chain-termination method. The cDNAs were subcloned into the shuttle plasmid vector pAdCMV-Link35 in appropriate orientation. The recombinant plasmids were cotransfected into 293 cells with DNA of H5.110CMVlacZ35 digested with ClaI. Transfected cells were overlaid with agar and kept at 32°C for 15 days, and positive plaques for apoE cDNA were subjected to a second round of plaque purification. Recombinant viruses were expanded, purified by CsCl ultracentrifugation, and stored in 10% glycerol at -80°C. These viral DNAs were subjected to polymerase chain reaction–restriction fragment length polymorphism analysis,36 and the genotypes were confirmed. Viruses are referred to as AdapoE2, AdapoE3, and AdapoE4. When injected into apoE-deficient mice, these viruses expressed the expected human apoE isoforms in vivo and resulted in comparable levels of the 3 different human apoE isoform mRNAs in the liver.33
Experimental Protocols in Animals
The first series of experiments were performed in 12-week-old,
male and female, apoE-deficient mice fed a chow diet. Groups of mice
were matched on the basis of plasma cholesterol levels
before initiation of the experiment to ensure similar mean
cholesterol levels in each group. At 12 weeks of age, 1
group of mice (n=13) was killed for baseline quantitation and
examination of atherosclerotic lesions. The remaining mice were
injected with 2.5x108 plaque-forming units per
gram of body weight of AdapoE2 (n=12), AdapoE3 (n=13), AdapoE4 (n=12),
a control adenovirus encoding the lacZ gene (AdlacZ, n=12), or sterile
saline (n=12). Mice were killed 6 weeks after injection (18 weeks of
age) for quantitation and morphological characterization of
atherosclerotic lesions. Plasma was obtained on days 0, 7, 14, 28, and
42 after adenovirus injection for analysis of transgene
expression and lipids. Males and females were analyzed
separately, but because there were no differences between the 2 groups,
the atherosclerosis data were therefore pooled for
final analysis.
To determine whether hepatic apoE expression would influence more advanced lesions, we performed a similar experiment in 26-week-old, female, apoE-deficient mice fed a chow diet. Mice were injected with the apoE3-expressing adenovirus (n=13) or control virus (n=13), killed 6 weeks later, and compared with the mice killed at baseline (n=13). Plasma was obtained on days 0, 7, 14, 28, and 42 after adenovirus injection for analysis of transgene expression and lipids. Mice were obtained fom Jackson Laboratories (Bar Harbor, ME). Mice were cared for and handled according to accepted ethical practices.
En Face Quantitation of Atherosclerotic Lesions in the Aortic
Arch
Mice were anesthetized with Rompun/ketamine, ip,
and after the aorta was gently perfused with saline via the left
ventricle, the heart was cut off at the base and embedded in OCT. The
rest of the aorta was removed and fixed in 10% formalin/PBS for at
least 3 days. Aortic arch lesion area was quantitated using a method
similar to that previously described.37 38 After the
adventitial and adipose tissue was removed, the aortic arches were
stained with oil red O solution (1.8% oil red O, wt/vol, in 60%
isopropanol, filtered twice through a 0.2-µm filter) for 15 minutes
and destained with 60% isopropanol for 5 minutes to eliminate
background staining. The outer curvature of the arch was cut
longitudinally, and the arch was laid open on a glass slide and mounted
in Supermount (BioGenex). The image was captured with the use of
a Leica MZIZ microscope and digitized, and the oil red
O–stained lesion area was quantitated using the Quantimet 500 image
analysis system. All data capture and quantitation were
performed in a blinded fashion.
Immunohistochemistry and Quantitation of Atherosclerotic Lesions in
the Aortic Root
The fresh-frozen, OCT-embedded hearts were used for
immunohistochemistry and quantitation of lesions in the aortic root.
Serial sections (8 µm) of the aortic root were mounted on masked
slides. Sections were fixed in acetone, air-dried, rehydrated in PBS
containing 0.02% NaN3, and blocked with 1% BSA
in PBS/NaN3. For detection of human apoE,
ß2-integrin, and vascular cell adhesion
molecule (VCAM)-1, sections were reacted with polyclonal goat
anti-human apoE antibody (Jackson Immuno Research Labs), monoclonal
hamster anti-murine CD18 antibody (clone 2E6),39 or
monoclonal rat anti-murine VCAM-1 (clone 429, Pharmingen), followed by
incubation with biotinylated mouse anti-goat IgG antibody, goat
anti-hamster IgG antibody, or mouse anti-rat IgG antibody,
respectively, in the presence of 200 µg/mL normal mouse IgG. Antibody
reactivity was detected with horseradish peroxidase–conjugated
biotin-streptavidin complexes and developed with diaminobenzidine
tetrahydrochloride as the substrate. Immunostained sections
were photographed on a Leica microscope.
For quantitation of lesions in the aortic root, 5 sections stained with antibody to ß2-integrin, each separated by 40 µm (thus spanning a total of 200 µm of the root), were analyzed from each mouse by using methods similar to those previously described.40 41 42 43 The entire intimal lesion area in each section was manually traced and quantitated using a Quantimet 500 image analysis system in a blinded fashion. The mean lesion area per section was determined for each mouse.
Analytical Methods
The plasma total cholesterol levels were measured in
individual mice at each time point with an enzymatic assay on a Cobas
Fara II (Roche Diagnostic Systems Inc) with the use of
Sigma reagents (Sigma Chemical Co). Plasma human apoE levels were
quantitated in individual mice at each time point by using an
immunoturbidimetric assay (Sigma). Plasma samples from groups of 6 mice
were pooled at each time point and subjected to fast protein liquid
chromatography (FPLC) gel filtration (Pharmacia LKB
Biotechnology) on two Superose 6 columns as described.44
Cholesterol concentrations in the fractions were determined
with an enzymatic assay (Wako Pure Chemical Industries, Ltd). The
cholesterol in the HDL fractions was divided by the total
cholesterol recovered from the column, and this fraction
was multiplied by the total plasma cholesterol
concentration to determine the plasma HDL cholesterol
concentration in each pooled sample.
Statistical Analysis
Atherosclerotic lesion area data were subjected to a 1-way
ANOVA. Experimental groups were compared with the baseline group by
using the Dunnett test. Repeated-measures ANOVA was used to compare
cholesterol levels among different groups of mice over time
after gene transfer. Statistical significance for all comparisons was
assigned at P<0.05. Graphs represent mean±SEM
values.
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Results |
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Liver-Directed Gene Transfer of ApoE-Influenced Plasma Lipids in ApoE-Deficient Mice
After injection of adenoviruses into 12-week-old, apoE-deficient mice fed a chow diet, the plasma concentrations of apoE2 were considerably higher than those of apoE3, which in turn were somewhat higher than those of apoE4 over the course of the 6-week experiment (the Table
). These differences in plasma
concentrations were not due to differences in the abundance of hepatic
mRNA and may have been due to the known differences in plasma
catabolism among the apoE isoforms.24 26 As expected,
expression of apoE3 and apoE4 resulted in sustained, marked reductions
in plasma cholesterol levels (Figure 1A) in chow-fed, apoE-deficient mice.
Cholesterol levels in apoE3- and apoE4-expressing mice were
not significantly different at any time point. Expression of apoE2
reduced cholesterol levels for a sustained period (Figure 1A), but levels were significantly higher than in the apoE3 and
apoE4 mice at all time points except at 14 days (P<0.05),
despite much higher plasma apoE concentrations in the apoE2 mice (the
Table). FPLC gel filtration of plasma revealed substantial reductions
in VLDL and remnant lipoproteins after gene transfer in mice expressing
apoE3 and apoE4 and somewhat less but nevertheless significant
reductions in mice expressing apoE2 (data not shown). HDL
cholesterol levels were increased to a similar degree by
expression of all 3 apoE isoforms (Figure 1B).
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Liver-Derived ApoE Accumulated Within Established Atherosclerotic
Lesions in ApoE-Deficient Mice
Immunostaining of aortic root sections with
antibody against human apoE established that liver-derived apoE
accumulated in substantial amounts within the vessel wall,
predominantly localized within atherosclerotic lesions (Figure 2). This was true of all 3 isoforms,
although lesions were rare and very small in mice expressing apoE3 (see
below). Lesions from mice injected with AdlacZ had no evidence of apoE
immunoreactivity (Figure 2). Importantly, there was no evidence
of lacZ expression in the vessel wall, suggesting that
intravenous injection of the adenovirus vectors did not
result in direct transduction of macrophages or other cells in
the arterial wall. To definitively test whether
macrophages expressed apoE after apoE adenovirus injection,
apoE-deficient mice were injected with the apoE3 adenovirus, and
peritoneal macrophages and aortas were harvested 2, 4, and 6
weeks after adenovirus injection for reverse transcription–polymerase
chain reaction analysis of human apoE mRNA. We found no
evidence of human apoE mRNA in peritoneal macrophages or in the
vessel wall at any of the time points after virus injection (data not
shown). We conclude that intravenous administration of
recombinant, replication-defective adenovirus to apoE-deficient mice
did not result in expression of apoE by macrophages or other
cells in the vessel wall. Instead, liver-derived apoE gained access to
the intima via the plasma compartment and was deposited at sites of
preexisting atherosclerotic lesions, where it may have been retained
through binding to components of the extracellular matrix.
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Liver-Directed ApoE Gene Transfer Caused Regression and
Morphological Changes of Preexisting Fatty Streak Lesions
Atherosclerosis was quantified using 2 independent
methods: quantitation of lesion area in multiple sections through the
aortic root (Figure 3A) and en face
quantitation of lesion area in the aortic arch (Figure 3B).
Compared with mice killed at baseline, significant lesion progression
occurred in mice injected with the control AdlacZ virus. There were no
significant differences in lesion area between the AdlacZ- and
saline-injected mice (data not shown). In contrast, hepatic expression
of apoE3 resulted in virtually complete regression of atherosclerotic
lesions at both sites within the aorta. Very little residual lesion
could be identified in the AdapoE3-injected mice. In contrast, AdapoE4
did not induce statistically significant lesion regression, despite the
fact that it reduced cholesterol levels to the same extent
as did apoE3. ApoE4 did, however, prevent further progression of
lesions. Finally, expression of apoE2 did not induce regression but
also prevented lesion progression. For both
atherosclerosis assays, 1-way ANOVA indicated that the
groups were significantly different (P<0.0001). In pairwise
comparisons with the baseline group, the AdlacZ-injected group was
significantly higher (P<0.01) and the AdapoE3-injected
group significantly lower (P<0.05), but the AdapoE4 and
AdapoE2 groups were not significantly different from baseline.
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Hepatic expression of apoE had a substantial impact on the morphology
of atherosclerotic lesions. OCT-embedded, frozen sections were
analyzed for reactivity with an antibody against
ß2-integrin (Figure 4), which is expressed on
macrophages and macrophage-derived foam cells. In
the mice examined at baseline, the lesions were rich in
macrophage-derived foam cells and had few smooth muscle
cells, but no fibrous caps. In the control mice injected with AdlacZ,
lesions progressed to larger, more complex, foam cell–rich lesions
that still lacked fibrous caps in most cases. In striking contrast, the
residual lesions in the AdapoE3-injected mice had almost no detectable
foam cells; only layers of residual dense reactivity with the
anti–ß2-integrin that was not associated with
discrete cells were observed. Interestingly, although expression of
apoE4 and apoE2 did not induce regression of total lesion area, the
morphology of the lesions revealed reduced numbers of foam cells and
development of prominent, smooth muscle–derived fibrous caps that
stained for antibodies to laminin (data not shown) as well as VCAM-1
(Figure 5). Control virus–injected mice
had an abundance of typical foam cells (which do not stain for VCAM-1),
whereas apoE virus–injected animals had a substantial increase in
VCAM-1–positive smooth muscle cells, including cells within thick,
fibrous caps (Figure 5). Only 6% of lesions from mice injected
with AdlacZ had fibrous caps, whereas 68% of the detectable residual
lesions in the AdapoE3-injected mice, 42% of the lesions in the
AdapoE4-injected mice, and 39% in the AdapoE2-injected mice had
discrete fibrous caps.
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Liver-Directed Gene Transfer of ApoE Influenced the Extent and
Morphology of Advanced, Complex Lesions in 6-Month-Old
ApoE-Deficient Mice
To determine whether gene transfer of apoE would induce regression
of advanced, complex lesions, we performed a similar experiment with
AdapoE3 in 26-week-old, chow-fed, apoE-deficient mice. By this age,
chow-fed apoE-deficient mice develop complex lesions containing
smooth muscle cells, extracellular matrix, and necrotic lipid
cores.10 11 12 13 Hepatic expression of apoE3 in 26-week-old,
apoE-deficient mice resulted in a significant decrease in plasma
cholesterol levels comparable to those in younger mice
(data not shown). Significant regression of atherosclerotic lesion area
in the aorta (Figure 6A) and the aortic
root (Figure 6B) in the AdapoE3-injected mice was noted,
compared with mice killed at baseline. For both
atherosclerosis assays, 1-way ANOVA indicated that the
groups were significantly different (P<0.0001). In
comparison with the baseline group, the control adenovirus–injected
group was significantly higher (P<0.01) and the
AdapoE3-injected group significantly lower (P<0.01). The
magnitude of regression of advanced lesions was somewhat less than that
seen with fatty streak lesions.
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Hepatic apoE3 expression also induced morphological changes in advanced
lesions (Figure 7). In mice killed at
baseline, lesions contained abundant foam cells and necrotic lipid
cores in addition to smooth muscle cells and extracellular matrix.
Lesions from AdlacZ-injected control mice generally progressed in size
and abundance of ß2-integrin–positive foam
cells compared with lesions in baseline mice. In contrast, lesions in
mice expressing apoE3 had substantially fewer foam cells, smaller lipid
cores, and a relatively higher fibrous content, similar morphological
changes to those seen in the younger mice. Therefore, the reduction in
lesion size associated with apoE3 expression was primarily due to the
loss of foam cell mass. As was noted in younger mice, substantial
liver-derived apoE was found within atherosclerotic lesions 6 weeks
after adenovirus injection (data not shown). These results indicate
that liver-derived apoE induced regression of advanced, complex lesions
and accumulated in lesions in aging apoE-deficient mice as it did in
younger apoE-deficient mice.
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Discussion |
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Liver-directed gene transfer and hepatic expression of human apoE3 in chow-fed, apoE-deficient mice reduced plasma cholesterol levels and resulted in rapid and marked regression of preexisting atherosclerotic lesions. Six weeks after gene transfer of apoE3, virtually complete regression of preexisting fatty streak atherosclerotic lesions at 2 different sites within the aorta was noted. Substantial regression of advanced, complex lesions in older mice was seen as well. Regression occurred despite the absence of macrophage-derived apoE, indicating that macrophage-derived apoE is not required for induction of atherosclerotic lesion regression. Although regression of atherosclerosis has been demonstrated in other animal models,1 2 3 4 5 6 those studies mostly involved dietary manipulations over several months to years, and the degree of quantitative regression was generally modest. In contrast, regression induced by hepatic expression of apoE3 in apoE-deficient mice was rapid and the extent of regression was marked. Similar to previous regression studies, regression in this model was characterized by loss of macrophage-derived foam cells and a relative increase in smooth muscle cells and extracellular matrix. Therefore, this approach provides a model for rapid regression of preexisting atherosclerotic lesions in mice and may be useful in elucidating cellular and molecular events associated with regression and morphological changes in atherosclerotic lesions.
The reduction in plasma cholesterol induced by hepatic expression of apoE undoubtedly played a major role in mediating the regression seen in these experiments. However, macrophage-specific transgenic expression of apoE in apoE-deficient mice reduced progression of atherosclerosis even after controlling for changes in plasma cholesterol levels.18 Furthermore, when apoE-deficient bone marrow was transplanted into wild-type mice, atherosclerotic lesion formation was increased despite a lack of effect on plasma cholesterol levels.19 Thus, macrophage-derived apoE appears sufficient to inhibit the progression of atherosclerotic lesions, independent of its effects on plasma lipoproteins. In the current study, liver-derived apoE gained access to and accumulated within atherosclerotic lesions in the absence of macrophage-derived apoE. Therefore, it is possible that apoE deposited within the lesions may have had additional direct effects that, together with the reduction in plasma cholesterol, promoted regression of lesions.
That liver-derived apoE can access the vessel wall via the plasma
compartment is itself not surprising, given that many plasma proteins
are known to gain access to the arterial
intima.45 Plasma apoE may gain access to the vessel wall
in the context of small lipoprotein particles such as
-LpE20 21 and pre-ß-LpE.21 Plasma apoE
is an important contributor to the ability of plasma to induce
cholesterol efflux from cells in vitro.46 47
Once within the intima, apoE would be expected to associate with
components of the extracellular matrix, such as heparan sulfate
proteoglycans48 49 and laminin,50 for which
it has known affinity. In fact, the distribution of the plasma-derived
apoE detected by immunohistochemistry in lesions in these experiments
was consistent with its being primarily extracellular.
Consistent with the concept that plasma apoE may gain access to
the vessel wall is the report by Fazio et al,19 in which
wild-type mice reconstituted with apoE-deficient bone marrow were found
to have some apoE present within the vessel wall despite the lack
of macrophage production of apoE.19
Although the direct antiatherogenic mechanisms of apoE are poorly understood, apoE has been shown in vitro to have a variety of direct cellular effects, including stimulation of cellular cholesterol efflux,29 51 52 53 54 inhibition of platelet activation,55 inhibition of T-lymphocyte proliferation,56 inhibition of proliferation and chemotaxis of tumor cells,57 effects on neurite outgrowth,30 31 32 and antioxidant effects.58 ApoE4 is less effective at promoting cholesterol efflux than is apoE3 in vitro,29 accumulates in cells via a heparan sulfate proteoglycan–mediated pathway to a lesser extent than does apoE3,49 and differs from apoE3 in other cellular effects in vitro.9 30 31 32 In the current experiments, expression of apoE4 reduced plasma cholesterol levels to the same extent as did apoE3 but did not result in significant regression of atherosclerotic lesions over a 6-week time period. Therefore, differences between apoE3 and apoE4 in their cellular effects could potentially explain some of the differences in their effects on atherosclerotic lesions. However, further experiments will be required to definitively test this hypothesis.
In summary, regression of preexisting atherosclerotic lesions in apoE-deficient mice was rapidly induced by hepatic expression of apoE3, despite the absence of macrophage-derived apoE. The morphological changes in lesions seen in this model of regression resemble those in other animal models, induced over longer periods of time. Liver-derived apoE gained access to and was retained by intimal atherosclerotic lesions. ApoE4 was less effective in inducing regression despite similar effects on plasma lipoproteins as apoE3. The rapid regression of preexisiting atherosclerotic lesions induced by apoE gene transfer in apoE-deficient mice provides a murine model for the investigation of the cellular and molecular events associated with atherosclerosis regression.
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Acknowledgments |
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This work was supported in part by grants HL55323-01 and HL/AG57811-01 from the National Institutes of Health, Bethesda, Md (to D.J.R.); a grant-in-aid from the American Heart Association (to D.J.R.); and a grant from the W.W. Smith Charitable Trust (to D.J.R.). K.T. was supported in part by The Mochida Memorial Foundation For Medical and Pharmaceutical Research. We are indebted to Pearle Smith and Allison Hanlon for excellent technical assistance and to Dr Jane Glick for helpful discussions.
Received April 27, 1998; accepted January 20, 1999.
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