(Circulation. 1997;96:2722-2728.)
© 1997 American Heart Association, Inc.
Articles |
Hypercholesterolemia Exacerbates Transplant Arteriosclerosis via Increased Neointimal Smooth Muscle Cell Accumulation
Studies in Apolipoprotein E Knockout Mice
From the Cardiovascular Biology Laboratory, Harvard School of Public Health (C.S., W.-S.L., M.E.R., D.Z., D.L.F., E.H.), the Department of Medicine, Harvard Medical School (W.-S.L., M.E.R., E.H.), the Cardiovascular Division, Brigham and Women's Hospital (M.E.R.), and the Cardiac Computer Center, Massachusetts General Hospital (J.B.N.), Boston.
Correspondence to Edgar Haber, MD, Harvard School of Public Health, 677 Huntington Ave, Boston, MA 02115-6028. E-mail haber{at}cvlab.harvard.edu.
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Abstract |
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Background Hypercholesterolemia is thought to be a significant risk factor for coronary vasculopathy in cardiac transplant recipients.
Methods and Results We examined the development of
arteriosclerosis in mouse carotid artery loops
allografted from B.10A(2R) (H-2h2) donors to
normocholesterolemic C57BL/6J (H-2b)
recipients and hypercholesterolemic C57BL/6J recipients
in which the apolipoprotein (apo) E gene had been knocked out. Luminal
occlusion and cross-sectional neointimal area were greater
in arteries allografted into hypercholesterolemic
recipients at 15 and 30 days after transplantation. We also measured
cellular and extracellular matrix components of the
neointima by computerized planimetry of the fractional
areas subtended by smooth muscle cells (anti–
-actin stain),
collagen (Masson's trichrome), lipid (oil red O), and leukocytes
(anti-CD45). The neointimal area stained for smooth muscle
cells was significantly greater in hypercholesterolemic
recipients than in normocholesterolemic recipients at
15 and 30 days after allografting. Lipid contributed to
neointimal area to a lesser degree, and there was no
significant increase in the contribution of collagen or leukocytes.
Conclusions Smooth muscle cell accumulation appears to be the principal contributor to the increase in neointimal area observed in arteries allografted into hypercholesterolemic mice.
Key Words: vasculature • transplantation • muscle, smooth • hypercholesterolemia
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Introduction |
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Although graft rejection and infection remain the major predictors of mortality during the first year after heart transplantation, accelerated coronary artery disease becomes the chief impediment to survival thereafter.1 Hypercholesterolemia has been shown by some investigators to be a significant risk factor for the development of coronary vasculopathy in transplant recipients.2 3 4 5 Kobashigawa and colleagues6 showed that treatment with a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (pravastatin) reduces the incidence of coronary vasculopathy, yet they found no correlation between cholesterol levels and development of coronary vasculopathy during a 12-month period of observation. This paradoxical finding suggests that pravastatin may ameliorate vasculopathy through a mechanism other than the lowering of serum cholesterol. Other studies in rabbits, rats, and mice have yielded variable results.7 8 9 10 11 Thus, both the role and the mechanism of hypercholesterolemia in transplant vasculopathy remain open questions.
We have used a mouse carotid artery transplantation model12 to identify immunologic components of transplant arteriosclerosis.13 In the work reported here, we used the same model to study the role of hypercholesterolemia in transplant arteriosclerosis. These experiments allowed us to compare the tissue components contributing to arteriosclerotic lesions and the magnitude of lesion development in carotid artery loops allografted into hypercholesterolemic mice (in which the apolipoprotein E [apo E] gene had been deleted14 15 ) with that in loops allografted into normocholesterolemic control mice of the same genetic background. We found that a neointima developed faster and that luminal occlusion increased in arteries allografted into hypercholesterolemic mice. Smooth muscle cell accumulation appeared to be the principal cause of this increase in neointimal size.
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Methods |
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Animals
Carotid artery transplantation was performed on 8-week-old male mice weighing 28 to 32 g (Jackson Laboratory) in the following donor-recipient combinations: B.10A(2R) (H-2h2) into C57BL/6J (H-2b) (allograft, normocholesterolemic recipient); B.10A into C57BL/6J in which the apo E gene had been deleted14 15 (allograft, hypercholesterolemic recipient); and C57BL/6J apo E knockout into C57BL/6J apo E knockout (isograft, hypercholesterolemic recipient). The apo E knockout mice had been backcrossed over six generations to C57BL/6J (Jackson Laboratory). The animals were maintained on a normal mouse chow diet (No. 5010, Purina Mills) before and after surgery and were not treated with an immunosuppres-sive agent.
Transplantation
A donor carotid artery segment was anastomosed onto the carotid
artery12 of a histoincompatible (or compatible) recipient
in which the apo E gene had been deleted (or not, control). The
operation was performed on anesthetized mice under a dissecting
microscope (Wild, model M3Z). A midline incision was made on the
ventral side of the neck from the suprasternal notch to the chin. The
left carotid artery was dissected from the bifurcation in the distal
end toward the proximal end as far as was technically possible. The
artery was then occluded with two microvascular clamps (8 mm long,
ROBOZ Surgical Instruments), one at each end, and two longitudinal
arteriotomies (0.5 to 0.6 mm) were made with a fine needle (30
gauge) and scissors. In the donor mouse, both the left and the right
carotid arteries were fully dissected from the arch to the bifurcation.
The graft was then transplanted paratopically into the recipient in an
end-to-side anastomosis with an 11/0 continuous nylon suture under x16
or x25 magnification, and the skin incision was closed with a 4/0
interrupted suture. The time during which the graft was
ischemic did not exceed 60 minutes. Grafts were harvested 15 or
30 days after transplantation.
Histology and Morphometry
The proximal half of the transplanted loop (
2.5 mm) was
fixed for 3 to 4 hours in methyl Carnoy's solution at 4°C and
embedded in paraffin. The other half was fixed in 4%
paraformaldehyde for 3 hours, dehydrated in 30%
sucrose for 48 hours, embedded in medium (OCT Compound, Miles), frozen
in powdered dry ice, and kept at -80°C. Consecutive serial sections
were obtained from the center of the donor loop to the site of the
anastomosis with the recipient carotid artery. Tissue samples fixed in
methyl Carnoy's solution were stained with Verhoeff's
elastin,12 anti–-actin,12
anti-CD45,12 Masson's trichrome,13 or
anti-macrophage antibody F4/80 (Caltag Laboratories). Frozen
samples were stained with oil red O. Histological,
morphometric, and immunocytochemical analyses were performed as
described.12
The areas of the neointima, the media, and the lumen were
measured by computerized planimetry of sections obtained 150 and
300 µm from the junction of the donor carotid loop and the
recipient carotid artery. Color video images were recorded from a
Nikon Labophot-2 light microscope equipped with a Sony DXC-760MD video
camera and control unit. The images were captured on a Power Macintosh
7100/80 computer (Apple Computer) with the Scion LG3 frame grabber
(Scion, Inc) program. Image analysis routines were created
within a modified version (Organize-It Software) of the NIH Image 1.1
program. After tracing the area to be measured with the cursor, the
operator sampled five pixels of the color that defined a given stain.
The area encompassed by pixels (not always contiguous) in the color
range for anti–-actin, Masson's trichrome, oil red O, or anti-CD45
was then computed automatically by the software. The
neointima was defined as the area bounded by the internal
elastic lamina and the lumen. The media was defined as the region
between the internal and external elastic laminae. The lumen was
defined as the clear part of the vessel in the center of the
section.
Statistical Methods
The results of Wilks-Shapiro tests of normality were
consistent with gaussian distributions for all measurements of
area subtended by the four assay reagents (Fig 4
; anti–-actin for
smooth muscle, Masson's trichrome for collagen, oil red O for lipid,
and anti-CD45 for leukocytes) when the areas were normalized against
each corresponding total intimal area. These normalized measurements
were then entered into a MANOVA with two grouping factors: apo E status
(with the levels normocholesterolemic control and
hypercholesterolemic apo E knockout) and day (with the
levels day 15 and day 30).16 The four variates in the
MANOVA design were the normalized areas subtended by each of the four
stains. Because the apo E status/day data cells represented
3 mice in the control/day 15 combination, 4 mice in the
knockout/day 15 combination, 4 mice in the control/day 30
combination, and 5 mice in the knockout/day 30 combination, the design
was slightly unbalanced. Only the oil red O variate (lipid)
demonstrated a significant apo E status x day interaction
(P=.0012). Therefore, the oil red O data were
analyzed separately in two univariate ANOVAs.
Because eight distinct null hypotheses were tested (linear additive
parameters for apo E status=0 and for day=0 for each of the
four variates), the Bonferroni method was used to adjust the nominal
-value for statistical significance from .05 to .05÷8=.00625 to
correct for spurious significance due to multiple
comparisons.17 Therefore, comparisons were regarded as
significant only at P<.00625. All estimates of variability
are stated as mean±SD. The data presented in Fig 4 were also
analyzed with a stepwise multiple regression
program18 to explore interdependencies among the four
stain variates and overall intimal area.
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P=.0002, hypercholesterolemic recipient
vs normocholesterolemic control recipient, day 15 and
day 30; P=.002, hypercholesterolemic day 15
vs hypercholesterolemic day 30. §P=.002,
hypercholesterolemic day 15 vs
hypercholesterolemic day 30. ¶P=.0001,
hypercholesterolemic vs control, day 30;
P=.0006, hypercholesterolemic day 15 vs
hypercholesterolemic day 30. ||P=.0006,
normocholesterolemic day 15 vs
normocholesterolemic day 30. |
Results |
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Carotid artery loops from B.10A donors were allografted into C57BL/6J recipients (normocholesterolemic mice) and C57BL/6J recipients in which the apo E gene had been deleted (hypercholesterolemic apo E knockouts). As a control, loops from apo E knockout donors were isografted into apo E knockout recipients.
Serum Cholesterol Levels
Mean serum cholesterol levels measured by enzymatic
assay (Boehringer-Mannheim) were 102±17 (SD) mg/dL
(n=6) in B.10A mice and 90±8 mg/dL (n=4) in
normocholesterolemic C57BL/6J mice before
transplantation. Mean serum cholesterol levels were
652±187 mg/dL (n=9) in hypercholesterolemic
C57BL/6J apo E knockout mice after transplantation.
Region of Donor Loop Sampled
In hypercholesterolemic apo E knockout mice not
subjected to surgery, atherosclerotic lesions develop preferentially at
arterial bifurcations,19 an observation
consistent with the classic appreciation of
atheromatous lesion distribution in humans. Therefore,
in the present study we examined cross-sectional
neointimal area in sections from the center of the
transplanted carotid artery loop, as we had in our previous
studies,12 13 as well as neointimal area in
sections close to the junction between the transplanted
arterial loop and the recipient carotid artery. This
examination allowed us to determine the possible effects of turbulent
blood flow at bifurcations, which exacerbates
arteriosclerotic lesion formation in the setting of
hypercholesterolemia.
We found no significant difference at 15 days after transplantation between the intimal area [expressed as (intimal area/intimal+medial area)x100] in the junctional region and that in the center in arterial loops allografted into normocholesterolemic mice [40.6±15.1 (SD) versus 38.5±17.8, P=.70]. In loops allografted into hypercholesterolemic animals, however, the neointimal cross-sectional ratio at 15 days was substantially greater in the junctional region (not including the suture line) than in the center (69.9±9.1 versus 25.7±8.9, P<.0001). This observation suggests that turbulent blood flow has a substantial effect on lesion development in hypercholesterolemic but not normocholesterolemic mice. We subsequently measured differences between normocholesterolemic and hypercholesterolemic mice in sectionsof donor loop obtained 150 and 300 µm from the anastomosis.
Size of Neointima and Degree of Luminal
Occlusion
Carotid artery allotransplantation into
hypercholesterolemic mice resulted in a higher degree
of luminal occlusion and formation of neointimas with a
larger cross-sectional area in the region near the anastomosis than did
allotransplantation into normocholesterolemic mice at
both 15 (Figs 1 through 3) and 30 (Figs 2 and 3)
days after transplantation. As expected, intimal area increased and
residual luminal area decreased between days 15 and 30 (Figs 2 and 3)
in loops allografted into normocholesterolemic as well
as hypercholesterolemic recipients. An alternative
analysis of these comparisons that normalized for vessel
diameter [(intimal area/intimal+medial area)x100] gave similar
results: loops allografted into normocholesterolemic
recipients versus hypercholesterolemic recipients at 15
days, 40.6±15 versus 50.5±14, P=.04; at 30 days, 56.8±6.8
versus 70.7±4.8, P<.0001.
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We found previously that isotransplantation of carotid artery
loops into normocholesterolemic mice results in minimal
formation of neointima.12 Isotransplantation
of nonatherosclerotic arteries from 8-week-old apo E knockout donors
(an age at which the vessels are still clear in these
hypercholesterolemic mice) into 8-week-old apo E
knockout recipients (Fig 2
) did result in formation of a modest,
eccentric neointima [11.8±16x103 (SD)
µm2] at 30 days after transplantation that was much
smaller than the neointimas formed in arterial
loops allografted into apo E knockouts at both 15 and 30 days after
transplantation (63.09±13.62x103 and
112.51±27.68x103 µm2, respectively).
Although a neointima was evident on
histological examination in five of the eight apo E
knockout arteries isografted into apo E knockout recipients, there was
no neointima whatsoever in the remaining three. The
8-week-old apo E knockout mice used as donors did not manifest
atherosclerosis in the carotid artery (data not shown).
It is likely that the lesions observed in the apo E isografts proceeded
independently of the transplantation operation.
There was little variability in cholesterol values in apo E knockout mice (as indicated under "Serum Cholesterol Levels," the SD was small: 652±187 mg/dL). There was also no significant correlation between cholesterol values in individual animals and intimal lesion size (r=.12), probably because the range of variability in both cholesterol values and cross-sectional lesion areas was small.
Number of Nuclei in the Neointima
To determine whether the increase in the size of the
neointima in arteries allografted into
hypercholesterolemic mice was due to an increase in
cellularity or to an increase in cell size or deposition of
extracellular matrix, we compared the number of nuclei in sections of
B.10A arterial loops that had been allografted into
normocholesterolemic C57BL/6J recipients with that in
sections that had been allografted into
hypercholesterolemic C57BL/6J apo E knockout
recipients. Although there was no significant difference in the number
of nuclei at 15 days, at 30 days the number of nuclei in the intimas of
arteries allografted into apo E knockouts was significantly higher
[814±158 (SD) versus 565±221 nuclei/1000 µm2,
P=.037]. These findings suggest that cellular proliferation
or migration in hypercholesterolemic mice contributes
at least in part to the increase in neointimal size.
Contributions to Neointimal Areas of Component
Cells
The cellular and extracellular matrix components contributing to
the increase in neointimal area were measured by
computerized planimetry of the areas subtended by colors
representing anti–-actin (which stains for smooth
muscle cells), Masson's trichrome (which stains for collagen), oil red
O (which stains for lipid), and anti-CD45 (which stains for
leukocytes). Each area measurement was made independently in separate
and consecutive vessel sections. These measurements permitted
quantification of cellular components and matrix substances (smooth
muscle cells, lipid, and collagen) that are not amenable to
conventional cell counting.
On day 15 after transplantation, smooth muscle cells made the major
contribution to the neointimal area in arterial
loops allografted into hypercholesterolemic mice (Figs 1, right, and 4). The contributions of
collagen (Fig 4), lipid (Figs 4 and 5,
center), and leukocytes (Fig 4) were but trivial. By day 30, smooth
muscle cells remained the major contributor to intimal area (Figs 4 and 6, right), but the contribution of lipid
became more significant (Figs 4 and 5, right). On
histological examination, lipid was deposited both
intracellularly and extracellularly. Double staining with
macrophage-specific antibody F4/80 and anti–-actin,
as well as single staining with the two antibodies on adjacent
sections, showed that foam cells in the neointima arose
from both the smooth muscle cell and the macrophage lineages.
Approximately 90% of the foam cells were stained with anti–-actin
and 10% were stained with F4/80 (data not shown).
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There was no significant difference between collagen deposition in
loops allografted into normocholesterolemic and
hypercholesterolemic mice at day 15 or day 30 (Fig 4).
However, a comparison between day-15 and day-30 neointimas
in loops allografted into hypercholesterolemic as well
as normocholesterolemic mice indicated a significant
increase in collagen deposition (Fig 4). In
hypercholesterolemic mice at day 30 after
transplantation, collagen was the third-most-prevalent component
studied (behind smooth muscle and lipid) of the neointima
in the allografted artery (Fig 4). Leukocytes were significantly more
prevalent in arterial loops allografted into
normocholesterolemic recipients than in those
allografted into hypercholesterolemic recipients, at
both 15 and 30 days after transplantation (Fig 4). Because the major
contribution to the enlarged neointimas in arteries
allografted into hypercholesterolemic mice came from
smooth muscle cells, smooth muscle cell accumulation appears to be a
consequence of hypercholesterolemia, lipid
deposition in the vessel wall, or chemotactic (or growth) factors
secreted by foam cells.
The sum of the areas subtended by the four stains studied did not equal
the total area of the neointima. For example, the areas
stained by anti–-actin (smooth muscle), Masson's trichrome
(collagen), oil red O (lipid), and anti-CD45 (leukocytes) accounted for
only 75% of the measured neointimal area in
arterial loops allografted into apo E knockout recipients
at 30 days after transplantation. The presence of immature smooth
muscle cells, which do not contain -actin,20 21 and the
fact that extracellular matrix contains many substances other than
collagen are two possible explanations for this difference. It should
also be noted that the antibody to CD45 stains the leukocyte cell
membrane, in contrast to the antibody to -actin, which stains the
smooth muscle cell cytoplasm. This difference in staining would result
in an underestimation of the area occupied by leukocytes. These
disparities notwithstanding, however, smooth muscle cell accumulation
appears to be the principal reason for the larger
neointimas and greater degree of luminal occlusion in
arteries allografted into hypercholesterolemic as
opposed to normocholesterolemic recipients.
Lipid-filled foam cells or extracellular lipid (revealed by oil red O)
made a smaller contribution to neointimal size, and
extracellular matrix (Masson's trichrome) and inflammatory cells
(anti-CD45) made negligible contributions.
A stepwise regression analysis showed that changes in the
intimal area correlated best with changes in the area stained by
anti–-actin (P<.001 compared with the areas stained by
oil red O, Masson's trichrome, and anti-CD45) and that changes in the
area stained by anti–-actin in turn correlated best with changes in
the area stained by oil red O (P<.001 compared with the
areas stained by Masson's trichrome and anti-CD45). By indicating that
smooth muscle cells accounted for most of the increase in intimal area
and that lipid accounted for most of the increase in smooth muscle
cells, this analysis confirms the comparative analysis
presented in Fig 4.
Characteristics of the Media
There was significant deposition of lipid in the media
[10±4% (SD) of the medial area] by 15 days after transplantation in
B.10A arterial loops allografted into C57BL/6J apo E
knockout (hypercholesterolemic) recipients (Fig 5, center). Little lipid deposition was visible in B.10A
arterial loops allografted into
normocholesterolemic C57BL/6J recipients (not shown).
By 30 days after transplantation, medial lipid deposition had increased
to 24±13% in loops allografted into
hypercholesterolemic animals (Fig 5, right) compared
with 3.4±2.5% in loops allografted into
normocholesterolemic controls (Fig 5, left). Lipid
particles deposited in the medias of B.10A arteries allografted into
normocholesterolemic C57BL/6J recipients (Fig 5, left)
were smaller than lipid particles deposited in those allografted into
C57BL/6J apo E knockout recipients (Fig 5, center and right). This
difference may reflect the presence of fragmented extracellular
membrane rather than foam cells. The larger globules observed in the
B.10A to C57BL/6J apo E knockout combination are more
consistent with foam cells.
Arterial Remodeling
If the intima were to expand and simply impinge on the lumen
without affecting the cross-sectional area of the vessel overall, then
the sum of the intimal and residual luminal areas should not differ
between arteries allografted into normocholesterolemic
and hypercholesterolemic mice, despite any difference
in intimal area. If there was arterial remodeling, however,
the overall area inside the internal elastic lamina should increase. We
studied this question by comparing the cross-sectional vessel area
(intimal+luminal area). At 30 days after transplantation (Fig 6), there
was a significant increase in cross-sectional vessel area in
arterial loops allografted into
hypercholesterolemic in comparison with
normocholesterolemic recipients
[110.0±21.9x103 (SD) versus
76.7±21.7x103 µm2,
P=.0006]. At 15 days (not shown), there was no significant
difference (P=.13). These findings suggest a degree of
arterial remodeling caused by massive formation of
neointima in hypercholesterolemic
recipients that does not occur in normocholesterolemic
recipients.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Although clinical and experimental observations indicate that hypercholesterolemia accelerates transplant arteriosclerosis,2 3 4 7 8 the mechanism of this acceleration has not been documented. We developed a mouse model of carotid artery allotransplantation that permits quantification of the cellular composition and size of arteriosclerotic lesions.12 We have used the model to define components of the immune system responsible for transplant arteriosclerosis13 and apply it here to an examination of accelerated arteriosclerosis in the presence of hypercholesterolemia. At both 15 and 30 days after transplantation, the area of neointima formed in arterial loops allografted into hypercholesterolemic apo E knockout recipients was significantly greater than that in loops allografted into normocholesterolemic recipients (Fig 2
), as was the
degree of luminal occlusion (Fig 3).
We noted that the neointimas formed in
hypercholesterolemic mice contained a larger number of
nuclei than those formed in normocholesterolemic
controls, which suggested that cellular proliferation was in part
responsible for the increase in area. To determine the cellular and
extracellular matrix components contributing to this increase in
neointimal size, we stained sections of allografted
arteries individually with anti–-actin (to show smooth muscle
cells), Masson's trichrome (to show collagen, an extracellular matrix
component), oil red O (to show extracellular lipid as well as lipid
within foam cells), and anti-CD45 (to show inflammatory cells).
Automated computerized planimetry specific to each stain permitted an
objective evaluation of the fractional area of neointima
subtended by that stain. We found that the area occupied by smooth
muscle cells made a major contribution to the increase in
neointimal size overall (Fig 4), although intracellular
lipid deposition contributed as well. Changes in collagen and
inflammatory cells made but minor contributions to overall
neointimal area, and they did not account for the increased
neointimal size in arteries allografted into
hypercholesterolemic recipients. Rather than just
impinging on the lumen, the very large neointima that
formed 30 days after transplantation in arteries allografted into
hypercholesterolemic recipients (Fig 6) caused an
overall expansion of the vessel's diameter, in essence an example of
arterial remodeling.22
It is not surprising that foam cells should occur in arteriosclerotic lesions developing in hypercholesterolemic animals. Indeed, lipid deposition frequently accompanies transplant arteriosclerosis in human specimens.23 What is surprising is the prominent role played by smooth muscle cells in the augmented lesion in hypercholesterolemic animals. Smooth muscle cell accumulation is a characteristic of human transplant arteriosclerosis,23 and we have also observed it in carotid arteries allografted into normocholesterolemic mice.12 Smooth muscle cell accumulation is probably mediated by an immunologic process in which helper T cells, humoral antibody, and macrophages participate.13 The stimulus for the substantial additional smooth muscle cell accumulation seen in arteries allografted into hypercholesterolemic mice is not apparent. One possibility is that the additional accumulation derives from the action of a growth factor secreted in greater amounts by macrophage-derived foam cells than by non–lipid-containing macrophages. Wang et al24 showed that interleukin (IL)-8 mRNA and protein levels are three to four times higher in human foam cells than in non–lipid-containing macrophages. IL-8 is a potent stimulus to DNA synthesis and cell migration in vascular smooth muscle.25 Although IL-8 has not been identified yet in the mouse, there are two forms of the IL-8 receptor in this species.26 Because these receptors are present in the mouse, ligands to them, such as N51, may play the role of IL-8.27 28 Our studies do not address the issue of whether HMG-CoA reductase inhibitors manifest their beneficial effects in humans by lowering plasma cholesterol or by inhibiting smooth muscle cell proliferation directly.6 To differentiate between the two possibilities, it will be necessary to repeat the studies in LDL receptor–deficient mice, a model in which HMG-CoA reductase inhibitors have no effect.
Another unusual finding, generally not observed in transplant
arteriosclerosis in humans, is that lipid
deposition was significant in the medias of arterial loops
allografted into hypercholesterolemic animals,
particularly at 30 days after transplantation (Fig 5). The very high
levels of lipid in apo E knockout mice may have accounted for this
difference, because oxidation of the abundant lipid29
could have contributed additionally to medial smooth muscle cell
proliferation and migration to the neointima.
In conclusion, hypercholesterolemia appears to increase the rate of neointima formation and vascular occlusion in experimental transplant vasculopathy by a mechanism that depends on smooth muscle cell accumulation. This observation should lead to a reevaluation of the importance of controlling cholesterol and lipid levels after clinical cardiac transplantation.
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Acknowledgments |
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This work was supported by a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute. We are grateful to Mark A. Perrella and Choon-Joo Kho for their advice about the design of our studies.
Received April 21, 1997; revision received June 4, 1997; accepted June 6, 1997.
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