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From the Department of Pathology and Laboratory
Medicine,*
University of Cincinnati College of Medicine; and
the Division of Pathology,
Children’s
Hospital Research Foundation, Cincinnati, Ohio
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-actin-specific antibodies revealed that the neointima contained
proliferating smooth muscle cells. Neointimal area was 3.4-fold
greater, and the intima/medial ratio as well as stenotic
luminal area was more pronounced in apoE(-/-) mice than those
observed in control mice (P < 0.05). The human
apoE3 transgenic mice in FVB/N genetic background were then used to
verify a direct effect of apoE in protection against neointimal
hyperplasia in response to mechanically induced vascular injury.
Results showed that neointimal area was reduced threefold to fourfold
in mice overexpressing the human apoE3 transgene (P
< 0.05). Importantly, suppression of neointimal formation in
the apoE transgenic mice also abolished the luminal stenosis observed
in their nontransgenic FVB/N counterparts. These results documented a
direct role of apoE in modulating vascular response to injury,
suggesting that increasing apoE level may be beneficial in protection
against restenosis after vascular surgery.
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Introduction |
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Accelerated coronary arteriosclerosis because of restenosis after surgical operations is different from progressive atherosclerosis in that lipid deposition and macrophage foam-cell appearance are late events. Pathological studies revealed that narrowing of the coronary vessels in both restenosis and allograft arteriosclerosis are related to intimal hyperplasia, with abnormal proliferation and migration of vascular smooth muscle cells from the tunica media to the intima.9-11 One hypothesis suggests that these accelerated forms of arteriosclerosis are because of immune-mediated endothelial injury, thus exposing the underlying vascular smooth muscle cells to mitogenic growth factors, which induces phenotypic conversion of the smooth muscle cells from the contractile-nonproliferating phenotype to the secretory proliferating phenotype.12-15 However, more recent data revealed that differences in abnormal vascular remodeling, associated with inefficient compensatory enlargement of the arterial wall, is the contributory factor toward restenosis.16,17 Although coronary stents have been used successfully to reduce vascular wall remodeling with a decrease in the rate of restenosis,18,19 restenosis because of smooth muscle cell hyperplasia occurs after stenting in 20 to 30% of patients.20,21 The key factors that are important for regulating smooth-muscle cell proliferation and in determining the severity of neointimal hyperplasia have not been completely elucidated.
Recent studies revealed that apolipoprotein (apo) E4 homozygosity is
associated with increased risk of restenosis after percutaneous
transluminal coronary angioplasty in human patients.22,23
The relationship between the 
4/4 genotype and restenosis seemed to
be independent of serum cholesterol and apo(a) levels.23
These results suggested a lipid transport-independent role of apoE in
protection against vascular disease. Our previous studies showed that
apoE inhibits oxidized low-density lipoprotein- and platelet-derived
growth factor-induced smooth-muscle cell migration and proliferation
in vitro.24
Thus, apoE may have direct cell
regulatory functions in the vessel wall. ApoE-deficient mice have been
used previously to assess neointimal formation after various
treatments.25
However, the direct role of apoE in
dictating the severity of the neointima in vivo remains
unclear. The current study used apoE-deficient mice as well as mice
with transgenic overexpression of human apoE3 to explore the importance
of apoE level in dictating neointimal hyperplasia after mechanically
induced injury of the vessel wall.
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Materials and Methods |
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Male apoE-null mice back-crossed to a C57BL/6 genetic background
were obtained from The Jackson Laboratory (Bar Harbor, ME). The human
apoE-transgenic mice were generously provided by Dr. John Taylor
(Gladstone Institute, San Francisco, CA). Detailed characterization of
these apoE transgenic mice was reported previously by de Silva et
al.26
These apoE transgenic mice were originally produced
in the ICR strain background and were back-crossed with FVB/N mice in
our institutional facility for seven generations to >99% genetic
homogeneity in FVB/N background before experiments. The wild-type
C57BL/6 and FVB/N mice were obtained initially from The Jackson
Laboratory and were maintained as breeding colonies in our institution.
These animals were used as controls for the apoE(-/-) and apoE
transgenic mice in all experiments. The animals were maintained on a
12-hour light/12-hour dark cycle and were fed a normal mouse chow diet
(Harlan Teklad, Madison, WI). Food and water were available ad
libitum. The animals were used for experimentation when they
reached 6 to 8 weeks of age, weighing 
25 to 30 g. All animal
experimentation protocols were performed under the guidelines of animal
welfare by the University of Cincinnati, in accordance with National
Institutes of Health guidelines.
Human ApoE Assay
Human apoE level in the transgenic mice was measured by enzyme-linked immunosorbent assay. A 96-well microtiter plate was incubated overnight with 100 µl of a 2 µg/ml solution of mouse anti-human apoE monoclonal antibody 1D7 (Ottawa Heart Institute Research Corporation, Ottawa, Ontario, Canada). Nonspecific sites were then blocked with phosphate-buffered saline (PBS) containing 5 mg/ml Tween-20 and 5 mg/ml bovine serum albumin. One hundred µl of mouse serum was added to each well and the incubation was continued for 2 hours, followed by a 2-hour incubation with a 1:500 dilution of rabbit anti-human apoE polyclonal antibody (DAKO, Carpinteria, CA). The plates were washed and then incubated for an additional 2 hours with a 1:5,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG obtained from Sigma Chemical Co. (St. Louis, MO). Immunoreactivity was determined by addition of ALP 10 substrate (Sigma Chemical Co.) and measuring absorbance at 405 nm. Purified human apoE isolated according to Rall et al27 was used as the standard.
Carotid Artery Injury
Mechanically induced endothelial denudation was performed by modification of the method originally described by Lindner et al.28 In this modification,29 an epon resin probe made by forming an epon bead slightly larger than the diameter of the carotid artery (0.45 mm) on a 3-0 nylon suture instead of a guide wire was used for the arterial injury. The animals were anesthetized by intraperitoneal injection with a solution composed of ketamine (80 mg/kg body weight; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and xylazine (16 mg/kg; The Butler Co., Columbus, OH) diluted in 0.9% NaCl. The mice were immobilized and the fur covering the neck from sternum to chin were removed with lotion hair remover (Nair; Carter-Wallace, Inc., New York, NY). Surgery was performed using a dissection microscope (Leica GZ6; Leica, Buffalo, NY). The entire length of left carotid artery was exposed and the artery was ligated immediately proximal from the point of bifurcation with a 7-0 silk suture (Ethicon, Inc., Somerville, NJ). Another 7-0 suture was placed around the common carotid artery immediately distal from the branch point of the external carotid. A transverse arteriotomy was made between the 7-0 sutures and the resin probe was inserted and advanced toward the aorta arch and withdrawn five times. The probe was removed and the proximal 7-0 suture was ligated. Once restoration of blood flow through the carotid branch points was confirmed, the incision was closed with a 5-0 sterile surgical gut (Ethicon, Inc.). All these procedures were performed within 20 minutes. Animals were allowed to recover in a 37°C heat box. The identical surgical procedure was applied to each animal to assure reproducibility of the results.
Tissue Preparation and Histological Staining
Fourteen days after inducing arterial injury, animals were anesthetized and perfused with 0.9% NaCl by placement of a 22-gauge butterfly angiocatheter in the left ventricle. The mice were subsequently perfusion-fixed in situ by infusion with 10% buffered formalin (pH 7.0) for 20 minutes at a constant pressure of 100 mmHg. The entire neck was dissected from each mouse and fixed in 10% buffered formalin for an additional 48 hours. The whole neck was decalcified for 48 hours before embedding in paraffin. Identical whole-neck cross-sections of 5 µm were made from the distal side of the neck beginning at the point of the distally ligated 7-0 suture. The whole-neck sections were used to evaluate both the injured and the uninjured control vessels on the same section. For each mouse, four levels of serial sections at 500-µm intervals were made, and the data collected were averaged which allowed for the measurement to represent lesion formation along the entire length of the artery. Parallel sections were subjected to routine hematoxylin and eosin (H&E) staining as well as to Verhoeff Van-Gieson staining of elastic lamina. Four unstained sections from each level were used for immunohistochemistry.
Morphometry
Morphometric analyses were performed on elastin-stained tissue. For each animal, four whole-neck cross-sections with both injured left and uninjured-control right carotid arteries were measured. Images were digitized and captured using a Sony video camera (Sony, New York, NY) connected to a personal computer. Measurements were performed at a magnification of x200 using a Scion Image analysis computer program (Scion, Frederick, MD). For each artery, luminal area, area inside the internal elastic lamina, and the area encircled by external elastic lamina were measured. Medial area was calculated as area encircled by external elastic lamina-area inside the internal elastic lamina and intimal area was calculated as area inside the internal elastic lamina-luminal area. To calculate the medial thickness for each vessel cross-section, the linear distance between internal elastic lamina and external elastic lamina was measured independently in four places, each at 90° apart and averaged. From these measurements, the ratio of intimal area and medial area, and the percent of luminal stenosis (100 x intimal area/area inside the internal elastic lamina) were calculated.
Immunohistochemistry
For all staining, sections were deparaffinized with xylene by incubating for 10 minutes three times and then dehydrated with a series of graded ethanol from 70 to 100% for 10 minutes each. Slides were then washed in distilled water for 5 minutes, and endogenous peroxidase activities were blocked by incubating for 30 minutes with 0.5% hydrogen peroxide in PBS containing Triton X-100 (Sigma Chemical Co.). Slides were then washed three times in the same solution without H2O2 for 15 minutes each. Nonspecific binding sites were blocked by incubation for 30 minutes with 1.5% serum in PBS containing Triton X-100.
For the identification of smooth muscle cells and endothelial cells,
sections were incubated overnight at 4°C with anti-smooth muscle
-actin (Clone 1A4; Sigma Chemical Co.) at 1:3,000 dilution or
anti-Von Willebrand Factor (DAKO, Carpinteria, CA) at 1:100 dilution,
respectively. The slides were washed three times for 15 minutes each
with PBS containing Triton X-100 and then incubated for 1 hour at
23°C with 0.5% biotinylated anti-mouse IgG (Vector Laboratories,
Inc., Burlingame, CA) for anti-smooth muscle cell -actin or 0.5%
biotinylated anti-rabbit IgG (Vector Laboratories, Inc.) for Von
Willebrand Factor in the same solution containing 1.5% normal serum.
Slides were then washed as described above, and then incubated with the
avidin-peroxidase complex reagent (Peroxidase Vectastain Elite ABC kit,
Vector Laboratories, Inc.) for 1 hour at 23°C. The reaction was
visualized with 3,3'-diaminobenzidine.
Identification of proliferating cells in the S phase of the growth cycle was accomplished by injecting the mice with three doses of 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg; Sigma Chemical Co.) intraperitoneally at 24, 8, and 1 hour before their sacrifice, followed by immunohistochemical analysis with mouse monoclonal anti-BrdU (Clone BU33, diluted 1:300; Sigma Chemical Co.). Cells in the G1 growth phase were identified by immunohistochemical staining with the mouse monoclonal anti-cyclin D1 antibody, DCS-6 (Oncogene Research Products, Boston, MA) at a dilution of 1:20. The sections were pretreated by incubation with 4 mol/L HCl for 30 minutes at 37°C and neutralized in 0.2 mol/L borate buffer, pH 9.0. After a 15-minute washing with PBS containing Triton X-100, the sections were further incubated with 0.1% trypsin for 30 minutes at 37°C, followed by blocking endogenous peroxidase and nonspecific binding sites as described above. The reaction was visualized using the Vectastain Elite ABC kit as described above.
Statistical Analysis
All values were expressed as mean ± SEM. When only two groups (injured arteries and contralateral control arteries) were compared, differences were assessed by a paired Student’s t-test. Multiple comparisons were first tested by analysis of variance. When the analysis of variance demonstrated significant differences, individual mean differences were analyzed by the Student-Newman-Keuls test. Statistical software SigmaStat (Version 2.0, Jandel Co., San Rafael, CA) was used in statistical analysis. For all statistical analyses, P < 0.05 was considered significant.
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. Importantly, endothelial denudation
by resin probe did not result in damage of the elastic lamina (Figures 2 and 3)
.
Thus, this procedure is ideally suitable to evaluate neointimal
hyperplasia as a consequence of endothelial denudation with minimal
trauma to the underlying medial smooth muscle cells. Animals with
damaged elastic lamina after mechanically induced injury were excluded
from subsequent characterization.
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. Representative histology data
comparing injured and uninjured carotid arteries from a control and two
apoE-null mice are shown in Figure 3
. Although little or no cells could
be observed in the intima, within the innermost internal elastic
lamina, of either apoE(+/+) or apoE(-/-) mice before injury (Figures 2 and 3)
, a significant number of cells was found to be accumulated in
the intima 14 days after injury of the carotid arteries (Figures 2 and 3)
. Immunohistochemical analysis with smooth muscle cell-specific
-actin antibodies identified the majority of cells in injured and
uninjured media, as well as in the neointima after arterial injury, as
smooth muscle cells (Figure 4)
.
|
. Interestingly, the severity of
neointimal formation in response to arterial injury was found to be
3.4-fold greater in apoE(-/-) mice in comparison with that in the
C57BL/6 control mice (11,223.75 ± 2,715.4
µm2
versus 3,323.98 ± 2,231.65
µm2, P < 0.05) (Figure 5C)
.
Analysis of BrdU-labeled cells revealed an eightfold and fivefold
increase in the number of proliferating cells in the media and intima
of apoE(-/-) mice compared to that in
control animals (P < 0.05) (Figure 6a
and
Table 1
). The greater neointimal
formation in apoE(-/-) mice also resulted in the significant increase
of intima/media ratio in apoE-null mice in comparison with that
observed in the C57BL/6 control mice (0.11 ± 0.073
versus 0.359 ± 0.091, P < 0.05)
(Figure 5D)
. In addition, luminal stenosis was found to be more severe
in the apoE(-/-) mice after arterial injury (32.8 ± 8.01%
versus 7.0 ± 3.58% in control mice, P
< 0.05) (Figure 5E)
.
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. Representative histology
data after arterial injury of FVB/N control mice and apoE transgenic
mice are shown in Figure 7
.
Interestingly, the FVB/N mice were found to have a more pronounced
arterial response to injury, with more severe neointimal formation,
than those observed with the C57BL/6 control mice (compare Figures 3 and 7
). However, neointimal formation in response to arterial injury
was significantly reduced in the apoE-transgenic mice (Figures 7 and 8)
. Note that the thickening is because
of cellular accumulation in the neointima and not thrombus formation as
shown by H&E staining (Figures 3 and 7)
. Corresponding morphometric
analysis of the complete set of data showed that medial thickness and
medial area were significantly increased in both wild-type FVB/N and
apoE transgenic mice after arterial injury. No significant difference
was observed in these parameters between the wild-type and apoE
transgenic mice (Figure 8, A and B)
. However, neointimal area as well
as the intima/media ratio in apoE transgenic mice were significantly
lower than their wild-type counterparts after arterial injury
(P < 0.05) (Figure 8, C and D)
. Importantly, in
the FVB/N mice, luminal stenosis of the injured carotid artery was
found to be 16.5 ± 6.75%, whereas stenosis of the injured artery
in the apoE transgenic mice was 4.3 ± 1.69%
(P < 0.05) (Figure 8E)
. The decrease in
neointimal area and intima/media ratio observed in the apoE transgenic
mice coincided with a threefold to fivefold decrease in the number of
BrdU-labeled cells in these animals in comparison with that observed in
the wild-type FVB/N mice (P < 0.05) (Figure 6
and Table 1
).
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. The
anti-cyclin D1-positive cells were clearly more abundant in the injured
arteries of apoE(-/-) mice compared to that observed in their control
C57BL/6 counterparts (Figure 9, b and d)
. In contrast, anti-cyclin
D1-positive cells were more abundant in the injured arteries of FVB/N
mice than the apoE transgenic mice (Figure 9, f and h)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The animals used in the current study were maintained on a low-fat diet
without cholesterol supplementation. Although the plasma cholesterol
levels in the apoE knockout mice were significantly higher than that
observed in the C57BL/6 control mice, the plasma cholesterol levels of
the human apoE3 transgenic mice were not significantly different from
that in their control FVB/N counterparts (Table 2)
. Thus, the effect of
apoE on vascular homeostasis cannot be fully explained by its effect on
plasma cholesterol level. Although the plasma cholesterol level may be
one factor that modulates arterial response to injury, the protective
effect of the apoE transgene against neointimal hyperplasia suggests
that the level of apoE in the circulation may also be an important
determinant in vascular response to injury. Although the apoE transgene
does not alter plasma cholesterol level, it may have lipid-related
effects in the vessel wall. These may include its effect of cellular
cholesterol efflux and/or in routing of lipoproteins through
receptor-mediated clearance mechanisms. However, the data are also
consistent with the hypothesis that apoE may be protective in the
vasculature via a process that is independent of its lipid transport
properties.
A lipid-lowering independent role of apoE in protection against vascular occlusive diseases has been proposed previously based on several indirect observations. These include the ability of intravenously injected apoE to inhibit atheroma formation in Watanabe heritable hyperlipidemic rabbits without affecting their plasma cholesterol concentrations,34 and the ability of arterial wall-derived apoE to inhibit diet-induced atherosclerosis without affecting plasma cholesterol levels in transgenic mice.35 The direct effect of apoE in limiting neointimal hyperplasia is also consistent with previous in vitro observations which showed that apoE has additional functions independent of cholesterol transport. For example, apoE has been shown to be an anti-oxidant capable of protecting cells against oxidative insults.36 Because reactive oxygen species such as hydrogen peroxide seemed to be required for the activation of quiescent smooth muscle cells,37 apoE may limit neointimal hyperplasia by reducing the level of reactive oxygen species in the arterial wall. More recently, we showed that apoE also has direct cell regulatory functions, including the ability to inhibit smooth-muscle cell migration and proliferation in response to oxidized low-density lipoprotein and platelet-derived growth factor.24 Medial and intimal staining for BrdU-labeled cells suggests that apoE can reduce cellular proliferation in vivo, whereas the absence of apoE allows for increased proliferation and subsequent intimal thickening. Similar to the in vitro effects of apoE on smooth muscle cell functions, the cytostatic functions of apoE in vivo also seemed to be mediated via its inhibition of cyclin D1 gene expression in response to mechanically induced injury of the vasculature.
The results of the current study emphasized a direct cytostatic effect of apoE on the vessel wall and underscored the potential for using apoE gene therapy as treatment for vascular diseases. Kashyap et al38 have already shown that intravenous infusion of recombinant adenovirus containing the human apoE gene can effectively reduce plasma cholesterol level and suppress atherosclerosis in apoE-deficient mice. Hasty et al39 showed that transduction of bone marrow with apoE-expressing retrovirus was also effective in reducing early foam-cell lesion formation in apoE-deficient mice. Although the latter study showed that arterial macrophage expression of apoE had no beneficial effects during the later stages of atherosclerosis, the results of the current study suggest the potential benefit of apoE gene therapy in combination with vascular surgery for the treatment of vascular occlusive diseases. The ability of apoE to inhibit neointimal formation and to decrease luminal stenosis highlighted the potential of apoE therapy as a viable option in lowering the risk of restenosis after vascular surgery.
Although the current study is focused on comparing the effect of apoE deficiency or its enrichment on vascular response to injury, it is noteworthy that the two groups of control mice, namely the C57BL/6 and the FVB/N mice, displayed significant difference in the severity of neointimal formation in response to arterial injury. This observation is consistent with the recent report of strain differences in neointimal hyperplasia in rats.40 These two studies indicated a possible genetic influence on the development of neointima after vascular injury. Interestingly, our data showed that neointimal hyperplasia was more severe in FVB/N mice than in C57BL/6 mice. Thus, the difference in severity of neointimal hyperplasia in response to arterial injury between the two inbred strains of mice diverse from their documented susceptibility to diet-induced atherosclerosis, in which C57BL/6 mice were consistently found to be the most susceptible strain.41 This observation, along with observation of apoE protection against both atherosclerosis and vascular response to injury, suggests that pathogenesis of these vascular diseases may be controlled by distinct and overlapping genetic factors. With the identification of inbred mouse strains differing in their vascular response to injury, we can now take advantage of advances made in the development of high-resolution genetic linkage maps in the mouse model42 to identify gene(s) that are important in determining the risk and severity of neointimal formation in response to arterial injury. Such information will be useful to identify patients at risk for restenosis after vascular surgery.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant HL-61332 (to D. Y. H.) and an American Heart Association grant-in-aid SW-96-43-S (to D. P. W.).
Accepted for publication August 21, 2000.
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