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Animal Models |
From the Institute for General and Experimental
Pathology,*
University of Innsbruck Medical School,
Innsbruck; the Institute for Biomedical Aging
Research,
Austrian Academy of Sciences,
Innsbruck; and the Division of Cardiology,
Department of Internal Medicine, University Hospital of Innsbruck,
Innsbruck, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypercholesterolemia has been shown to be a significant risk factor for the development of vein graft atheroma.4 Evidence indicates that rates of obstructive atheroma in grafted veins were highly correlated to preoperative serum cholesterol levels,5 and that the lesion development was predicted by higher levels of plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL).6 Accordingly, patients with familial hypercholesterolemia exhibit a high incidence of late graft occlusion after bypass surgery.7 However, the molecular mechanism of vein graft atherosclerosis by which hypercholesterolemia initiates, promotes, or perpetuates the lesion remains largely unknown.
Several animal models manifesting lesions resembling neointimal hyperplasia of human vein grafts have been developed8-12 and have helped address specific interventional issues, but no spontaneous atheromatous lesions in their vein grafts have been observed. The apolipoprotein E-deficient (apoE-/-) mouse generated by gene targeting13 provides an appropriate animal model for such a study. In apoE-/- mice, blood cholesterol is markedly elevated because of increased levels of VLDL and LDL.13
In our previous study, we established a new model for the study of neointima formation of venous bypass grafts in mice, and demonstrated the presence of abundant MAC-1-positive monocytes/macrophages and smooth muscle cells (SMCs) in the early stages of lesions in vein grafts.14 In the present study, we developed and characterized an animal model of vein graft atheroma in apoE knockout mice, and demonstrated that hypercholesterolemia promotes the formation of typical atherosclerotic plaques, ie, atheroma in vein bypass grafts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animal experiments were performed according to protocols approved by the institutional committee for use and care of laboratory animals. ApoE-deficient mice of the C57BL/6J strain13 were purchased from The Jackson Laboratory (Bar Harbor, ME). Three genotypes of apoE-/-, apoE+/-, and apoE+/+ mice were identified using The Jackson Laboratory’s polymerase chain reaction (PCR) protocol (primers: oIMR180 5'-GCC TAG CCG AGG GAG AGC CG-3', oIMR181 5'-TGT GAC TTG GGA GCT CTG CAG C-3' and oIMR182 5'-GCC GCC CCG ACT GCA TCT-3'). The mice were maintained on a 12 hours light/12 hours dark cycle at 22°C, receiving food and water ad libitum.
The procedure used for vein grafts was similar to that described previously.14 Briefly, 3-month-old mice were anesthetized with pentobarbital sodium (50 mg/kg body weight, i.p.). The vena cava vein was harvested and washed with saline solution containing 100 U/ml of heparin. The right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal, cut in the middle, and a cuff was placed at the end. The cuff was made of an autoclavable nylon tubing 0.63 mm in diameter outside and 0.5 mm inside (cat. no. 800/200/100/200, Portex Ltd., Hythe-Kent, UK). The artery was turned inside out over the cuff and ligated. The vein segment was grafted between the two ends of the carotid artery by sleeving the ends of the vein over the artery-cuff and ligating them together with the 8–0 suture. Vigorous pulsations in the grafted vessel confirmed successful engraftment.
Tissue Preparation
Vein grafts were harvested at 2, 4, and 8 weeks postoperatively (4 to 8 mice at each time point/group) by cutting the implanted segments from the native vessels at the cuff end. For histological analysis, perfusion with 4% phosphate-buffered formaldehyde was performed, as described previously.14 For frozen section preparation, vein grafts were harvested without perfusion and immediately frozen in liquid nitrogen.
Histology and Lesion Quantification
The grafts were dehydrated in graded ethanol baths, cleared in xylol, embedded in paraffin, and sectioned. The thickness of the vessel wall was determined by measuring 4 regions of a section along a cross, and recorded in micrometers (means ± SEM). The procedure for lesional area measurement is similar to that described elsewhere.15 Briefly, the lesion was defined as the region between the lumen and the adventitia. Using a transmission scanning microscope (LSM 510, Zeiss, Jena, Germany) vessels were scanned, saved, and then overlaid by different linings to trace the lumen and adventitia. The lesion area was determined by subtracting the area of the lumen from the area enclosed by the line inside of adventitia. Six to eight cross-sections were obtained by selecting the first of every three sections from each graft. Areas were measured and recorded in square micrometers.
Immunohistochemical Staining
The procedure used in the present study was similar to that
described previously.14,15
Briefly, serial 5-µm-thick
frozen sections were overlaid with rat monoclonal antibodies against
mouse MAC-1 leukocytes (CD11b/18), CD4, or CD8 (PharMingen, San Diego,
CA). Sections were visualized with alkaline phosphatase-anti-alkaline
phosphatase complex (Dakopatts, Copenhagen, Denmark) and
developed using a substrate solution containing 9.8 ml Tris buffer (0.1
mol/L, pH 8.2), 0.2 ml dimethylformamide, 8 mg naphthol AS-MX
phosphate, 3 mg levamisole, and 10 mg Fast Red TR salt (Sigma). For SMC
staining, a mouse monoclonal antibody against 
-actin (Sigma) labeled
with phosphatase was used. Semiquantitive evaluation was performed at
10 x 40 magnification. Positive stained cells in the intima
and media were counted on two regions of each section and expressed as
the range of the cell number or the percentage of total nuclei per 100
micrometers of the vessel wall.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay
Accumulated internucleosomal DNA fragments (apoptosis) were detected using an in situ apoptosis detection kit (Boehringer Mannheim, Mannheim, Germany) as described previously.16 Sections were developed with Fast Red substrate and counterstained with hematoxylin. Percentages of positive-stained cells were determined by counting the numbers of labeled and total cells using a light microscope. Positive cells of two regions of each section of 4- and 8-week grafts were counted. Morphological features of apoptosis were assessed on light microscope.
Immunofluorescence Double Staining
For double staining, sections were incubated with the rat
monoclonal antibody against MAC-1 and visualized with swine anti-rat Ig
conjugated with fluorescein isothiocyanate (FITC, Dakopatts). Sections
were rinsed and stained with rabbit anti-proliferating cell nuclear
antigen (PCNA) antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
developed with swine anti-rabbit Ig-TRITC (Dakopatts). For SMC and PCNA
staining, a mouse monoclonal antibody against -actin (Sigma) labeled
with FITC was used. For visualization of nuclei, sections were
counterstained with the DNA stain Hoechst 33258.
LDL Isolation and Oxidation
EDTA plasma was pooled from normolipemic, fasting (12–14 hours) humans, aged 20 to 30 years. Lipoproteins were prepared by differential centrifugation using solid KBr to adjust the density, as described previously.17,18 LDL were obtained in fractions between 1.020 and 1.050 g/ml. Concentrations of LDL were determined gravimetrically by aliquot weight after drying, and quantities of lipoproteins were expressed as total weights.17,18 LDL oxidation was performed by incubation of LDL (1 mg/ml phosphate-buffered saline) with 10 µmol/L CuCl2 at 37°C for 18 hours.19 The extent of oxidation was assessed by measurement of thiobarbituric acid reactive substances (9.8 ± 1.3 nmol/mg).20
SMC Culture
Vascular SMCs from apoE-/- and apoE+/+ mice were cultivated from
their aortas, as described elsewhere.21,22
Cells were
incubated at 37°C for 7 to 10 days and passaged by treatment with
0.05% trypsin/0.02% EDTA solution. The purity of SMCs was routinely
confirmed by immunostaining with antibodies against -actin.
Experiments were conducted on SMCs that had just achieved confluence.
Cell Proliferation and Viability Assays
For proliferation assays, SMCs cultured in 96-well plates in medium containing 10% fetal calf serum at 37°C for 24 hours were serum-starved for 2 days. Platelet-derived growth factor-AB, angiotensin II, LDL, oxidized LDL, and fetal calf serum were added, respectively, and incubated at 37°C for 24 hours. For cell viability assay, SMCs were plated at a density of 2 x 103 cells/well (96-well plate) in the medium containing 10% fetal calf serum and incubated at 37°C for 48 hours. Oxidized LDL, LDL, H2O2, and sodium nitroprusside (SNP) were added to the culture, respectively, and incubated at 37°C for 24 hours. A solution from a proliferation/apoptosis kit (Promega) was added 4 hours before measurement. The optical density at 490 nm was recorded with a photometer.
Annexin V/Propidium Iodide Double Staining and Fluorescence-Activated Cell Sorter (FACS) Analysis
Annexin staining was performed according to the manufacturer’s instructions (PharMingen) and as described previously.16 The cellular fluorescence signal was recorded on the FL1 and FL2 channel of a FACS scan flow cytometer (Program Cell Quest, Becton Dickinson, Mountain View, CA) and expressed on a logarithmic scale. After appropriate markings for negative and positive populations, the percentage of annexin V+/PI- or V+/PI + cells was determined and compared with unstressed controls.
Statistical Analysis
Statistical analyses were performed using the Mann-Whitney U test and analysis of variance, respectively. Results are given as mean ± SEM or ± SD. A P value < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The venous wall of apoE-/- and wild-type mice is composed of
intima, a monolayer of endothelium, media, 1- or 2-layered SMCs, and
adventitia, a small amount of connective tissue. Vein grafts of
wild-type mice at 8 weeks (Figure 1a)
showed neointimal hyperplasia, ie, thickening of the vessel wall up to
10 or 20 layers of cells. Interestingly, intimal lesions of vein grafts
in apoE-/- mice showed a marked increase at 8 weeks (Figure 1b)
. The
lumen of vein grafts reduced up to 70% compared to that of wild-types.
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summarizes statistical data of
lesion thickness and areas measured with a laser microscope in
transmission scan mode. A significant difference between groups of
apoE-/- and wild-type mice was found at 4 and 8 weeks
(P < 0.05 and P < 0.0001,
respectively). Likewise, two- to threefold increases in lesion areas of
vein grafts in apoE-/- mice were observed (Figure 2
, low panel).
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, but no fully maturated atheroma
could be found even at 16 weeks.14
In apoE-/- mice,
typical atheroma in 8-week vein grafts displayed the classical complex
morphological features and heterogeneous cellular compositions.
Atheromatous lesions consisted of the fibrous cap (Figure 3, b and c)
,
infiltrated mononuclear cells (Figure 3b)
, foam cells (Figure 3c)
,
cholesterol crystal structure (Figure 3d)
, necrotic core with
calcification (Figure 3e)
, and neovasculature (Figure 3f)
. In addition,
the cell density in apoE-null vein graft atheroma was much lower than
that of apoE+/+ neointimal lesions. These complicated lesions closely
parallel those characteristic of human vein graft
atherosclerosis.
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We have provided evidence that lesions of vein graft-induced
neointima from wild-type mice contain mainly macrophages and
SMCs.14
Figure 4
and Table 1
demonstrate the presence of abundant
SMCs in venous bypass graft lesions 4 and 8 weeks after surgery.
Initially, a monolayer of SMCs in normal vein walls stained strongly
positive (data not shown), and no staining was seen in vein segments
stained with normal rat serum as a negative control (Figure 4a)
.
Importantly, -actin-positive SMCs distributed in the most area of
neointima of 4- and 8-week vein grafts from wild-type mice (Table 1)
,
whereas a majority of these positive cells localized in the cap region
of vein graft atheroma in apoE-/- mice (Figure 4b
and Table 1
). A
significant difference in SMC numbers in 8-week graft lesions between
apoE-/- and apoE+/+ was found (33 vs. 60%; Table 1
).
Abundant infiltration of MAC-1-positive cells was found in intima
and/or adventitia of 4-week vein grafts of both types of mice (Table 1)
. MAC-1-positive cells remained predominant in atheroma of 8-week
grafts (Figure 4c)
, whereas they were decreased in neointima of
wild-type mice 8 weeks after the operation (Table 1)
. Most foam cells
displayed MAC-1 positivity, indicating a mononuclear cell origin.
MAC-1-positive cells could be found around the necrotic core and the
most area under the fibrous cap. Thus, mononuclear cells constituted a
large portion of atheroma in vein grafts of apoE-deficient mice.
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, Figure 4d
). Interestingly, the distribution
of CD4+ lymphocytes was patchy, with small foci of cells generally
located beneath the fibrous cap of atheroma (Figure 4d)
. Few CD8+ T
cells were found in neointima or atheromatous lesions. Increased Cell Death in Vein Graft Atheroma
Because of rapid development of atheromatous lesions, it would be
interesting to determine cell turnover in vein grafts. PCNA is widely
used as a cell replicating marker, and was also used for labeling of
vein grafts from both groups. Data summarized in Figure 5
show a similarity in the percentage of
PCNA+ cells in neointima and atheromatous lesions of apoE+/+ and
apoE-/- mice. Double-labeling studies revealed that most PCNA+ cells
were -actin-positive (82 ± 15%), whereas MAC-1 and PCNA
double-positive cells were rare (<5%). These results indicate that
SMC replications in situ in the intima of vein grafts occur,
and that there is no significant difference between apoE-/- and
apoE+/+ mice.
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shows
typical examples of TUNEL-positive cells in both neointima and
atherosclerotic lesions of vein grafts. Although TUNEL-positive cells
were found throughout the lesions, they were more abundant around the
necrotic core (Figure 6e)
. Quantitative analysis revealed that
apoptotic/necrotic cells in atheroma of apoE-/- mice were
significantly higher than those in neointima from the wild-types
(Figure 7
; 11 vs. 5.6%;
P < 0.05). To study the mechanism of higher rate of
cell death in atheroma, vein isografts between apoE-/- and apoE+/+
mice were performed. Figure 8
shows data
indicating that hyperlipidemia is largely responsible for increased
numbers of cell death as well as atheroma formation. However, apoE-/-
veins grafted into apoE+/+ mice showed that numbers of
apoptotic/necrotic cells were not significantly different from veins
grafted among apoE+/+ mice (Figure 8b)
.
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Because of increased cell death in apoE-/- grafts, we compared
the proliferative ability and viability of cultivated arterial SMCs
from apoE-/- and apoE+/+ mice. In response to serum, platelet-derived
growth factor-AB, angiotensin II, LDL, and oxidized LDL, SMCs from both
types of mice replicated markedly, but no significant differences in
cell numbers were observed (Figure 9a)
.
Interestingly, apoE-deficient SMCs revealed a significant decrease in
cell viability when stimulated by a NO donor sodium nitroprusside
(SNP), free radical H2O2, and
oxidized LDL (Figure 9b)
. Low concentrations (up to 50 µg/ml) of
oxidized LDL-induced SMC replication were observed, whereas higher
concentrations (>100 µg/ml) resulted in cell death (Figure 9, a and b)
, similar to the findings obtained in SMCs from other species. To
further determine whether oxidized LDL stimulates SMC apoptosis or
necrosis, FACS analysis of double-stained SMCs was performed. The data
shown in Figure 8, c
-f, indicate that the main population of dying SMCs
underwent apoptosis. ApoE-deficient SMCs had a higher rate of both
spontaneous and oxidized LDL-stimulated apoptosis/death (Figure 9, c
-f).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1
). Second,
both the human and mouse atheromas are characterized by concentric
intimal proliferation, with a dominance of SMCs in the cap
region27
(Figure 4
and Table 1
). Third, lipid deposition,
including foam cells and cholesterol crystal, can be found in
atherosclerotic lesions of both species. Finally, apoE-knockout mice
develop complicated atheromatous lesions, including necrotic core,
calcification, and lumen occlusion that also occur in vein bypass
grafts in humans. Thus, our model could be useful in the study of human
venous bypass graft atherosclerosis. This model offers the potential
for significant progress in the near future in understanding the
pathogenesis and treatment of the vein graft disease. The rabbit is the first and most frequently used animal at all points of atherosclerosis research, including vein bypass grafts.28,29 Atheromatous lesions analogous to those in humans have been induced in vein grafts of rabbits by administration of a cholesterol-enriched diet.30 However, our animal model of vein graft atheroma in apoE-deficient mice has several advantages. First, the number of animals used for the graft experiments can be reduced, since vein graft lesions show very low viability among apoE-/- inbred mice. Second, there is a large disparity in blood cholesterol levels of rabbits fed with a cholesterol-enriched diet, whereas blood lipid concentrations are similar in apoE-deficient mice of the same age. Third, when vein isografts between apoE-/- and other knockout mice are performed, the role of the specific gene or protein can be clarified. For example, if vein segments from ICAM-1-deficient C57BL/6J mice are grafted to carotid arteries of apoE-/- C57BL/6J mice, the role of ICAM-1 in vein grafts can be identified.21
What are the initial factors stimulating the development of vein graft lesions? We hypothesize that biomechanical stress is an initiator for altered gene expression in the early stage of vein grafts, followed by vascular cell apoptosis/death, inflammation, SMC proliferation, and lipid deposition.16,21,31,32 Support for this hypothesis includes the fact that neointima hyperplasia in vein segments grafted to veins does not occur in mice14 and that atherosclerotic lesions in arteries grafted to arteries are very rare in apoE-null mice 8 weeks after surgery (data not shown). In grafted veins, mechanical force on the vessel segment suddenly increases more than 10-fold (arterial versus venous blood pressure), which provides a strong stimulus to vascular endothelial and SMCs.32 We previously demonstrated that acutely elevated blood pressure and mechanical stress activate growth factor receptor-MAPK signal pathways,33-36 which are critical in mediating mechanical stress-induced cell apoptosis and proliferation. Hyperlipidemia may be an amplifying factor that exerts its effects on vein graft atherosclerosis in the presence of elevated biomechanical stress.
One of the most important observations made in the current study is
that cell death in vein graft atheroma of apoE-deficient mice was
markedly increased and that increased cell death in apoE+/+ veins
grafted into apoE-/- mice could also be observed. SMCs from apoE-/-
mice had a higher rate of spontaneous apoptosis/death and were
sensitive to a variety of stimuli leading to cell death. A possible
explanation for this phenomenon is that high blood cholesterol levels
may lead to SMC differentiation to a phenotype sensitive to stress
stimuli. Such alterations in SMCs can be maintained for a period of
time, because the rate of cell death in apoE-/- veins grafted into
apoE+/+ mice and cultured apoE-/- SMCs is still higher (Figures 8 and 9)
. Concomitantly, Bennett et al37,38
provided evidence of
increased sensitivity of human vascular SMCs from atherosclerotic
plaques. These findings indicate that sustained exposure of SMCs to a
hypercholesterolemic environment alters the cell phenotype and promotes
cell differentiation. For example, they found elevated p53 protein
levels in SMCs from atherosclerotic lesions, which may be responsible
for increased sensitivity to apoptosis.37,38
Importantly,
increased cell death coincides with rapid development of atheroma in
apoE-null mice. Although apoptosis is believed to evoke inflammation
responses rarely in other tissues,39,40
apoptotic cells
release the S19 ribosomal protein dimer,41
a chemokine for
macrophages/monocytes, by molecular mimicry to complement
C5a.42
Necrotic cells in atheroma may release oxidized
lipids, resulting in continuous recruitment of blood monocytes, ie,
inflammatory responses. We found that the number of
monocytes/macrophages in 8-week atheroma is significantly higher than
in neointima (Table 1)
, suggesting that cell recruitment from blood is
a key event in maintaining the enlargement of atheromatous lesions.
In summary, we have developed and characterized a new animal model of vein graft atheroma in apoE-deficient mice. Atheromatous lesions bear many similarities to vein graft disease in humans. The mechanism of the pathogenesis of vein graft atheroma involves increased cell death and continuous recruitment of blood cell, by which biomechanical stress may serve as an initiator, and hypercholesterolemia synergistically enhances or amplifies the progress of lesions. This mouse model has several advantages over other animal models and, therefore, is highly recommended for use in the study of the pathogenesis and therapeutic interventions in vein graft disease.
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Acknowledgements |
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Footnotes |
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Supported by grants P12847-MED from the Austrian Science Fund, and P7917 from the Jubiläumsfonds of the Austrian National Bank. Dr. Hu is a recipient of APART Award from the Austrian Academy of Sciences.
Accepted for publication May 19, 2000.
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References |
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in vascular smooth muscle cells by mechanical stress. FASEB J 1998, 12:1135-1142This article has been cited by other articles:
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-interferon (100
ng/ml). Solution of proliferation/apoptosis kit
was added 4 hours before measurement. The optical density at 490 nm was
recorded with a photometry. Data are means ± SD of three
experiments. *Significant difference between apoE-/- and apoE+/+
SMCs. c–f: FACS analysis of annexin V/propidium iodide
double-stained SMCs. SMCs were treated with oxidized LDL
(d and
f) for 24 hours and labeled with
annexin V and propidium iodide. Cellular fluorescence signal was
recorded on FL1 and FL2 channels of a FACS scan flow cytometer and
expressed on a logarithmic scale. Note the higher number of spontaneous
cell death (c and
e) and oxidized LDL-induced apoptosis
(d and
f) in apoE-/- SMCs.