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(Circulation Research. 2001;88:780.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Macrophage p53 Deficiency Leads to Enhanced Atherosclerosis in APOE*3-Leiden Transgenic Mice

Bart J.M. van Vlijmen, Gery Gerritsen, Arthur L. Franken, Lianne S.M. Boesten, Mark M. Kockx, Marion J. Gijbels, Michel P. Vierboom, Miranda van Eck, Bob van de Water, Theo J.C. van Berkel, Louis M. Havekes

From the Departments of Biopharmaceutics (B.J.M.v.V., G.G., L.S.M.B., M.v.E., T.J.C.v.B.) and Toxicology (A.L.F., B.v.d.W.), Leiden/Amsterdam Center for Drug Research, Leiden/Amsterdam Center for Drug Research, Leiden, the Netherlands; TNO Prevention and Health (B.J.M.v.V., G.G., A.L.F., L.S.M.B., L.M.H.), Gaubius Laboratory, Leiden, the Netherlands; Department of Pathology AZ-Middelheim (M.M.K.), University of Antwerp, Antwerp, Belgium; and Departments of Human Genetics (M.J.G.), Immunohematology and Blood Bank (M.P.V.), and Cardiology and Internal Medicine (L.M.H.), Leiden University Medical Center, Leiden, the Netherlands.

Correspondence to Bart J.M. van Vlijmen, TNO Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, the Netherlands. E-mail bjm.vanvlijmen{at}pg.tno.nl

	Abstract

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Abstract—Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (p53^-/-) or control (p53^+/+) donor mice. Reconstitution of mice with p53^-/- bone marrow did not result in any hemopoietic abnormalities as compared with p53^+/+ transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with p53^-/- bone marrow showed a significant (P=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with p53^+/+ bone marrow. Although likely a secondary effect of the increased lesion area, p53^-/- transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1±1.3 versus 0.2±0.7; P=0.04) and lesion macrophages (macrophage area, 79.9±40.0 versus 39.7±27.3x10³ µm² per section; P=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]–positive nuclei going from 0.42±0.39 to 0.14±0.15%, P=0.071), whereas the number of proliferating cells (5'-bromo-2'-deoxyuridine–positive nuclei) was not affected (3.75±0.98 versus 4.77±2.30%; P=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.

Key Words: p53 • macrophages • atherosclerosis • transgenic • apoptosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cellular composition of an atherosclerotic plaque is an important determinant of plaque stability. For instance, lesions with a prominent presence of fibroblasts and smooth muscle cells and calcification are relatively stable and resistant to rupture. In contrast, plaques rich in cholesterol-loaded macrophages and extracellular lipid deposits are relatively soft and are considered to be highly unstable and vulnerable to rupture.¹ Therefore, modulation of the (cellular) composition of the latter plaques is important to reduce the risk of acute coronary syndromes. Cell infiltration, cell proliferation, and cell death (either necrosis or apoptosis) are key processes in determining the cellular composition of atherosclerotic tissues.^{2 3} Proliferation of smooth muscle cells and endothelial cells and infiltration of macrophages contribute to lesion development.³ Cell death (including apoptosis) has been postulated to play an important role in the development and homeostasis of complicated lesions.^{4 5 6 7} The tumor suppressor protein p53 is an essential molecule in both cell proliferation and apoptosis. p53 is a transcription factor that is upregulated after various conditions of cellular stress, including DNA damage, nitrogen oxide, hypoxia, and oxidative stress, as well as stress caused by oxidized lipoproteins (for review, see Ko and Prives⁸ ). p53 expression is increased in human atherosclerotic lesions, both in lipid-loaded macrophages and vascular smooth muscle cells.^{9 10} The exact role of p53 in atherosclerosis has remained, however, largely unclear.

Recent studies using mice lacking both p53 and apolipoprotein E (ApoE) (p53^-/-apoE^-/- mice) indicate that p53 is important for atherogenesis; p53^-/-apoE^-/- mice developed larger atherosclerotic lesions as compared with apoE^-/- mice.¹¹ In vitro studies showed that p53 facilitates the apoptosis of smooth muscle cells isolated from human atherosclerotic lesions^{12 13} and that p53 is involved in oxidized LDL-induced apoptosis of human macrophages.¹⁴ However, no information is available on the cell-specific role for p53 in the modulation of atherosclerotic plaques in vivo. Thus, it is unclear whether the accelerated aortic plaque formation in the absence of p53 is related to altered proliferation and/or cell death of either smooth muscle cells or lipid-loaded macrophages. In the present study, we have investigated the cell-specific role for p53 in macrophages using p53^-/- bone marrow transplantation into APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis.^{15 16}

Reconstitution of APOE*3-Leiden mice with p53^-/- bone marrow resulted in a significant increase in atherosclerotic lesion size, with increased necrosis and decreased apoptosis. These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerotic plaques.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Male mice homozygous for inactivation of the p53 gene (originally generated by Donehower et al¹⁷ ) were obtained by crossing p53 heterozygous–deficient (p53^+/-) female with p53-deficient (p53^-/-) male mice, both at the 10th backcross into the C57BL/kh background. Mice were analyzed for their p53 status by polymerase chain reaction (PCR) analysis.¹⁸ Control male C57BL/kh mice were derived from our own stocks and were originally obtained from Charles River (Sulzfeld, Germany). Transgenic mice expressing the human APOE*3-Leiden gene¹⁹ were at the 16th backcross into the C57BL/6J background and identified with an ELISA for human apoE.¹⁵ Mice were bred and housed under specific pathogen–free conditions. Bone marrow–transplanted mice were housed in sterilized filter-top cages and fed sterilized regular chow diet or a semisynthetic high-fat/high-cholesterol (HFC) diet containing 1% cholesterol, 15.0% fat, and 0.5% cholate, composed according to Nishina et al.²⁰ Diets were purchased from Hope Farms. Drinking water was supplemented with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sugar.

All animal work was approved by the regulatory authority of Leiden University and carried out in compliance with guidelines issued by the Dutch government.

Irradiation and Bone Marrow Transplantation
To induce bone marrow aplasia, male APOE*3-Leiden mice (age 8 to 10 weeks) were exposed to a single dose of 13 Gy (0.28 Gy/min, 200 kV, 4 mA) x-ray total-body irradiation using an Andrex Smart 225 x-ray machine (Andrex Radiation Products AS) with a 4-mm aluminum filter 1 day before transplantation. Bone marrow cell suspensions were isolated by flushing the femurs and tibias from male p53^-/- mice or p53^+/+ control mice with PBS. Single-cell suspensions were prepared by passing the cells through a 30-µm nylon gauze. Irradiated recipients received 1.5x10⁷ bone marrow cells by intravenous injection into the tail vein. After transplantation, the mice were placed on a regular chow diet for 4 weeks and then on the HFC diet for 12 weeks.

Blood Sampling and Plasma Cholesterol Analysis
Blood samples were collected in EDTA-coated vials by retro-orbital bleeding under halothane anesthesia. Plasma cholesterol levels were measured enzymatically using a commercially available kit (No. 236691, Boehringer Mannheim). White blood cells were isolated as described by Miller et al,²¹ lysed, and used directly for checking the absence or presence of the p53 knockout allele by PCR analysis.¹⁸

Quantification of Atherosclerotic Lesion Area
After 12 weeks on the HFC diet, the mice were euthanized. Hearts and aortas were dissected and stored overnight in ice-cold phosphate-buffered 2% paraformaldehyde (pH 7.4) and embedded in paraffin. Serial cross sections (5 µm thick) were taken throughout the entire aortic valve area (according to Paigen et al²² ). Sections were routinely stained with hematoxylin-phloxine-saffron (HPS). For quantification of atherosclerosis, for each animal, full-color images of 4 sections of the aortic valve area were acquired at 40-µm intervals with a charge-coupled device camera (HV-C10, Hitachi) that was connected to a light microscope (Microphot-FXA, Nikon) equipped with a 4x objective. The charge-coupled device camera was connected to a personal computer running Qwin image-analysis software (Leica Imaging Systems Ltd). The images were all captured under identical lighting and stored in TIFF format. Thereafter, atherosclerotic lesion areas in the vascular region were measured manually using a pen and Drawing Board III (Calcomp Digitizer Products division analyzer) that was connected to a personal computer, and total lesion areas were determined using Qwin image-analysis software. The same operator, who was blinded for the group allocation, performed all analyses.

Semiquantitative Analysis of Necrosis
For semiquantitative analysis of necrosis, lesions were classified into 4 categories, with the following scores: 0, absence of necrosis; 1, few necrotic cells (few cells show pyknosis or karyorrhexis or are completely deleted); 2, a necrotic core (area without cells covering up to 10% of the total lesion area; presence of necrotic debris); and 3, bulbs of necrosis (large area without cells covering 10% to 50% of the total lesion area; presence of necrotic debris). Necrosis was scored in the aorta valve region in 3 serial sections at 40-µm intervals and is expressed as the average score per mouse (ie, necrotic index).

Quantification of Macrophages
After deparaffinization and rehydration, sections were incubated overnight at 4°C with a rabbit antibody to mouse macrophages (AIA-312040, 1/1500, Accurate Chemical and Scientific). Subsequently, sections were incubated with goat biotinylated antibodies to rabbit IgG (Vector Laboratories) for 45 minutes at 37°C followed by incubation with avidin-biotin complex labeled with horseradish peroxidase (1/3200, Vector Laboratories). Peroxidase was viewed with diaminobenzidine (DAB; Sigma) as a chromogen. For quantification of macrophage lesion area, images of the aorta for computer analysis were captured as described above. Threshold values that discriminated between macrophage and nonmacrophage lesion areas were selected. Extravascular and other nonlesion areas also identified by the threshold setting were edited out before lesion areas were computed. Macrophage lesion area was quantified in the aorta valve region in 4 serial sections at 40-µm intervals and expressed either as total macrophage lesion area per section or as a percentage of total lesion area.

Macrophage numbers in atherosclerotic lesions (ie, number of AIA-312040–positive cells) were quantified at the level of individual lesions (30 individual lesions per group, 2 lesions per mouse) ranging in size from 0 to 75 000 µm² and is expressed as the number of macrophages per 10 000 µm².

Quantification of T Cells
After deparaffinization and rehydration, sections were incubated for 1 hour at 37°C with a rabbit antibody to human CD3 that cross-reacts with mouse CD3 (No. A452, 1/100, DAKO). Subsequently, sections were incubated with goat biotinylated antibodies to rabbit IgG (1/200, Vector Laboratories) for 30 minutes at 37°C, followed by 30 minutes of incubation with avidin-biotin complex labeled with horseradish peroxidase (1/100, BioGenex). Peroxidase was viewed with 3-aminoethyl-9-carbazole (Sigma) as a chromogen. T-cell numbers in atherosclerotic lesions (ie, CD3-positive cells) were quantified at the level of individual lesions exactly as described above for macrophage numbers.

Quantification of Macrophages and T Cells in Spleen and Blood
For quantification of monocyte/macrophage and T-cell area in spleens of bone marrow–transplanted mice, images of either AIA-312040– or anti-CD3–stained spleen sections were captured and analyzed as described above for the aorta. Spleen macrophage/T-cell area was quantified in a random section for each individual mouse and expressed as percentage of total spleen area.

For quantification of the percentage of monocytes and T cells in blood, blood smears were stained with either AIA-312040 or anti-CD3 and lightly counterstained with hematoxylin. Using a light microscope at x100 magnification for each individual mouse, 150 white blood cells were scored for either AIA-312040 or anti-CD3 positivity. Monocyte and T-cell numbers were expressed as percentage of total white blood cells.

Human ApoE Staining
Sections were incubated overnight at 4°C with a rabbit polyclonal antibody anti-human apoE (1/1000) followed by 2 hours of incubation at room temperature with peroxidase-conjugated goat anti-rabbit IgG (1/500, Nordic Immunology). Peroxidase was viewed with DAB as a chromogen.

5'-Bromo-2'-Deoxyuridine (BrdU) and Terminal Deoxynucleotidyl Transferase End-Labeling (TUNEL) Staining
To label DNA-synthesizing cells, 6 transgenic animals per group received BrdU (Sigma; 60 mg/kg IP) 1 day before they were euthanized. Serial sections of heart and aorta were stained for BrdU as described previously²³ using a monoclonal mouse anti-BrdU antibody (DAKO) and DAB as a chromogen.

Serial sections of heart and aorta were stained for apoptosis using the TUNEL technique exactly as described by Kockx et al²⁴ with 3-aminoethyl-9-carbazole as a chromogen. Only those TUNEL-positive nuclei were included that displayed morphological features of apoptosis including cell shrinkage, aggregation of chromatin into dense masses, and nuclear fragmentation.

Numbers of BrdU- or TUNEL-positive nuclei in lesions were quantified in the aorta valve region in 4 serial sections at 40-µm intervals and are expressed as a percentage of the total number of nuclei in the lesion for each individual mouse.

Statistical Analysis
Data were analyzed using nonparametric Mann-Whitney rank sum tests. P values less than 0.05 were regarded as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bone Marrow Transplantation
Male APOE*3-Leiden mice were lethally irradiated (13 Gy) and transplanted with 1.5x10⁷ bone marrow cells derived from either p53^+/+ or p53^-/- donor mice. Four weeks after bone marrow transplantation, the regular chow diet was changed to a HFC diet. After 12 weeks of the HFC diet, mice were weighed and subsequently bled and euthanized for collection of spleen, heart, and aorta. As shown in Table 1, mean body weight was not different for p53^+/+APOE*3-Leiden and p53^-/-APOE*3-Leiden mice. PCR analysis on genomic DNA from white blood cells of each mouse revealed that for all p53^-/-APOE*3-Leiden mice (and p53^-/- donors), the genotype of the bone marrow was p53^-/- (Figure 1). As shown in Table 1, the 12-week challenge with a HFC diet resulted in high plasma cholesterol levels that were not different between p53^+/+APOE*3-Leiden and p53^-/-APOE*3-Leiden mice. In addition, distribution of cholesterol among the plasma lipoprotein fractions, as determined by gel filtration chromatography using Superose 6B column (SMART system), revealed similar lipoprotein profiles for the 2 groups with accumulation of cholesterol mainly in VLDL- and IDL-sized fractions (data not shown).

View this table: [in this window] [in a new window]   Table 1. Physical Condition of APOE1 3-Leiden Mice Reconstituted With p53+/+ or p53-/- Bone Marrow

View larger version (47K): [in this window] [in a new window]   Figure 1. Detection of the 120-bp PCR product representing p53 knockout allele in isolated white blood cells. Bands of the 120-bp PCR product are shown for white blood cells of 1 representative control p53+/+ mouse (lane 1) and 1 p53-/- donor mouse (lane 2) and for white blood cells of 2 representative control p53+/+ transplanted (lanes 3 and 4) and 2 p53-/- transplanted (lanes 5 and 6) APOE*3-Leiden (L) mice at the end of the experiment.

To determine whether the absence of p53 affects the monocyte/macrophage and T-cell population, blood smears and spleens (as a representative white blood cell–rich organ) were stained either with the anti-mouse macrophage polyclonal antibody AIA-312040 or an anti-human CD3 antibody that cross-reacts with mouse CD3. As shown in Table 1, p53^+/+ and p53^-/-APOE*3-Leiden mice had comparable levels of AIA-312040– and CD3-positive cells at the level of both blood smears and spleen, indicating that the absence of p53 did not affect monocyte/macrophage and T-cell numbers.

Analysis of Atherosclerosis
The comparable physical condition (body weight and monocyte/macrophage and T-cell numbers) and plasma cholesterol levels for the p53^-/-APOE*3-Leiden and p53^+/+APOE*3-Leiden mice allow us to dissect selectively the effect of macrophage p53 deficiency on the atherosclerotic process in these mice.

The histopathology of atherosclerotic lesions in p53^-/-APOE*3-Leiden and p53^+/+APOE*3-Leiden mice was analyzed (Figure 2). In both groups, atherosclerotic lesions were in the aortic intima, ranging from foam-cell–rich fatty streaks to fibrous plaques with a cap and lipid core underneath with necrotic material, cholesterol clefts, extracellular lipid, and some foam cells. Such plaques are typical for APOE*3-Leiden mice after a 12-week dietary challenge.^{15 16 23 25} On gross inspection of HPS sections of the proximal aorta, p53^-/-APOE*3-Leiden mice had more extensive lesions than p53^+/+APOE*3-Leiden mice, which were more fibrous and had larger cores, more necrotic material, and more cholesterol clefts (Figures 2A and 2B). The total lesion area was quantified in each group by computer-assisted morphometry (Figure 3 and Table 2). Mean lesion area in p53^-/-APOE*3-Leiden mice was significantly 2.3-fold higher than that in p53^+/+APOE*3-Leiden mice (P=0.006). In addition, blind semiquantificative evaluation revealed that plaques in p53^-/-APOE*3-Leiden mice had a significant (P=0.04) 5-fold increase in necrotic index, with an average index of 1.1, representing plaques with mild to moderate necrosis (Table 2). This increase in necrosis in p53^-/-APOE*3-Leiden mice likely results from the observed increase in lesion size in these mice, because p53^+/+APOE*3-Leiden mice with large lesions (mean lesion area ranging from 100 to 330 000 µm² per section) display necrosis at a level comparable with that of p53^-/-APOE*3-Leiden mice in the same lesion size range (necrotic index of 1.2±1.3 [n=4] and 1.0±1.2 [n=10] for p53^+/+ and p53^-/-APOE*3-Leiden, respectively; Table 2).

View larger version (78K): [in this window] [in a new window]   Figure 2. Typical lesions in APOE*3-Leiden mice reconstituted with either p53+/+ (A, C, and E) or p53-/- bone marrow (B, D, and F). Serial 5-µm sections were either stained with HPS (A and B), macrophage-specific antibody AIA-312040 (C and D), or T-cell–specific anti-human CD3 antibody (E and F; arrow indicates CD3-positive cell). Sections were counterstained with hematoxylin.

View larger version (15K): [in this window] [in a new window]   Figure 3. After 12 weeks of HFC feeding, the extent of atherosclerosis in APOE*3-Leiden mice reconstituted with either p53+/+ () or p53-/- bone marrow (•) was quantified at the level of aortic valves. Each data point represents mean lesion area per mouse. Line represents mean area for each group. P value given vs p53+/+APOE*3-Leiden mice.

View this table: [in this window] [in a new window]   Table 2. Characteristics of Atherosclerotic Lesions in APOE3 3-Leiden Mice Reconstituted With p53+/+ or p53-/- Bone Marrow

To determine whether macrophage p53 deficiency affects macrophages in lesions, we quantified the lesion area positive for the anti-mouse macrophage polyclonal antibody AIA-312040 (Figures 2C and 2D). Macrophage lesion area showed a significant (P=0.02) 2-fold increase, parallel with the increase in plaque area (Table 2). When expressed as percentage of lesion covered by macrophages, no effect of p53 deficiency on macrophage area was observed (Table 2), indicating that macrophage p53 deficiency did not result in enrichment of plaques with macrophages. Similarly, p53 deficiency did not affect the relative number of macrophages (ie, AIA-312040–positive nucleated cells) as analyzed for individual lesions ranging in size from 0 to 75 000 µm² (Table 2). Irrespective of the p53 genotype, macrophage numbers show a relative decrease in large lesions (lesion size range 30 to 75 000 µm²), which reflects the increase in fibrosis and necrosis in lesions within this size range (Table 2).

To determine whether reconstitution with p53-deficient bone marrow affects T-cell numbers in lesions, we quantified the number of cells positive for the anti-human CD3 polyclonal antibody (Figures 2E and 2F) in individual lesions ranging in size from 0 to 75 000 µm². As shown in Table 2, T-cell numbers per 10 000 µm² were not affected by p53 deficiency or by the size of the lesion.

Although PCR analysis on genomic DNA from white blood cells revealed that for all p53^-/-APOE*3-Leiden mice the genotype of the white blood cells was p53^-/-, some original p53^+/+ cells may have survived the lethal body irradiation and become part of the atherosclerotic lesion. Using an anti-human apoE antibody (staining human apoE-producing cells of the original recipient genotype, ie, APOE*3-Leiden.p53^+/+ and not donor-derived APOE*3-Leiden-negative.p53^+/+ or APOE*3-Leiden-negative.p53^-/- cells), all macrophages in lesions of nontransplanted APOE*3-Leiden mice were stained positive for human apoE, whereas only incidentally (<10%) human apoE-positive macrophages were observed in lesions of the bone marrow–transplanted mice (both p53^-/- and p53^+/+APOE*3-Leiden mice, Figure 4). This indicates that the change of genotype by bone marrow transplantation was successful also at the level of the atherosclerotic plaque to an extent comparable with earlier reported levels.^{26 27}

View larger version (44K): [in this window] [in a new window]   Figure 4. Lesions of nontransplanted APOE*3-Leiden mice (A) and lesions of bone marrow–transplanted mice (reconstituted with either p53+/+ [B] or p53-/- bone marrow [C]) were stained with a human apoE-specific antibody. In lesions of nontransplanted APOE*3-Leiden mice, macrophages all are strongly positive for human apoE, whereas in lesions of bone marrow–transplanted mice, a macrophage incidentally shows apoE positivity (arrows). Sections were lightly counterstained with hematoxylin.

Proliferation and Apoptosis
p53 is an essential molecule in both cell proliferation and apoptosis. To investigate whether macrophage p53 deficiency increased the size of atherosclerotic lesions by increased proliferative activity, we injected mice with BrdU 1 day before the animals were euthanized and quantified the percentage of BrdU-positive nuclei in aortic atherosclerotic lesions. In p53^-/-APOE*3-Leiden mice, lesions had a frequency of 4.8±2.3% of BrdU-positive nuclei (Figure 5C), of which 71.0±11.5% were morphologically identified as macrophages and 29.0±11.5% as smooth muscle cells (Figure 5A). This is in line with values reported earlier for nontransplanted APOE*3-Leiden mice.²⁵ In addition, the frequency of BrdU-positive nuclei was not different from p53^+/+APOE*3-Leiden mice (3.7±1.0% of total nuclei, of which 75.2±13.3% and 24.8±13.3% were morphologically identified as macrophages and smooth muscle cells, respectively [Figures 5B and 5C]), indicating that macrophage p53 deficiency did not affect proliferation of the different cells in the plaque, including that of macrophages.

View larger version (64K): [in this window] [in a new window]   Figure 5. Lesions of p53+/+ and p53-/-APOE*3-Leiden mice were stained for proliferative and apoptotic activity using BrdU and TUNEL labeling, respectively. Representative images of BrdU-positive and TUNEL-positive nuclei in lesions of p53+/+ (A and D, respectively) and p53-/- transplanted mice (B and E, respectively) are shown. Sections were counterstained with light green or hematoxylin for BrdU and TUNEL staining, respectively. Arrows indicate BrdU- or TUNEL-positive nuclei. C and F, Subsequently, incidence of BrdU-positive (C) and TUNEL-positive (F) nuclei in lesions of p53+/+ () or p53-/- (•) transplanted mice was quantified. Data are mean percentage of BrdU/TUNEL-positive nuclei per mouse. Line represents mean percentage of positive nuclei for each group. P value given vs p53+/+APOE*3-Leiden mice.

Cell death plays an important part in the formation of atherosclerotic plaques. Apoptotic cell death occurs in plaques, both in humans and in animal models.^{4 5 6 7 25} To investigate whether macrophage p53 deficiency increased the size of atherosclerotic lesions by decreased apoptotic activity, we determined apoptosis by the TUNEL technique. TUNEL was carried out using citric acid pretreatment and careful titration of proteolytic pretreatment and terminal deoxynucleotidyl transferase concentration to avoid labeling of nonnuclear structures and a high fraction of nonapoptotic nuclei.²⁴ In combination with morphological criteria (see Materials and Methods), the TUNEL technique has become a reliable means of detecting the execution phase of apoptosis. The majority of apoptotic nuclei were found in the core region (91±13.4% and 93.6±15.6% of the TUNEL-positive nuclei were morphologically identified as macrophages and 8.7±13.4% and 6.4±15.6% of TUNEL-positive nuclei were identified as smooth muscle cells, for p53^+/+ and p53^-/-APOE*3-Leiden mice, respectively [Figures 5D and 5E]). The average frequency of apoptotic nuclei in the lesions was 0.42±0.39% in p53^+/+APOE*3-Leiden mice (Figure 5F). Although the P value of 0.071 can be considered as borderline (in)significant, macrophage p53 deficiency resulted in a 75% decrease in TUNEL-positive nuclei (0.42±0.39 and 0.14±0.15% of total nuclei for p53^+/+ and p53^-/-APOE*3-Leiden mice, respectively), suggesting that decreased apoptosis may contribute to the pathogenic mechanism leading to the increase in atherosclerotic lesions in p53^-/-APOE*3-Leiden mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerotic lesions using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. Reconstitution of APOE*3-Leiden mice with p53^-/- bone marrow resulted in a significant 2.3-fold increase in mean total lesion area as compared with mice reconstituted with p53^+/+ bone marrow. In addition, p53^-/- transplanted mice demonstrated a significant 2-fold increase in mean lesion macrophage area and a significant increase in necrosis. These observations coincided with a tendency toward decreased apoptosis (TUNEL-positive nuclei), whereas the number of proliferating cells (BrdU-positive nuclei) was not affected. These studies indicate that macrophage p53 is a suppressing factor in the progression of atherosclerotic plaques, establishing, in addition to its suppressing function in cancer, a suppressing function for atherosclerosis.

Using apoE-deficient (apoE^-/-) mice as atherosclerotic background, Guevara et al¹¹ already demonstrated that whole-body p53 deficiency leads to an increase in atherosclerotic lesion area. Lesions in the p53^-/-apoE^-/- mice grossly had the same appearance when compared with lesions in p53^-/-APOE*3-Leiden mice, although in our case, macrophage p53 deficiency led to more prominent necrosis (Figure 2B and Table 2) when compared with whole-body p53 deficiency. Combining our results with those of Guevara et al¹¹ suggests that the effect of macrophage p53 deficiency on atherosclerosis can, at least in part, explain the effects of overall deficiency on atherosclerosis development.

Reconstitution of APOE*3-Leiden mice with p53^-/- bone marrow resulted in an increase in lesion area, increase in macrophage area, and increase in necrosis as compared with mice reconstituted with p53^+/+ bone marrow. However, the number of macrophages and extent of necrosis were not different between the 2 groups when comparing lesions within the same size range (Table 2), which indicates that absence of macrophage p53 affects the rate of atherosclerosis development rather than the composition of lesions.

In a parallel study, we observed that female APOE*3-Leiden mice reconstituted with p53^+/- bone marrow exhibited a 1.6-fold increase in atherosclerotic lesion size as compared with p53^+/+ transplanted controls (155.9±102.0 [n=16] versus 94.1±48.6 µm² [n=10], P=0.186). These data strongly suggest that macrophage p53 influences atherosclerosis development in a gene-dose–dependent matter.

In our study of p53 deficiency specifically in macrophages, no effect on proliferation rate (or DNA synthesis) in the plaque was seen, whereas whole-body p53 deficiency led to a 3-fold increase in cell proliferation rate, mainly confined to macrophages.¹¹ These differences may result from the cell-type–specific approach used in the present study, suggesting that p53 deficiency in other cell types in the plaque (ie, endothelial and smooth muscle cells) may lead to increased proliferation of monocytes/macrophages. One can speculate that p53-dependent effects on cell cycle and/or death of smooth muscle and/or endothelial cells affects cytokine production and thereby proliferation of macrophages. On the other hand, the basic level of BrdU-positive nuclei in p53^+/+apoE^-/- control mice is already lower than observed in APOE*3-Leiden mice (1.31±0.45% versus >3.7%) and may contribute to the observed differences. In contrast to apoE-deficient mice (atherosclerotic background used in the study by Guevara et al¹¹ ), lesion macrophages in transplanted APOE*3-Leiden mice still synthesize functional apoE, which is known to strongly modulate the atherosclerotic process.²⁸ In addition, in APO*3-Leiden mice, atherosclerosis is induced using a cholate-containing diet. Cholate is known as an inflammatory stimulus²⁰ and may thereby affect the histopathological and molecular events during atherogenesis. Possibly the presence of apoE-producing macrophages and the use of dietary cholate contributes to the observed differences between our study and that of Guevara et al.¹¹

p53 is a known inducer of apoptosis,⁸ and absence of the protein can result in resistance to apoptosis.^{29 30} Plaques in p53^-/-APOE*3-Leiden mice had a tendency to have decreased frequency of TUNEL-positive cells, indeed suggesting a p53-dependent decrease in apoptosis of macrophages in the plaque. Downstream apoptosis targets of p53, such as BAX, a proapoptotic protein of the BCL-2 family, and the death receptor FAS and its ligand, are expressed in macrophages in the plaque.^{31 32 33} Likely, absence of p53 may result in decreased apoptosis of macrophages via these pathways. Remarkably, whole-body p53 deficiency did not decrease apoptosis rates in atherosclerotic plaques in apoE-deficient mice, in which apoptosis rates even tended to increase.¹¹ Whether this is due to the cell-type–specific approach, the presence of apoE in macrophages, or the use of dietary cholate in the present study, as outlined above for the effect of absence of p53 on proliferation rate, is subject to speculation. In vitro studies using isolated lipid-laden p53-deficient macrophages may shed more light on the mechanistic aspects of how p53 may affect macrophage turnover.

The number of circulating monocytes and T cells may be an important determinant of the level of atherosclerosis.^{34 35} p53 deficiency in bone marrow may affect the monocyte and T-cell population by increasing proliferation rate and decreasing apoptosis of these cell types. Immunohistological evaluation of blood smears and spleens of p53^-/-APOE*3-Leiden and p53^+/+APOE*3-Leiden mice revealed comparable levels of monocytes and T cells in the control p53^+/+ and p53^-/- transplanted groups. Hence, the observed increase in atherosclerosis in p53^-/-APOE*3-Leiden mice cannot be explained by a p53-related increase in number of circulating blood monocytes and T cells.

Because the present study shows that p53 deficiency specifically in macrophages leads to a significant doubling of atherosclerotic lesion size, one may speculate that drugs that stimulate p53 expression in macrophages may lead to a reduction in atherosclerosis. Future studies are required to demonstrate whether p53 or its downstream targets can be used to modulate the cellular composition of plaques, thereby enhancing plaque stability and reducing acute coronary syndromes.

	Acknowledgments

 
This work was supported by the Netherlands Organization for Scientific Research (Projects 902-26-207 and 902-21-208), the Netherlands Heart Foundation (Project 2000.051), and the Royal Netherlands Academy of Arts and Sciences. We thank Sophie Bos for technical assistance.

	Footnotes

 
Original received September 18, 2000; revision received February 23, 2001; accepted February 23, 2001.

	References

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