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The Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK.
Abstract
Recent studies have indicated that the tumor suppressor gene p53 limits atherosclerosis in animal models; p53 expression is also increased in advanced human plaques compared with normal vessels, where it may induce growth arrest and apoptosis. However, controversy exists as to the role of endogenous levels of p53 in different cell types that comprise plaques. We examined atherosclerotic plaque development and composition in brachiocephalic arteries and aortas of p53eC/eC/ApoEeC/eC mice versus wild type p53 controls. p53eC/eC mice demonstrated increased aortic plaque formation, with increased rates of cell proliferation and reduced rates of apoptosis in brachiocephalic arteries. Although most proliferating cells were monocyte/macrophages, apoptotic cells were both vascular smooth muscle cells (VSMCs) and macrophages. Transplant of p53 bone marrow to p53eC/eC/ApoEeC/eC mice reduced aortic plaque formation and cell proliferation in brachiocephalic plaques, but also markedly reduced apoptosis. To examine p53 regulation of these processes, we studied proliferation and apoptosis in macrophages, bone marrow stromal cells and VSMCs cultured from these mice. Although endogenous p53 promoted apoptosis in macrophages, it protected VSMCs and stromal cells from death, a hitherto unknown function in these cells, in part by inhibiting DNA damage response enzymes. p53 also inhibited stromal cell expression of VSMC markers. We conclude that endogenous levels of p53 protect VSMCs and stromal cells against apoptosis, while promoting apoptosis in macrophages, and protect against atherosclerosis development.
Key Words: atherosclerosis apoptosis cell proliferation
Introduction
The advanced atherosclerotic plaque is composed of a variety of different cell types. Thus, many plaques consist of a fibrous cap, composed of vascular smooth muscle cells (VSMCs), collagen, and extracellular matrix, which separate a lipid-rich core from the lumen. The core comprises intra and extracellular lipid and foam cells of predominantly macrophage origin. VSMC number is determined by the net effect of VSMC proliferation, migration, death, and recruitment from circulating precursors. Low levels of VSMC proliferation are found in advanced human lesions, even in culprit sites in unstable angina1,2 or after plaque rupture.3 In contrast, apoptosis of VSMCs occurs in advanced plaques at higher levels than in normal vessels,4eC6 and is increased in unstable versus stable angina patients.7 In addition, direct induction of apoptosis induces rupture of mouse plaques.8 The regulation of VSMC apoptosis is therefore of considerable significance.
The tumor suppressor gene p53 encodes a transcription factor that activates genes involved in growth arrest (p21, GADD45) and apoptosis (eg, Bax, Fas, IGF-bp3, PAG608, p53-induced genes , PUMA, NOXA; see review9). p53 induces apoptosis via both transcriptional activation of proapoptotic genes/repression of antiapoptotic genes and nontranscriptional mechanisms. Thus, transcriptional activation by p53 is necessary for apoptosis in some cell types,10eC12 but p53-mediated apoptosis can occur without de novo RNA and protein synthesis in others.13eC15 p53 can form inhibitory complexes with Bcl-XL and Bcl-2, directly promoting apoptosis at the mitochondrion.16 Although much of p53-mediated apoptosis signals through mitochondrial pathways, p53 inducible genes also regulate sensitivity to death receptoreCmediated apoptosis (eg, Fas/DR5).17eC19 The cellular response to p53 depends both on the cell type, the level of p53 expression, and the presence of other apoptotic stimuli. Thus, low-level p53 expression often induces growth arrest, with apoptosis only induced by higher levels. The induction of growth arrest may also reduce the sensitivity of cells to apoptosis (see review9).
The role of p53 in VSMC proliferation and apoptosis in atherosclerosis is controversial. p53 expression is negatively correlated with markers of cell proliferation in human atherosclerosis,20 suggesting that p53 inhibits cell proliferation in vivo. Indeed, adenovirus expression of p53 (Ad-p53) reduces cell proliferation in the rat carotid artery21 or migration in the human saphenous vein22 and conversely, antisense oligonucleotides to p53 increase proliferation.23 p53 expression is associated with increased apoptosis in vivo and in vitro24eC26 and Ad-p53 induces plaque rupture and apoptosis in a collar model of atherosclerosis in ApoE knockout mice8 and apoptosis in human saphenous vein intima and media.22
These studies all suggest that p53 inhibits cell proliferation and promotes VSMC apoptosis in atherosclerosis. However, direct in vivo evidence from separate studies is contradictory.27eC29 p53eC/eC/ApoEeC/eC, /ApoE3-Leiden, or /LDL-ReC/eC mice develop accelerated atherosclerosis compared with p53+/+ mice. However, two studies from the same group found that p53eC/eC mice show increased cell proliferation but no change in apoptosis,27,29 whereas the third demonstrated reduced apoptosis, but no change in cell proliferation.28 Although studies have demonstrated effects of p53 on macrophages by bone marrow transplant,28,29 none of the studies identified the lineage of the proliferating/dying cells. In addition, none of the studies examined the specific role of p53 knockout in VSMCs, as transplants were performed of p53+/+ or p53eC/eC cells into p53+/+ mice, so that VSMCs derived from the vessel wall were all p53+/+. Finally, the effects of p53 were not examined in more advanced lesions. Compared with normal vessels, p53 expression is increased in advanced human plaques, such as those seen in mouse brachiocephalic vessels, but not detectable in early lesions.
We therefore examined the role of endogenous levels of p53 in macrophages and VSMCs in atherosclerosis in advanced lesions in ApoEeC/eC mice. We identify that endogenous p53 reduces atherosclerosis, with markedly different effects on macrophages, VSMCs, and bone marrow stromal cells that may also contribute to plaque VSMCs. Endogenous p53 protects VSMCs and stromal cells against apoptosis, a hitherto unknown role in these cells, and also inhibits stromal cell expression of VSMC markers.
Materials and Methods
Experimental Atherosclerosis Protocols
All animal experimental procedures conformed to animal Ethical Committee approval and UK Home Office licensing. p53 and ApoE knockout mice, both on the C57Bl6/J background, were purchased from Jackson Laboratories (Bar Harbor, Maine). Double null animals were generated, weaned at 6 weeks, and genotyped using the Tyler Jacks p53 and JAX ApoE PCR protocols. Male and female mice were fed a Western high-fat diet comprising 19.5% Casein milk fat and 0.05% cholesterol for 14 weeks in an isolated pathogen-free environment. Mice were euthanized by CO2 overdose and perfusion-fixed with 5% paraformaldehyde through cardiac puncture. For bone marrow transplantation experiments, 6-week-old mice received 7.5Gy total body irradiation. Tail bleeds were taken 24 hours after irradiation to confirm ablation of peripheral mononuclear cells and subsequently to determine extent of peripheral cell reconstitution. Bone marrow cells for repopulation were harvested from either p53+/+ or p53eC/eC mouse femurs, and 5x106 cell were injected, 48 hours after irradiation.
Characterization and of Primary Cell Cultures
p53+/+ or p53eC/eC VSMCs were explant-cultured from descending aorta. Cells were identified as VSMCs by immunocytochemistry for -SM-actin (SMA) and calponin. Macrophages were isolated by peritoneal lavage with PBS/BSA and identified using antibodies to mac-3. Bone marrow stromal cells were isolated by excising whole femurs from donor mice, removing proximal and distal trabecular heads, and flushing with 1 mL of medium. Cells were subsequently washed with PBS at 24 and 48 hours to remove nonadherent cells. Stromal cell cultures were examined for expression of SMA and Calponin at 5 and 30 days after isolation.
Statistical Analysis
Student t test was used for data following a Gaussian distribution and the Mann-Whitney rank sum test used under nonbinominal conditions.
An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org.
Results
To examine the effects of global p53 deficiency on atherosclerotic plaque formation, we generated p53eC/eC/ApoEeC/eC mice (all on C57Bl6/J background). Double null animals were weaned at 6 weeks and fed an atherogenic diet to 20 weeks of age. Littermate controls that were wild type for p53 underwent the same regime. There was no difference in weight gain between experimental groups, and mice did not develop evidence of neoplasia by time of euthanasia. We examined plaque development in aortas of these animals using Oil Red O (ORO) staining and computerized planimetry. Brachiocephalic plaques were used to examine plaque cellular composition after staining for SMA, mac-3, CD3, or Sirius Red (for collagen). Proliferation or apoptosis were identified using antibodies to Ki67, or cleaved caspase 3 expression in cells demonstrating pyknotic nuclei, respectively, with double labeling to identify the cell of origin.
p53 knockout resulted in a 102% increase in total aortic plaque area (Figure 1; Table 1) with an increase in percentage of aorta covered with plaques from 5.9% to 11%. p53 knockout did not significantly increase brachiocephalic plaque area. There was no significant change in cellular composition in p53eC/eC versus p53+/+ brachiocephalic plaques, as detected by SMA, mac-3, or Sirius Red staining (Table 1; Figure 1; and online data supplement). Less than 1% of cells in plaques were CD3 positive in either group (supplemental Figure I). However, p53eC/eC lesions showed 57% increase cell proliferation and a 70% reduction in apoptosis when compared with p53+/+ mice. Double labeling indicated that the majority of proliferating cells were mac-3eCpositive. Although most apoptotic cells were identified as monocyte/macrophages (Figure 1D and 1E), SMA-positive cells also expressed cleaved caspase 3 in p53eC/eC mice (Table 1).
We also examined brachiocephalic plaques for evidence of plaque rupture including the presence of fibrous cap discontinuity, intraplaque hemorrhage, and luminal thrombosis, and studied buried fibrous caps/multilayering, the etiology of which is currently controversial. Although some plaques (<5%) demonstrated evidence of intraplaque hemorrhage, there was no significant difference between groups and luminal thrombus and fibrous discontinuity were not seen in either group. p53eC/eC plaques showed a significant increase in the presence of buried fibrous caps/multilayering (mean 0.86 versus 0.5 buried caps/plaque) (Figure 1F and 1G).
Replacement of p53 in Bone MarroweCDerived Cells Inhibits Atherosclerosis
To examine the role of p53 in bone marroweCderived cells and VSMCs separately in vivo, we transplanted p53+/+/ApoEeC/eC or p53eC/eC/ApoEeC/eC bone marrow cells into p53eC/eC/ApoEeC/eC mice. Unlike earlier studies28,29 where transplants were performed into p53+/+/ApoEeC/eC or p53+/+/LDLReC/eC mice, our studies do not correct the p53 deficiency in VSMCs derived from the host vessel wall. This allows us to study the effect of replacing p53 in the bone marroweCderived cells, but not the vessel walleCderived VSMCs, which remain p53eC/eC.
Six-week-old p53eC/eC/ApoEeC/eC mice were lethally irradiated, transplanted with p53+/+/ApoEeC/eC or p53eC/eC/ApoEeC/eC bone marrow cells after 48 hours, fed an atherogenic diet, and euthanized at 20 weeks as before. Cell counts and reconstitution were followed by Coulter counts of peripheral blood (supplemental Figure IIA). p53eC/eC/ApoEeC/eC mice were susceptible to irradiation, exhibiting a 96% suppression of peripheral mononuclear cells. There were no differences in either time or extent of reconstitution of peripheral blood counts in mice transplanted with p53eC/eC or p53+/+ marrow, with an average reconstitution of 66% by 5 weeks and 97% by the end of the study. Littermates irradiated but not reconstituted had marked bone marrow suppression with significantly delayed recovery, further confirming the successful bone marrow reconstitution in the transplanted mice. There was no difference in cholesterol/triglyceride levels between p53eC/eC or p53+/+ transplanted mice (supplemental Figure IIB and IIC). Although aortic plaque development in transplanted mice was similar to that seen in untransplanted mice, brachiocephalic plaque development was significantly reduced in mice transplanted receiving either p53+/+and p53eC/eC marrow compared with untransplanted mice. This site-specific effect of irradiation/transplantation has been noted before,30 and means that although comparisons between transplanted mice of different genotypes are valid, comparison between transplanted and untransplanted mice need to be viewed with caution.
Transplant of p53+/+/ApoEeC/eC bone marrow cells reduced aortic plaque area by 39.9% (Figure 2) compared with p53eC/eC transplant, with no significant reduction in brachiocephalic lesions area (Table 2). There was again no significant change in the relative abundance of monocyte/macrophages in brachiocephalic plaques, but of note, the percentage of cells expressing SMA or mac-3 was >100%, suggesting that some cells expressed both markers. Transplant of p53+/+/ApoEeC/eC cells significantly reduced cell proliferation (Table 2), with double-labeling revealing the dominant proliferating cell type to be the monocyte/macrophages in both p53eC/eC and p53+/+ groups (Figure 2). Total apoptosis was markedly reduced in mice receiving p53+/+/ApoEeC/eC bone marrow. Although most apoptotic cells expressed monocyte/macrophage markers (Figure 2), VSMC apoptosis was not seen in mice receiving p53+/+ marrow. Transplant of p53+/+ bone marrow also decreased the presence of buried fibrous caps/multilayering (1.14 versus 1.67 buried caps/plaque), but again, fibrous cap discontinuity and luminal thrombosis were not seen in either group.
p53 Deficiency Has Differential Effects on Macrophages and Smooth Muscle Cells
The acceleration of atherosclerosis seen by global knockout of p53 does not fully elucidate the role of p53 in specific cell types comprising the atherosclerotic plaque. Recent studies suggest that bone marroweCderived cells can transdifferentiate into cells expressing VSMC markers.31 Thus, if p53 knockout increases bone marroweCderived cells in lesions, transdifferentiation may also increase cells expressing VSMC markers. To examine the role of p53 in VSMCs and macrophages separately and bone marrow stromal cells that have shown to transdifferentiate, we isolated VSMCs, bone marrow stromal cells, or peritoneal macrophages (a fully differentiated macrophage) from p53+/+ or p53eC/eC mice. Immunocytochemistry using SMA/calponin or Mac-3, respectively, was used to confirm cell lineage and purity, and examine transdifferentiation of stromal cells.
Peritoneal macrophage cultures were mac-3eCpositive/SMA-negative and VSMCs were SMA/calponin-positive/mac-3eCnegative immediately after isolation. We used time-lapse videomicroscopy over 96 hours to examine cell proliferation rates and morphology of cell death and vital dye exclusion to measure death rates in media containing 10% or 0% FCS (Figure 3). p53eC/eC VSMCs and stromal cells after 10 weeks of culture showed significantly increased rates of cell proliferation compared with p53+/+ cells, but contrary to expectations, both showed increased rather than decreased rates of cell death in 0% FCS when compared with p53+/+ cells (Figure 3; Table 3). Cell death was typically apoptotic as judged by videomicroscopy and vital dye exclusion. p53eC/eC VSMCs and stromal cells were also more sensitive to other stimuli inducing apoptosis including the DNA damaging drug etoposide or UV irradiation (Table 3). Peritoneal macrophages from either p53+/+ or p53eC/eC mice showed no cell division in culture, but, in contrast to VSMCs and stromal cells, p53eC/eC cells exhibited reduced apoptosis in 10% FCS at 48 hours (Figure 4). Both p53+/+ and p53eC/eC macrophages accumulated oxidized LDL to the same extent (Figure 4) and exhibited similar rates of death induced by oxidized LDL or low serum when assessed by dye exclusion.
At first isolation, there was no difference in percentage of cells expressing SMA or calponin between p53eC/eC and p53+/+ stromal cells. However, by 10 weeks in culture, p53eC/eC stromal cells showed a marked increase in SMA and calponin expression compared with p53+/+ cells (Figure 4). Thus, endogenous p53 protects VSMCs and bone marroweCderived stromal cells from apoptosis but promotes apoptosis in macrophages, and reduces transdifferentiation of the bone marroweCderived stromal cells to VSMCs.
p53 Inhibits Activity of DNA Damage Enzymes
p53 is a major effector of responses to DNA damage, inducing both growth arrest and apoptosis. DNA breaks activate a number of upstream protein kinases, including ATM (Ataxia Telangiectasia Mutated) and ATR (ATM-related kinase). These in turn phosphorylate-specific substrates, including the checkpoint kinases Chk-1 and Chk-2, which phosphorylate and activate p53. Although p53 is a substrate of ATM, ATR, Chk-1, and Chk-2, p53 can catalyze repair of many forms of DNA damage, including single strand breaks and homologous recombination,32,33 preventing the propagation of damaged DNA that leads to apoptosis. We find that p53 protects VSMCs from apoptosis induced by etoposide and UV irradiation, both of which induce DNA damage. We therefore examined whether p53+/+ and p53eC/eC VSMCs and macrophages had different responses to DNA damage that mediated different levels of apoptosis. Even under basal conditions, p53eC/eC VSMCs showed marked expression of both ATM/ATR substrates (a marker of activity of these enzymes) and P-Chk-1 (Figure 5). These differences were not seen between p53+/+ and p53eC/eC macrophages. To prove that p53 was responsible for these effects, we reintroduced a conditional allele of p53 (p53ERTM) into p53eC/eC VSMCs. p53ERTM is a p53-estrogen receptor fusion protein that is activated by 4-hydroxytamoxifen.34 p53 activation inhibited ATM/ATR substrates and P-Chk-1 expression, and also inhibited etoposide-induced apoptosis (Figure 5).
Discussion
The role of endogenous levels of p53 in atherosclerosis remains controversial, with studies differing as to whether p53 regulates cell proliferation or apoptosis (or both) in lesions and the cell type(s) affected. In addition, previous studies examined early lesions in the aortic root, whereas p53 expression is not detectable in human lesions until a relatively advanced stage.35 We examined the separate contributions of p53 to VSMCs and macrophage accumulation in the mouse brachiocephalic artery. The brachiocephalic artery develops advanced atherosclerosis reproducibly, including lumen narrowing and medial thinning,36,37 and shows evidence of possible plaque rupture,37eC39 although the appearances that define the latter are still controversial.
We find that endogenous p53 expression reduces aortic atherosclerosis in ApoEeC/eC mice. p53 may affect many processes associated with atherosclerosis, for example macrophage adhesion, trafficking, differentiation, and foam cell formation; we find no effect of p53 on macrophage lipid accumulation. However, plaques in p53+/+ mice showed both reduced cell proliferation and increased apoptosis compared with p53eC/eC mice. As most proliferating cells were macrophages, a major effect in vivo of p53 is limiting macrophages or macrophage-derived cells. Although buried fibrous caps (with a consequent multilayered appearance) were reduced in p53+/+ mice, other features of plaque rupture were not present, and no difference in plaque composition was noted. This suggests that, whereas overexpression of p53 induces plaque rupture,8 endogenous p53 limits atherosclerosis development without major changes in plaque vulnerability.
To examine the role of endogenous p53 expression in either bone marroweCderived or vessel walleCderived cells, p53eC/eC/ApoEeC/eC mice were lethally irradiated and transplanted with either p53+/+/ApoEeC/eC or p53eC/eC/ApoEeC/eC bone marrow, thus replacing p53 in bone marrow cells in one experimental group. p53eC/eC mice undergo marrow suppression with irradiation, engraftment is complete by 12 weeks and atherosclerosis at 20 weeks was still evident. p53+/+p53eC/eC transplant resulted in significantly less aortic atherosclerosis, but no change in brachiocephalic plaque composition compared with p53eC/eCp53eC/eC transplants. However, p53+/+p53eC/eC transplants showed significantly reduced cell proliferation and apoptosis. The effects of p53 deficiency on proliferation and apoptosis we observe differ from previous studies,28,29 which found reduced proliferation but no change in apoptosis,29 or vice versa,28 possibly because we transplanted into p53eC/eC/ApoEeC/eC mice, not correcting any effect of p53 deficiency in VSMCs from the host vessel wall. Thus, in our transplant studies, p53eC/eC VSMCs from the host vessel wall may contribute to increased cell proliferation and increased apoptosis. In addition, we have studied the effects of p53 knockout in brachiocephalic rather than aortic root lesions studied previously. The brachiocephalic lesions demonstrate a more advanced, complex morphology than aortic root lesions, mimicking a number of features of human plaques. This site specificity of effect of manipulating atherosclerosis, also described in a number of other models,40,41 may explain why we see significant reduction in plaque area in aortas but not brachiocephalic arteries. Although the measures of plaque area are different in each location (en face versus cross sectional area), manipulations affecting plaque initiation versus progression may differentially affect atherosclerosis in less advanced plaques in the aorta compared with brachiocephalic arteries.
An initially puzzling observation is apparent in the data from these transplant studies. Transplant of p53+/+ bone marrow decreased apoptosis in plaques, whereas overexpression of p53 in the vessel in previous studies induces apoptosis.8,22 To study p53 effects in VSMCs in isolation, we derived aortic VSMCs, peritoneal macrophages, or bone-marrow stromal cells from p53eC/eC or p53+/+ mice. p53+/+ macrophages underwent higher rates of apoptosis in standard culture conditions (10% FCS) than p53eC/eC macrophages, although there was no difference in low serum or after oxidized LDL treatment. In contrast, both p53+/+ VSMCs and stromal cells showed decreased rather than increased apoptosis, arguing that endogenous p53 in these cells is protective. Protection by p53 was found in response to a variety of proapoptotic stimuli, including low serum, UV irradiation, and etoposide treatment.
Although p53 has mostly been implicated as a proapoptotic gene, we have previously shown that a dominant-negative p53 or the Type 16 HPV gene E6 (which degrades p53) promotes apoptosis in human VSMCs.34 In addition, endogenous p53 has been shown to protect mouse embryo fibroblasts, a function that mapped to the C terminal 38 amino acids, but was independent of transcriptional activation.42,43 These studies indicate that although excess p53, induced in the context of DNA damage, for instance, induces apoptosis, endogenous p53 in VSMCs is protective. Although p53 may protect via many mechanisms, we find that p53eC/eC VSMCs (but not macrophages) show high basal activity of specific DNA damage response enzymes (including ATM/ATR and Chk-1). Reintroduction of p53 into these cells inhibited activity of these enzymes, and also inhibited DNA damage-induced apoptosis. Thus, the protective effect of p53 may be partly due to inhibition of DNA damage responses in VSMCs.
We also found that bone marroweCderived stromal cells show transdifferentiation to cells expressing VSMC markers, which may explain why some apoptotic cells appeared to express both SMA and Mac-3. This transdifferentiation was reduced by endogenous p53 expression. Some of the reduced apoptosis seen in mice receiving p53+/+ marrow may therefore be due to reduced apoptosis of bone marrow stromal cells that differentiate to SMA-positive cells in vivo. This data also suggest that the contribution of bone marrow cells to VSMCs in atherosclerotic plaques may vary significantly with the genotype of donor cells. However, it is currently not clear how p53+/+ transplant reduces apoptosis in cells expressing a macrophage marker in plaques.
In summary, we find that endogenous p53 expression limits atherosclerosis by inhibiting monocyte/macrophage cell division and increasing macrophage apoptosis. However, endogenous p53 expression affects VSMCs, bone marrow stromal cells, and macrophages very differently. We identify a novel role for endogenous levels of p53 in VSMCs and stromal cells, that of protection against apoptosis.
Acknowledgments
This study was supported by British Heart Foundation Grant RG98009. M.R.B. is a BHF Chairholder.
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