© 1997 American Heart Association, Inc.
Articles |
Topographical Association Between the Cyclin-Dependent Kinases Inhibitor P21, p53 Accumulation, and Cellular Proliferation in Human Atherosclerotic Tissue
From the Departments of Pathology (C.I., G.M., E.W., H.E.S.) and Medical Biometrics and Informatics (J.S.M.), University of Freiburg; and the Department of Internal Medicine IV, University of Frankfurt (A.M.Z.), Germany.
Correspondence to Christian Ihling, MD, Department of Pathology, University of Freiburg, Albertstraße 19, D-79104 Freiburg.
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Abstract |
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Abstract The cell cycle is controlled by cyclin-dependent protein kinases (CDKs). The activity of these enzymes is directed by inhibitors of CDKs. The 21-kD protein product (P21) of the WAF1/CIP1 gene, which can be transactivated by the protein product of the tumor suppressor gene p53, acts as an inhibitor of cyclin-dependent kinases. To assess whether both P21 and p53 may play a role in the control of cellular proliferation in atherosclerotic lesions, the topographical association between p53, P21, and the proliferation marker MIB1/Ki-67, was analyzed by immunohistochemistry in human carotid atheromatous plaques of 26 patients. p53 immunoreactivity (IR) was present in 26 of 26 cases in the nuclei of virtually all cell types (macrophages [MPs], smooth muscle cells [SMCs], endothelial cells [ECs]) in areas with chronic inflammation in 71.08±8.28% of the nuclei. p53 staining in the control tissue from human coronary arteries was present in 0.3±0.45% of the cells (P<.002). P21-IR was present in 24 of 26 specimens in 64.38±10.13% of the cells (controls: 3.8±1.85%, P<.002) and localized to nuclei of MPs (CD68 positive) and SMCs (
-actin positive), as well as ECs of microvessels
present in 21 specimens (21 of 21) and luminal ECs present in
18 specimens (16 of 18). As shown by double labeling, P21-IR
colocalized with p53-IR in most MPs (24 of 24), intimal SMCs (22 of
24), ECs of microvessels (19 of 21), and luminal ECs (10 of 16).
Interestingly, few p53-positive cells did not show
simultaneous P21-IR, and, conversely, not all P21-positive
cells demonstrated p53-IR. MIB1/Ki-67-positive cells were identified in
21 of 26 tissue specimens in 3.53±1.79% of the nuclei (controls: 0%,
P<.002) and localized principally to MPs bordering the
atheromatous lipid core (21 of 26) and to a few
scattered SMCs (16 of 26), ECs of microvessels (13 of 21), and luminal
ECs (2 of 18). Most importantly, none of the cells coexpressing P21 and
p53 were positive for MIB1/Ki-67-IR, indicating the absence of
proliferating activity. In summary, this study demonstrates that P21-IR
is present in the atherosclerotic plaque and colocalizes with p53
in most MPs, SMCs, and ECs. The lack of proliferation markers in cells
coexpressing p53 and P21 suggests that transcriptional activation of
the WAF1/CIP1 gene by p53 may be involved in the control of
cellular proliferation in advanced human atherosclerotic plaques.
Key Words: cell proliferation • p53 • atherosclerosis • P21 • WAF1/CIP1 gene
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Introduction |
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The cycle of cell division is controlled by periodic changes in the activity of CDKs.1 The catalytically operative units, which are composed of cyclins and cyclin-dependent kinases, exert their regulatory function by phosphorylation of key proteins involved in cell-cycle progression. The activity of these kinases is directed by inhibitors of CDKs. P21, the 21-kD protein product of the WAF1/CIP1 gene, which is localized to chromosome 6p21.2,2 acts as a universal inhibitor of cyclin-dependent kinases.3 In normal fibroblasts, P21 has been shown to be associated with every cyclin kinase complex examined, including those containing CDK4/cyclin D complexes.4 5 These complexes are of particular importance for the progression of the cell cycle through the G1 phase due to their ability to phosphorylate the product of Rb6 . Hypophosphorylated Rb inhibits the transcriptional activity of the cell cycle regulatory transcription factor E2F, which is the cellular target for Rb,7 through its direct association with E2F,8 thereby inhibiting the transcription of genes involved in the transition of cells from G1 to S phase.9 Thus, P21 may block cell-cycle progression by repressing phosphorylation of Rb.6 Furthermore, in addition to being a cyclin kinase inhibitor, P21 may negatively regulate the cell cycle by binding to and inhibiting the DNA polymerase
cofactor,
PCNA.10 The protein product of the tumor suppressor gene p53 accumulates in response to DNA damage, causing a G1 block of the cell cycle.11 Interestingly, the promoter of the WAF1/CIP1 gene has a binding site for p53, and the transcription of the gene is activated by wild-type (wt) p53, but not by mutated p53.2 4 Recent evidence suggests that P21 can be induced by wt p53 in response to radiation-induced or chemically induced DNA damage in a variety of cells and is necessary for the p53-mediated G1 arrest.12 13 14 15 P21 may thus play a role as a cellular mediator of p53 growth-suppressive function.
In addition, it has been shown that wt p53 expression was suppressed during the period of active proliferation after acute cutaneous injury in swine and reemerged during the stages of wound healing, linking the expression and accumulation of wt p53 to physiological processes of tissue regeneration in vivo.16 Atherosclerosis is an inflammatory fibroproliferative process of the arterial intima sharing many morphological features with healing wounds and chronic inflammatory processes. Consequently, wt p53 and P21 may also play a role in the regulation of cell growth that occurs in response to vascular injury. Indeed, it has recently been shown by several groups that wt p53 is present in human atherosclerotic tissue17 18 and that P21 is upregulated after mechanical injury in porcine iliofemoral arteries and rat carotid arteries.19 20
However, to our knowledge nothing is known about the simultaneous accumulation of wt p53 and expression of P21 in cells of vascular tissue, ie, MPs, mesenchymal cells, and endothelium, including human atherosclerotic tissue. Therefore, to address a potential role of the interaction of p53 with WAF1/CIP1 for the regulation of cell proliferation in atherosclerotic tissue, using immunohistochemical methods, we assessed the presence and distribution of p53-IR, P21-IR, and MIB1/Ki-67- expressing proliferating cells in carotid atheromatous plaques from patients undergoing endarterectomy.
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Methods |
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Tissue Specimens
Twenty-six atherosclerotic plaques of human carotid arteries with severe complicated atherosclerosis were obtained from patients undergoing carotid endarterectomy. Control experiments were performed by staining samples of coronary arteries with diffuse intimal thickening in the absence of any atherosclerotic lesions from six patients undergoing heart transplantation for idiopathic dilated cardiomyopathy. The specimens were immediately immersed in 4% unbuffered formalin and then prepared according to standard methods. Six serial sections were stained with hematoxylin and eosin and used for immunohistochemistry.
Immunohistochemistry
After the quenching of endogenous peroxidase with
1% H2O2 for 30 minutes, serial sections were
incubated with 0.5% normal bovine serum to reduce nonspecific
background staining. Thereafter, the slides were incubated with
monoclonal antibodies directed against smooth muscle -actin
(dilution 1:1000; Sigma); MPs (CD68 [KP1], dilution 1:100, DAKO); p53
(clone DO 7, dilution 1:50, DAKO; clone DO 1, 1:250, Oncogene Science)
recognizing both wild-type and mutant p53 protein; P21 (OP 64,
monoclonal, dilution 1:10; PC 55, polyclonal, dilution 1:50, Oncogene
Science); and MIB1/Ki-67 (MIB1, monoclonal, dilution 1:50, Dianova;
MIB1/Ki-67, polyclonal, dilution 1:100, DAKO), respectively. For
immunostaining of p53, P21, and MIB1/Ki-67, antigens
were either unmasked by incubating the sections in 6 mol/L urea
in distilled water (aqua dest) for 1 hour at 98°C (p53 DO 7) or by
pressure cooking in 10 µmol citric acid, pH 6, for 3 minutes
(P21, MIB1/Ki-67, p53 DO 1). As a positive control for p53 and
MIB1/Ki-67-IR, we used sections from a follicular hyperplasia of a
tonsil and a breast carcinoma with a well-known IR for both antigens.
As a positive control for P21 staining, we used normal human colon,
where P21 staining is restricted to the postreplicative compartment of
the crypt and the surface epithelium facing the
lumen.12 21 Negative control experiments were performed by
replacing the primary antibodies with preimmune serum of the
corresponding species (mouse, rabbit). All slides were then incubated
with biotinylated secondary antibody at room temperature, followed by
incubation with avidin and biotinylated horseradish peroxidase complex
(ABC method, Vector Labs). Peroxidase activity was visualized by
3-amino-9-ethylcarbazole (AEC, Sigma) to yield a brown reaction
product.22 The nuclei were slightly counterstained
with hematoxylin.
To identify specific cell types expressing p53-IR, P21-IR or
MIB1/Ki-67-IR double labeling was performed with the primary antibodies
and a cell-specific antibody. Briefly, the labeling procedure was as
follows: First, p53, P21, and MIB1/Ki-67 staining was performed using
the above-described three-step ABC method, visualizing peroxidase
activity with AEC, which resulted in a brown staining. After thorough
washing overnight, in a second staining procedure, -actin or CD68
staining was performed using the three-step APAAP
method.23 The presence of both antigens was identified by
a distinctly separate brown staining of the nuclei and dark blue
staining of the cytoplasm.
To demonstrate nuclear colocalization of p53 (DO1) and P21 and simultaneous nuclear staining for P21 and MIB1/Ki-67, immunofluorescence double labeling was performed using the direct tyramide multicolor signal amplification (DuPont) according the manufacturer's instructions. In brief, the procedure was as follows: The tissue was prepared as described above. After blocking the slides to reduce nonspecific background staining, the tissue was incubated sequentially with primary antibodies directed against P21, p53, and MIB1/Ki-67 overnight. After washing, the slides were incubated simultaneously with the appropriate biotin- or fluorescein-labeled secondary antibodies (biotin-labeled goat anti-rabbit antibody, 1:200, Vector; biotin-labeled goat anti-mouse antibody, 1:200, Vector; fluorescein-labeled goat anti-mouse antibody, 1:100, Dianova). After washing, the sections were incubated with streptavidin horseradish peroxidase for 30 minutes and then again thoroughly washed. Subsequently, 300 µL of a 1:50 dilution of tetramethylrhodamine tyramide in 1x amplification diluent were pipetted on the slides for 10 minutes at room temperature, resulting in a red fluorescence of P21 (polyclonal) IR. The remaining peroxidase was inactivated by adding 100 µL 1% H2O2 to each slide (15 minutes). Afterward, the sections were incubated with an anti-fluorescein antibody (1:100, DuPont) coupled to horseradish peroxidase. Then, the sections were incubated in 300 µL of a 1:50 dilution of fluorescein tyramide in 1x amplification diluent for 10 minutes at room temperature, resulting in a green fluorescence of p53-IR (monoclonal) and MIB1/Ki-67-IR (monoclonal). Afterward, sections were counterstained with DAPI (Sigma) with 0.02 mg/mL PBS; 15 minutes at 37°C, resulting in a blue fluorescence of the nuclei.
To be able to distinguish between the red tetramethylrhodamine tyramide fluorescence (absorption 550 nm, emission 570 nm), the green fluorescein tyramide fluorescence (absorption 494 nm, emission 517 nm), and the strong autofluorescence of lipopigment, which was present in abundance in the atherosclerotic tissue, in addition to monofilters a triple filter system from Zeiss was used (excitation triple band-pass filter 400 nm/495 nm/570 nm, beam splitter 410 nm/505 nm/585 nm, emission band-pass filter 460 nm/530 nm/610 nm). With this filter system, unspecific autofluorescence appeared grayish white and was clearly discernible from red, green, and blue fluorescence, indicating specific staining.
Histological Analysis
The specimens were analyzed by light-microscopy for the
presence and localization of antibody-mediated staining. Comparative
examination of serial sections and double labeling permitted the
assessment of colocalization of P21-positive cells with nuclear
staining for p53 and MIB1/Ki-67 in intimal SMCs (-actin positive) or
MPs (CD68 positive). Double-labeling was used to confirm the
simultaneous presence of the antigens in the nuclei and in
the cytoplasm.
Morphometric Analysis and Statistical Methods
To quantitatively study the distribution of cells with p53
accumulation and P21, as well as MIB1/Ki-67 expression, adjacent
sections from all 26 cases were evaluated. Using a morphometric
software (analySIS, Softimaging Software GMBH), 5 random microscopic
high-power fields at x400 from areas with severe chronic inflammation
were analyzed, scoring at least 500 cells as positive or
negative for nuclear staining. All data are reported as mean±SD. The
Wilcoxon two-sample test was applied for comparison of the
atherosclerotic tissue with the controls.
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Results |
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Histology
The atherosclerotic tissue exhibited features of advanced complicated plaques with a heterogenous composition including focal areas rich in SMCs (
-actin positive) or MPs (CD68
positive) between dense fibrous areas or loose connective tissue, as
well as focal areas of necrosis and calcification. Around the periphery
of the lesions in most cases, we found evidence of
neovascularization.
p53 Immunohistochemistry
Immunohistochemical staining disclosed evidence for p53 staining
in 26 of 26 specimens. p53 staining localized preferentially to
cell-rich areas surrounding atheromatous gruel (Fig 1A
) and was absent in cell-depleted,
fibrotic areas (26 of 26). The intensity of p53 staining in most of the
cells varied considerably. However, a large number of nuclei displayed
at least a distinct and finely granular staining, which is a sign of
p53 accumulation. As shown in serial sections and by double labeling,
nuclei of MPs and SMCs showed p53-IR in cell-rich regions (26 of 26).
Luminal ECs present in 18 specimens were positive for p53 in 10
cases (10 of 18), and ECs of newly formed microvessels present in
21 specimens showed p53-IR in 19 cases (19 of 21). Quantitative
analysis of p53 staining revealed that in cell-rich regions
71.08±8.28% of the cells exhibited nuclear accumulation of p53,
whereas in the control group, consisting of nonatherosclerotic
coronary arteries, p53 staining was restricted to 0.3±0.45%
of the cells (P<.002).
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P21 Immunohistochemistry and Relation to p53 Accumulation
P21-IR was present in 24 of 26 specimens and, as confirmed by
double labeling, localized to nuclei of MPs, MP-derived giant cells
(CD68 positive, Fig 2A) bordering
atheromatous gruel, and SMCs in the vicinity of the
atheromatous core (Fig 2B). Morphometric
analysis indicated that 64.38±10.13% of the cells were
positive for P21. By contrast, in the controls, 3.8±1.85% of nuclei
were positive for P21 (P<.002, Fig 3). Importantly, as shown in serial
sections (Fig 1A
and 1B) and double labeling, P21-IR colocalized with
p53-IR in all 24 cases in MPs (Fig 4A through
4D) and in 22 of 24 cases in intimal
SMCs. Luminal ECs present in 18 specimens were positive for P21 in
16 cases (16 of 18) and showed coexpression of P21/p53 in 10 cases (10
of 16). As shown in serial sections and by double labeling, ECs of
newly formed microvessels present in 21 specimens showed P21-IR in
all cases (21 of 21) and simultaneous P21-IR/p53-IR in 19
cases (19 of 21). It is interesting to note that not all p53-positive
cells showed simultaneous nuclear staining for P21 (about
20% of p53-positive cells did not stain for P21) and conversely, not
all P21-positive cells demonstrated nuclear accumulation of p53.
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MIB1/Ki-67 Immunohistochemistry and Relation to p53 and P21
Expression
MIB1/Ki-67-IR, indicating proliferative activity, was identified
in all specimens (26 of 26) in 3.53±1.79% of the cells and was absent
in the control tissue (P<.002). MIB1/Ki-67-IR localized
principally to foam cell–rich regions bordering on the
atheromatous lipid core (26 of 26, CD68 positive) and,
to a far lesser extent, in scattered SMCs (16 of 26) in the vicinity of
atheromas. ECs of newly formed microvessels, which were
present in 21 specimens, showed MIB1/Ki-67-IR in 13 specimens (13
of 21), and luminal ECs present in 18 specimens showed
MIB1/Ki-67-IR in two cases (2 of 18). Interestingly, although
MIB1/Ki-67-IR localized in all cases to plaque components showing at
the same time p53-IR and P21-IR, none of the P21-positive cells showed
simultaneous nuclear staining for MIB1/Ki-67, as shown by
double labeling (Fig 5).
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Discussion |
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Wild-type p53 is a nuclear phosphoprotein that mediates cell cycle arrest in the G1 phase in response to DNA damage, preventing the propagation of genetically damaged cells.11 As a transcription factor, wt p53 either activates or represses transcription of a series of genes including the WAF1/CIP1 gene.2 Interestingly, it has recently been demonstrated that wt p53 is present in human atherosclerotic tissue17 18 and that P21 inhibits proliferation of vascular SMCs in vitro and in vivo.19 20 The results of the present study considerably extend these previous observations by demonstrating that in contrast to nonatherosclerotic control arteries, the advanced atherosclerotic plaque is characterized by the presence of numerous MPs, SMCs, and ECs, with simultaneous p53 accumulation and P21 expression. Our novel findings suggest that the WAF1/CIP1 gene in cells of human atherosclerotic tissue may be transactivated in a p53-dependent fashion. Moreover, since none of the P21-positive cells exhibited signs of cellular proliferation, as indicated by the lack of MIB1/Ki-67-IR, we propose that P21 is involved in regulating cellular proliferation in human atherosclerotic lesions.
Accumulation of p53 was present in the nuclei of virtually all cell types in areas with signs of chronic inflammation. Chronic inflammatory processes within atherosclerotic lesions give rise to the production of cytokines, oxygen-derived free radicals (eg, O2-, H2O2, NO),24 25 26 and reactive intermediates (eg, peroxynitrite),27 28 which are all powerful DNA-damaging agents leading to the accumulation of p53.29 30 31 At the same time, plaque areas with signs of chronic inflammation were also characterized by dramatically increased expression of P21. Importantly, p53 and P21 IR colocalized in a substantial number of cells within inflammatory foci of the atherosclerotic plaque, suggesting a coordinated upregulation of both proteins. Thus, it is tempting to speculate that increased P21 expression is due to transactivation of the WAF1/CIP1 gene by wt p53, which accumulates after DNA damage.
However, colocalization of wt p53 and P21 does not prove a cause-and-effect relationship. In addition, the antibody used detects not only wt p53 but also the mutant forms. Nevertheless, several lines of evidence suggest a coordinated upregulation of p53 and P21 proteins. First, mutated or inactivated p53, which is the predominant form of p53 detected in proliferating tumor cells, is unable to transactivate the WAF1/CIP1 gene.2 4 Second, P21 expression was very weak in normal control tissue, which also demonstrated very weak p53 IR. Third, and most importantly, cells demonstrating simultaneous expression of p53 and P21 never exhibited positivity of the proliferation marker MIB1/Ki-67, indicating that both p53 and P21 appear to be functional in inhibiting cellular proliferation.
P21-IR colocalized with p53-IR in MPs, as well as in SMCs and ECs, in nearly all cases in a substantial number of cells. By contrast, and in accordance with the findings of previous reports,12 21 in segments of coronary arteries with diffuse intimal thickening and in the atherosclerotic tissue in a small number of cells, P21-IR did not colocalize with p53-IR, pointing out that P21 expression in medial SMCs, intimal cells, and ECs of coronary arteries, as well as in a few plaque cells, may be independent from p53 accumulation. Indeed, recently Parker et al32 demonstrated that WAF1/CIP1 expression during mouse development correlates with terminally differentiating tissue such as muscle. In addition, it has been shown that WAF1/CIP1 is induced at a late stage of differentiation, long after DNA synthesis is blocked, indicating that P21 may rather prevent division of terminally differentiated cells than participate in the induction of terminal differentiation.33 Interestingly, WAF1/CIP1 has further been identified as a gene involved in cellular senescence and is thus called senescent cell-derived inhibitor 1.34 Therefore, WAF1/CIP1 expression in coronary artery tissue without atherosclerosis, as well as in a few plaque cells, may reflect terminal differentiation or cellular senescence. In contrast to our results, Yang et al19 did not find WAF1/CIP1 expression either by immunohistochemistry or by Western blot in uninjured porcine iliofemoral arteries, whereas we found P21 expression in a small percentage of cells. This may either reflect species differences or may be due to different antibodies or staining methods used. Nevertheless, in agreement with our results, those authors found that after arterial injury, WAF1/CIP1 expression was present in the majority of SMCs.
Importantly, recent evidence suggests that adenovirus-mediated overexpression of human WAF1/CIP1 inhibits proliferation of rat vascular SMCsin vitro and in vivo by inhibiting phosphorylation of Rb, which was shown to be crucial for rat and pig vascular SMCs to enter S phase of the cell cycle and by the formation of complexes between P21 and PCNA.20 Furthermore, P21 may regulate cellular proliferation after arterial injury in porcine arteries.19 Consistent with these results, we show that MPs, SMCs, and ECs of human atherosclerotic tissue exhibiting P21-IR did not express MIB1/Ki-67, indicating that P21 may also be involved in mediating growth arrest in cells of human atherosclerotic plaques.
To avoid oversensitivity in measuring cell proliferation,35 we chose MIB1/Ki-67 as a marker of cell proliferation instead of PCNA, which is involved in DNA synthesis and whose activity is directly dependent on P21. The MIB1/Ki-67 antibody recognizes epitopes on two nuclear proteins with 345 kD and 395 kD, respectively, which are believed to sustain DNA structure during mitosis.36 Peak concentrations of the MIB1/Ki-67 antigen occur in the G2 and M phases, but it is not expressed in the G0 or early G1 phase. Thus, MIB1/Ki-67 expression correlates with DNA synthesis but does not reflect DNA repair synthesis, also detected by PCNA. MIB1/Ki-67 has a short half-life, and the level of MIB1/Ki-67 declines rapidly after mitosis.37 38 However, in accordance with the results from two recent reports by O'Brien et al35 and Pickering39 in which PCNA was used as a proliferation marker, we found similar low numbers of replicating cells.
In addition, in a recent report from Speir et al,18 immunodetectable p53 was absent in frozen sections of hypocellular primary coronary atherosclerotic plaques derived from patients undergoing atherectomy. Instead, p53 accumulation was restricted to cell-rich restenotic lesions with loosely arranged SMCs. In accordance with the observations by Speir et al,18 we found that p53 accumulation was most prominent in cell-rich areas and was very weak or absent in cell-depleted regions or in the control tissue in the absence of complicated atherosclerosis. Nevertheless, a possible source for minor discrepancies among these studies may result from the different immunohistochemical techniques used. p53 staining of tissue sections is critical and not only depends on tissue fixation but also requires thorough antigen retrieval. Stable and reproducible results using formalin-fixed and paraffin-embedded tissue were obtained in the present study applying the monoclonal antibodies DO1 and DO7 either after pressure cooking or antigen retrieval in 6 mol/L urea.
Furthermore, it has been shown that introduction of wt p53 gene by retroviral infection into cultured vascular SMCs from the rat thoracic aorta had an effect on neither the rate of cell proliferation nor the rate of apoptosis of this cell type despite high expression of the p53 protein.40 Therefore, one may assume that p53 may not influence growth of human vascular SMCs. However, recent studies indicated that p53 function in SMCs may be species specific: whereas adenoviral gene transfer of human p53 into cultured human and rabbit vascular SMCs resulted in a 95% decrease in cell number due to programmed cell death, there was only a moderate effect on cultured rat and pig vascular SMCs.41
In summary, the present study demonstrates that p53 accumulates in human atherosclerotic tissue in plaque areas with signs of chronic inflammation. In addition, p53 may be involved in the negative control of cell proliferation in advanced human atherosclerotic plaques by transcriptional activation of the WAF1/CIP1 gene. Therefore, our results suggest that chronic inflammation may contribute to the course of lesion evolution of human atherosclerotic plaques in an important manner through p53-dependent gene transactivation.
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Selected Abbreviations and Acronyms |
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) to the production of the
DNA-damaging agents O2-,
H2O2, NO, and ONOO-. DNA damage
causes ( |
Acknowledgments |
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We thank Stefanie Schöne and Gabriele von Kürthy for invaluable technical assistance in the performance of this study.
Received October 1, 1996; accepted March 20, 1997.
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References |
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