Elevated Levels of Oxidative DNA Damage and DNA Repair
Enzymes in Human Atherosclerotic Plaques
Wim Martinet, PhD; Michiel W.M. Knaapen, PhD; Guido R.Y. De Meyer, PharmD, PhD;
Arnold G. Herman, MD, PhD; Mark M. Kockx, MD, PhD
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Background—The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell
proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated
whether reactive oxygen species–induced oxidative damage to DNA occurs in human atherosclerotic plaques and
whether this is accompanied by the upregulation of DNA repair mechanisms.
Methods and Results—We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro8-oxo-2⬘-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and
nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque
including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques
contained 160⫾29 8-oxo-dG residues/105 dG versus 3⫾1 8-oxo-dG residues/105 dG in mammary arteries. Single-cell
gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei
was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (⬎90%). This suggests
that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision
repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair
pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear
antigen.
Conclusions—Our findings provide evidence that oxidative DNA damage and repair increase significantly in human
atherosclerotic plaques. (Circulation. 2002;106:927-932.)
Key Words: atherosclerosis 䡲 oxidative stress 䡲 apoptosis
N
elevated levels of oxidative DNA damage and repair in the
thoracic aorta of cholesterol-fed rabbits.10 The aim of the
present study was to investigate whether oxidative DNA
damage occurs in human atherosclerotic plaques and whether
DNA repair mechanisms are upregulated in response to DNA
damage.
umerous studies have linked excess generation of reactive oxygen species (ROS) with cellular damage and
atherogenesis.1,2 Although this notion is widely held, thorough factual evidence is lacking. ROS have been implicated
in a variety of distinct cellular processes, including initiation
of gene expression and promotion of cell proliferation,
hypertrophy, growth arrest, and/or apoptosis.1,2 On the other
hand, ROS are involved in oxidation of LDL, which is
considered a fundamental step in the initiation and progression of atherosclerosis. It is also tempting to speculate that
ROS may have some deleterious effects on DNA. Indeed,
ROS can provoke extensive oxidative DNA damage, DNA
strand breaks, and chromosomal aberrations.3 Significant
damage to DNA resulting from endogenous free radical
attack has already been suggested to contribute to the pathology of cancer4 and several neurodegenerative diseases.5,6 A
growing body of evidence indicates that oxidative DNA
damage is also a prominent feature of atherosclerotic
plaques.7–9 In light of this, we have recently described
Methods
Carotid Endarterectomy Specimens
Human carotid endarterectomy specimens (n⫽13) were obtained
from patients with a carotid stenosis ⬎70%, as demonstrated by
digital subtraction angiography and duplex ultrasonography. The
specimens were opened along their longitudinal axis. Half of the
specimen was fixed in 4% formalin within 2 minutes after surgical
removal. The other half was gently pressed against an agarose-coated
slide to examine DNA strand breaks by the alkaline single-cell gel
electrophoresis assay (see below). Tissue was frozen in liquid
nitrogen to be used as cryosections for RNA/DNA extraction and for
Western blotting. Complete longitudinal sections of formalin-fixed,
paraffin-embedded specimens contained the inner wall of the distal
Received March 28, 2002; revision received May 31, 2002; accepted May 31, 2002.
From the Division of Pharmacology, University of Antwerp, Wilrijk, Belgium (W.M., G.R.Y.D.M., A.G.H., M.M.K.); HistoGeneX, Edegem, Belgium
(M.W.M.K.); and the Cardiovascular Translational Research Institute Middelheim Antwerp (CATRIMA), Antwerp, Belgium (M.M.K.).
Correspondence to Dr Mark M. Kockx, Department of Pathology, AZ Middelheim, Lindendreef 1, B-2020 Antwerp, Belgium. E-mail
[email protected]
© 2002 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000026393.47805.21
927
928
Circulation
August 20, 2002
Patient Characteristics
Carotid Endarterectomy
Specimens
(n⫽13)
Mammary
Arteries
(n⫽8)
Age, y
72⫾1*
61⫾5
Women
3
2
Active smokers
7
3
Total cholesterol, mg/dL
226⫾10
266⫾23
LDL, mg/dL
147⫾10
173⫾21
Hypertension
10
4
6
3
Diabetes
Medication
subsequently homogenized.10 Immunoblot assays were performed as
previously described.10
Quantitative Measurement of
7,8-Dihydro-8-Oxo-2ⴕ-Deoxyguanosine
Genomic DNA was extracted from mammary arteries (n⫽5) and
carotid endarterectomy specimens (n⫽5) using the Wizard Genomic
DNA Purification Kit (Promega). DNA was digested with nuclease
P1, phosphodiesterase I, and alkaline phosphatase to yield free
deoxynucleosides.14 To determine the amount of deoxyguanosine
(dG), hydrolyzed samples and dG standards were introduced onto a
Prodigy ODS(3) HPLC column (150⫻1.0 mm). Deoxyguanosine
was detected at 254 nm (Waters CapLC system). The amount of
7,8-dihydro-8-oxo-2⬘-deoxyguanosine (8-oxo-dG) was measured by
using competitive ELISA (Highly Sensitive 8-OHdG ELISA Kit,
Japan Institute for the Control of Aging). Finally, the ratio of
8-oxo-dG/105 dG was calculated.
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NO donor
6
3
␤-Blocker
9
4
Ca antagonist
7
4
Alkaline Single-Cell Gel Electrophoresis Assay
Acetylsalicylic acid
7
5
The alkaline comet assay or single-cell gel electrophoresis assay was
performed as described.10 Peripheral blood monocytes were used as
negative controls.
Anticoagulant
2
1
Diuretic
6
2
Antidiabetic
5
2
Statistical Analysis
Statin
1†
4
ACE inhibitor
2
3
Age, total, and LDL cholesterol, as well as comet assay parameters
and 8-oxo-dG values were compared with the unpaired Student’s t
test. Other patient characteristics were evaluated by the Fisher’s
exact test. A value of P⬍0.05 was considered significant.
Data are expressed as number of specimens or mean⫾SEM.
*P⫽0.01, †P⫽0.047 vs mammary artery.
Results
common carotid artery, the proximal part of the external carotid
artery, and the carotid sinus.11 Nonatherosclerotic mammary arteries
(n⫽8) obtained during bypass surgery were used as negative controls
and were manipulated similarly. Characteristics of the study and of
the control group are summarized in the Table. Names of patients
and specimens were irreversibly encrypted. The local ethics committee approved this protocol.
Antibodies
Plaque Composition
The carotid endarterectomy specimens were in the form of
complete casts of carotid bifurcations. Thin fibrous cap
atheromata alternated with other stages of atherosclerosis
(fibrous cap atheromata and intimal xanthomata) in the same
specimen, in accordance with the adapted American Heart
Association classification scheme, as modified by Virmani et
The following mouse monoclonal antibodies were used: antiPARP-1 (clone C2-10; PharMingen), anti-7,8-dihydro-8-oxo-2⬘deoxyguanosine (clone N45.1; Japan Institute for the Control of
Aging), anti-p53 (clone DO-7), anti-CD68 (clone PG-M1), antiproliferating cell nuclear antigen (PCNA; clone PC10; DAKO),
anti-phospho-p53 (S392; clone SP2/0; Novocastra), anti-DNA-PK
(clone 42-psc), anti-DNA polymerase ␤ (clone 18S; Neomarkers),
and anti-␤-actin (clone AC-15), anti-smooth muscle cell actin (clone
1A4), and anti-SC-35 (clone SC-35; Sigma).
Polyclonal rabbit antibodies included anti-hNTH1, anti-MTH1,
and anti-hOgg1 from Novus Biologicals; anti-phospho-p53 (S15;
New England Biolabs); anti-active caspase-3 (PharMingen); antiinducible nitric oxide synthase (BIOMOL); anti-nitrotyrosine (Upstate Biotechnology); and anti-Ref-1 (C-20; Santa Cruz Biotechnology). Goat anti-mouse and sheep anti-rabbit peroxidase-conjugated
secondary antibodies were purchased from Jackson and DAKO,
respectively.
Immunohistochemistry and DNA In Situ End Labeling
The immunohistochemical reactions were performed by an indirect
peroxidase antibody conjugate method.12 For the detection of oligonucleosomal DNA cleavage, a stringent terminal deoxynucleotidyl
transferase end-labeling (TUNEL) technique was used.13 TUNEL
staining was combined with an immunohistochemical stain for
SC-35 to avoid aspecific labeling.13
Protein Isolation and Immunoblot Assays
Four human carotid endarterectomy specimens and 4 control samples
were pooled to reduce specimen-to-specimen variation and were
Figure 1. Immunohistochemical markers for oxidative stress and
apoptotic cell death in advanced plaques of carotid endarterectomy specimens. The regions shown are macrophage-rich and
located around the necrotic core (nc). A, Inducible nitric oxide
synthase; B, nitrotyrosine; C, cleaved caspase-3 (arrows); and
D, splicing factor SC-35 (blue) combined with TUNEL labeling
(brown). Arrowheads indicate one apoptotic body in the middle
of SC-35 positive cells. Scale bar⫽50 ␮m.
Martinet et al
DNA Damage and Repair in Atherosclerosis
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Figure 3. Quantitative measurement of 8-oxo-dG in carotid endarterectomy specimens (plaque; n⫽5) and mammary arteries
(control; n⫽5) by competitive ELISA. Test samples and standards were analyzed in triplicate. Total dG amounts in each
sample were determined by high performance liquid chromatography. **P⬍0.01 vs carotid endarterectomy specimens.
Figure 2. Localization of DNA damage marker 8-oxo-dG, Ref-1,
and DNA-PK in SMCs (␣-SMC actin–positive cells) and macrophages (CD68-positive cells) of advanced human atherosclerotic
plaques. A, Double staining for 8-oxo-dG (brown) and ␣-SMC
actin (blue). Besides SMCs, other cell types, including endothelial cells (arrowheads), stained for 8-oxo-dG. B, Double staining
for Ref-1 (brown) and ␣-SMC actin (blue). C, Double staining for
DNA-PK (brown) and ␣-SMC actin (blue). D, Double staining for
8-oxo-dG (brown) and CD68 (blue). E, Double staining for Ref-1
(brown) and CD68 (blue). F, Double staining for DNA-PK (brown)
and CD68 (blue). Scale bar⫽100 ␮m.
al.15 There were numerous foam cells of macrophage origin
(CD68-positive cells) present around the necrotic core of thin
fibrous cap atheromata. ␣-Smooth muscle cell actin immunoreactive cells were nearly absent in these regions (⬍0.2%
of the total area). Some foam cells surrounding the necrotic
core were strongly immunoreactive against inducible nitric
oxide synthase (Figure 1A). Nitrotyrosine was also detected
in this region (Figure 1B). Insoluble multilaminated material
composed of oxidized lipids and proteins was present in the
cytoplasm of many macrophages. We found some larger
extracellular lipid deposits in the necrotic core.
Oxidative DNA Modifications
Smooth muscle cells (SMCs) of mammary arteries showed
weak immunoreactivity for the oxidative DNA damage
marker 8-oxo-dG. In contrast, strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque (⬎90% of the
total area), including macrophages, SMCs, and endothelial
cells (Figures 2A and 2D). 8-oxo-dG immunoreactivity was
localized predominantly in the nucleus of the labeled cells.
Adjacent fragments of the inner media stained minimally or
did not stain at all. Plaque cells showed an ⬇55-fold higher
level of 8-oxo-dG in comparison with mammary arteries
(160⫾29 versus 3⫾1 8-oxo-dG residues/105 dG; Figure 3).
The majority of 8-oxo-dG immunoreactive cells in the plaque
(⬎95%) showed immunoreactivity for splicing factor SC-35
but were negative for active caspase-3 (Figure 1C) and were
not labeled by the TUNEL technique (Figure 1D). Immunoreactivity for 8-oxo-dG did not differ in intimal xanthomata
from thin fibrous cap atheromata (data not shown).
DNA Strand Breaks
Alkaline single cell gel electrophoresis revealed that the
number of DNA strand breaks was significantly higher in
cells of the carotid endarterectomy specimens compared with
cells derived from mammary arteries (Figures 4A and 4B).
Most cells in the plaque (⬎90%) contained DNA strand
breaks with varying outcomes (Figures 4A and 4B). However, only a minority of cells (1% to 2%) showed TUNEL
reactivity. Cells containing a massive amount of DNA strand
breaks were localized predominantly in regions of the plaque
that contained large amounts of oxidized lipid deposits
(Figure 4E). In mammary arteries (Figures 4A and 4C) and
peripheral blood monocytes (Figure 4D), DNA strand breaks
were scarce or completely absent.
Expression of DNA Repair Enzymes
Several proteins involved in DNA repair were upregulated in
plaques of carotid endarterectomy specimens when compared
with nonatherosclerotic vessels. Western blots revealed an
increased expression of p53, p53 phosphorylated at Ser15 and
Ser392, DNA-PK, redox factor/AP endonuclease Ref-1, and
PARP-1, but a constitutive expression of DNA polymerase ␤,
the N-glycosylases hOgg1 and hNTH1, and 8-oxoGTPase
(MTH1; Figure 5). Western blots were confirmed by immunohistochemistry. Strong nuclear immunoreactivity for Ref-1
and DNA-PK was present in the entire plaque (⬎90% of the
total area) in both macrophages and SMCs (Figures 2B, 2C,
2E, and 2F). The carotid artery endothelium and endothelial
cells from microvessels were negative for Ref-1 and
DNA-PK or showed occasional staining. SMCs from the
media adjacent to the plaque and from mammary arteries
stained minimally. In contrast with Ref-1 and DNA-PK
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Figure 4. Detection of DNA strand breaks in individual cells of
nonatherosclerotic mammary arteries versus plaque cells from
carotid endarterectomy specimens. A, Freshly isolated mammary arteries (control, n⫽8) and carotid endarterectomy specimens (plaque, n⫽13) were gently pressed against an agarosecoated microscope slide. Cells that adhered to the slide were
immediately covered with a second layer of low–melting-point
agarose and analyzed using the alkaline comet assay. Slides
contained both macrophages and SMCs in a ratio similar to the
one found in tissue sections. Tail length (TL) and tail moment
(tail length⫻%DNA in tail) increased significantly in all atheromas studied. B and C, Digitized comet images of cells isolated
from atherosclerotic plaques of the carotid artery (B) vs mammary arteries (C). D, Comet image of peripheral blood monocytes. E, Digitized image of a comet slide containing plaque
cells with an extensive amount of DNA strand breaks surrounded by autofluorescent lipid deposits (blue). The results are
expressed as mean⫾SEM. ***P⬍0.001 vs carotid endarterectomy specimens.
immunolabeling, only a subpopulation of cells in the plaque
stained for p53, phospho-p53, or PARP-1 (Figures 6A and
6B). Upregulation of p53 and PARP-1 occurred predominantly in the macrophage population of the plaque but was
undetectable in the adjacent media and mammary arteries.
Overexpression of DNA repair enzymes was associated with
elevated levels of PCNA (Figure 6C).
Immunoreactivity for Ref-1 and DNA-PK did not differ in
intimal xanthomata from thin fibrous cap atheromata. However, overexpression of PARP-1, p53, and PCNA was found
predominantly in macrophages around the necrotic core of
thin fibrous cap atheromata (Figure 6).
Figure 5. Western blot analysis of DNA repair enzymes in
pooled extracts from non-atherosclerotic control vessels (n⫽4;
lane 1) and from atherosclerotic plaques of the carotid artery
(n⫽4; lane 2). Experiments were done in duplicate. A representative immunoblot for each DNA repair enzyme is shown.
Discussion
In the present study, elevated levels of oxidative DNA
damage were found in human atherosclerotic plaques. This
finding supports the current assumption that oxidative tissue
injury is aggravated during plaque formation, most likely as a
result of oxidative stress. 8-oxo-dG is one of the most
abundant oxidative lesions in mammalian DNA.3 We observed an increased amount of 8-oxo-dG in human plaques
compared with the underlying media or nonatherosclerotic
mammary arteries. Strong immunoreactivity for 8-oxo-dG
was found in all cell types of the plaque, including macrophages, SMCs, and endothelial cells. Interestingly, 8-oxo-dG
is strongly mutagenic and leads to an increased frequency of
spontaneous G · C3 A · T transversion mutations in repairdeficient cells.4
Previous reports showed an increased mutation rate and
widespread microsatellite instability in human atherosclerotic
lesions.16,17 These may be related to the accumulation of
oxidized base residues. In addition, ROS may also generate
DNA strand interruptions.3 Single-cell gel electrophoresis
revealed that the number of DNA strand breaks was raised
significantly in the plaque compared with nonatherosclerotic
vessels. Because DNA strand breaks are fragile sites, they can
attract undesirable recombination events. Elevated levels of
Martinet et al
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Figure 6. Immunohistochemical stains of phospho-p53 (S392;
A), PARP-1 (B), and PCNA (C) in cells around the necrotic core
(nc) of carotid endarterectomy specimens. Scale bar⫽50 ␮m.
DNA strand breaks in the plaque may therefore account for
the chromosomal rearrangements described previously.18 It is
important to note that extensive chromosome abnormalities in
SMCs can be associated with transformation events (eg,
intimal uterine leiomyosarcomas).19 Atherosclerotic plaques
contain a large monoclonal population of SMCs20,21 and thus
may be regarded as monoclonal neoplasms of the arterial
wall. This is consistent with the findings that DNA extracted
from atherosclerotic plaques had a transforming ability when
transfected into NIH3T3 cells22 and that SMCs cultured from
plaque tissue retained transforming potential throughout
many cell passages.23 Monoclonality of plaque SMCs could
be caused by the expansion of a large, preexisting patch of
SMCs.20,21 An alternative mechanism for monoclonality that
corresponds with increased levels of oxidative DNA damage
is the creation of a new lineage of SMCs. This may occur
either through genetic mutation or through epigenetic
DNA Damage and Repair in Atherosclerosis
931
changes. If true, somatic mutations of SMCs may play an
important role in the pathogenesis of atherosclerotic plaques.
Severe oxidative DNA damage can induce apoptosis by
activating a variety of proapoptotic proteins. In the present
study, atherosclerotic plaques were characterized by the
appearance of apoptotic cell death, but the overall number of
TUNEL-positive nuclei was low (1% to 2%) and did not
correlate with the amount of 8-oxo-dG immunoreactive cells
(⬎90%). Therefore, we assume that cells in the plaque
contain a sublethal level of oxidative DNA modifications
and/or strand breaks and thus do not undergo the execution
phase of apoptosis. On the contrary, most cells surrounding
the necrotic core are labeled for splicing factor SC-35,
suggesting that these cells are viable and metabolically active.
However, we cannot rule out the possibility that DNA lesions
accumulate with time and eventually induce plaque cells to
undergo apoptotic cell death.24
DNA damage cannot be tolerated in mammals if left
unrepaired. Therefore, cells have developed many defense
systems to remove DNA damage. A major repair mechanism
for oxidative DNA damage, including 8-oxo-dG and DNA
strand breaks, is the base excision repair pathway.25,26 In the
present study, Western blot analyses and immunohistochemical stains indicated that there are several DNA repair
enzymes that are upregulated in the plaque, either those
involved in base excision repair (Ref-1, PARP-1) or in
nonspecific repair pathways (p53, DNA-PK). Furthermore,
we demonstrated that plaque p53 contains at least 2 phosphorylated residues, Ser15 and Ser392, which stabilize and
activate the protein in the plaque. Activation of p53 by
phosphorylation is an important regulatory event in the
arterial vessel wall because p53 deficiency, specifically in
macrophages, leads to a significant doubling of atherosclerotic lesion size.27 Overexpression of some base excision
repair enzymes does not always lead to increased protection
against DNA-damaging agents.26 For this reason, it remains
to be determined whether the capacity of the vascular cells to
repair endogenous lesions after upregulation of specific repair
enzymatic activity is increased. A nonphysiological level of
certain repair proteins may even prove detrimental to mammalian cells.26 This is particularly clear for PARP-1, whose
imbalanced production leads to necrotic cell death as a result
of NAD⫹ and ATP depletion.28 In this case, PARP-1 overexpression may also contribute to the formation of a necrotic
core, which is the hallmark of an atherosclerotic lesion.
Immunohistochemical analysis revealed the abundant expression of both DNA-PK and Ref-1 in the entire plaque.
DNA-PK is a serine/threonine kinase that serves as an
essential upstream activator of p53.29 Nevertheless, the significance of the upregulation of Ref-1 in atherosclerotic
plaques is unclear and remains to be elucidated. Herring et
al30 could not demonstrate increased resistance to DNA
damage in cells overexpressing Ref-1, indicating that increased levels of Ref-1 cannot improve the repair efficiency.
It is important to note that Ref-1 is also a redox factor and,
thus, a widespread regulator of transcription factors that
control multiple events in the life cycle of various cell types.
Downregulation of Ref-1 expression after induction of hypoxia into endothelial cells precedes DNA fragmentation and
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August 20, 2002
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apoptotic cell death. This suggests that Ref-1 is a crucial
enzyme that determines cell fate.31
Overexpression of DNA repair enzymes was associated
with elevated levels of PCNA. Because this protein is
essential for DNA replication, many groups have used it as an
important marker for cell proliferation. However, recent
evidence suggests additional roles for PCNA in chromatin
assembly, RNA transcription, and DNA repair.32 We assumed
that high levels of oxidative stress in the plaque could trigger
a unconventional repair pathway that modulates PCNA expression, given that mammalian cell extracts can repair
oxidative DNA modifications by an alternative PCNAdependent base excision repair pathway.33 Similar conclusions were reported for macrophages treated with oxidized
LDL, 34 as well as for cardiac myocytes in dilated
cardiomyopathy.35
In summary, our findings show that oxidative DNA damage and repair increase significantly in human atherosclerotic
plaques. There is currently a lack of essential information
regarding the functional consequences of damaged DNA in
human plaques, besides the potential role in carcinogenesis
and cell death. Of particular interest, however, is the finding
that several factors associated with the presence of DNA
adducts in aorta SMCs (ie, age, smoking habits, blood lipids,
and blood pressure) are also known to play a major role in
atherogenesis.7 In this context, detection of DNA damage
may provide a useful biomarker not only in carcinogenesis
but also in atherogenesis.
Acknowledgments
Research was supported by the Bekales Foundation and by the Fund
for Scientific Research-Flanders (project No. 1.5.206.00 and
G.0080.98). Dr Kockx is holder of a fund for fundamental clinical
research of the Fund for Scientific Research-Flanders. The authors
are indebted to Martine De Bie, Jeff Thielemans, and Walter Van
Dongen for excellent technical assistance and to Julia Zhyzko for
critical reading of the manuscript.
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Circulation. 2002;106:927-932; originally published online July 22, 2002;
doi: 10.1161/01.CIR.0000026393.47805.21
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