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Regular Articles |
From the Departments of Pathology,*
Medicine
(Cardiology),
and Molecular
Biotechnology,
University of Washington,
Seattle, Washington
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor
2 (TNF-R2) "death" receptor family.7
Transmission of a death signal via this cascade, even when the
receptors are ligated, depends on endogenous regulators of proteolytic
activation. For example, viral Fas-associated death domain-like
IL-1ß-converting enzyme (FLICE) inhibitory protein (v-FLIP), an
anti-apoptotic viral caspase antagonist, has homology to the death
effector domains (DEDs) in the prodomains of caspases 8 and
10.8
Like DED constructs of caspase 8, the viral FLIPs act
as dominant negatives for Fas-associating protein with death domain
(FADD)-mediated death, apparently by competitive binding of the
predomains of caspase 8 or 10 and thus the blocking of Fas-mediated
apoptosis.8 Recently, we and others9-16 have identified a mammalian molecule that is homologous to caspase 8. We named this molecule MACH-related inducer of toxicity (MRIT)13; however, it has also been called cellular FLIP (c-FLIP),9 Casper,10 FADD-like antiaptotic molecule 1 (FLAME-1),11 caspase homolog (CASH),12 I-FLICE,14 CLARP,15 and usurpin.16 For simplicity, we will use the term c-FLIP in this paper. c-FLIP is different from v-FLIP in containing a full-length sequence that includes, although in a distinct form, the active site of caspase 8. Interestingly, c-FLIP is very similar in structure to a dominant negative construct of caspase 8,9-16 and this has led to the presumption that c-FLIP is also an antiapoptotic molecule. In support of this hypothesis, studies in T cells show that c-FLIP levels are controlled during activation and go down when activated T cells become susceptible to Fas-mediated death,9 and the ability of Fas to kill T cells can be blocked if c-FLIP levels are restored using a chimeric form of c-FLIP ligated to viral membrane transport protein.17 Thus, although early studies, including our own,13 have shown that overexpressed c-FLIP induces cell death, the preponderance of the evidence is that this molecule is a natural inhibitor of death, mediated by those death receptors that are able to activate caspase 8 by Fas signaling or by other receptors. Fas is ubiquitously expressed in various tissues including the vessel wall.18,19 To explore possible functions of c-FLIP in atherosclerosis and vascular responses to injury, we have used in situ hybridization and immunohistochemistry to analyze the pattern of c-FLIP expression in human coronary arteries and in injured rat common carotid arteries. We find that c-FLIP is widely expressed in the normal vessel wall. c-FLIP is, moreover, up-regulated in rat neointimal lesions, as well as in the intima of nonatherosclerotic human coronary arteries. Double labeling for c-FLIP and terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) in the injured rat common carotid artery shows that loss of c-FLIP in the first few days after injury is correlated with the appearance of TUNEL-positive cells. For human atherosclerotic plaques, the majority of TUNEL-positive cells lack detectable c-FLIP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human coronary arteries were obtained from hearts removed from
patients with end-stage ischemic cardiomyopathy
(n = 6), idiopathic dilated cardiomyopathy
(n = 5), or congenital heart disease
(n = 2), a total of 9 men and 4 women, 26 to 63
years of age (see Table 2
below). Twenty-one coronary artery
segments were obtained from the hearts of these 13 subjects.
These segments were classified into two groups by classic histological
criteria: one comprising artery segments that demonstrated
nonatherosclerotic diffuse intimal thickening (DIT; n =
9) and the other comprising segments that demonstrated atherosclerotic
plaques (n = 12). Collection and use of these
tissues were approved by the University of Washington Human Subjects
Review Committee.
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The rat balloon injury model was described previously.20 Briefly, 3-month male Sprague-Dawley rats (around 450 g; Zivic Miller, Zelienople, PA) were used for these studies (n = 6 for each time point). All animals were given rat chow and water ad libitum. The animals were anesthetized with ketamine HCl (50 mg/kg), xylazine (5 mg/kg), and acepromazine (1 mg/kg) administered intramuscularly in the back. Osmotic minipumps containing bromodeoxy uridine (BrdU; 30 mg/ml) were implanted to measure DNA synthesis as described previously.20 The left common carotid artery was de-endothelialized by passage of a 2F Fogarty embolectomy catheter (Edwards Laboratories, Santa Ana, CA) that was inserted into the external carotid artery, advanced into the abdominal aorta, inflated, and withdrawn. This procedure was performed three times to ensure complete endothelial denudation of the common carotid artery. The external carotid was tied off, and blood flow was restored through the internal carotid artery. For morphological analysis and protein extraction, the rats were sacrificed at 2, 7, 14, 28, or 42 days after the injury by an overdose injection of sodium pentobarbital (intravenous Nembutal, Abbott Laboratories, North Chicago, IL). The animals were prepared by perfusion/fixation using 4% paraformaldehyde as described previously.20 Ten minutes before death by pentobarbital overdose, these rats received an intravenous injection of Evans blue (200 µL of 5% solution; Sigma Chemical Co., St. Louis, MO) to mark the de-endothelialized area. The carotid arteries were briefly flushed with ice-cold lactated Ringer’s solution (Baxter Healthcare Co.) at physiological pressure to remove blood, and the carotid arteries were excised. The adventitia was stripped (at the external elastic lamina) from the media. All specimens then were snap-frozen in liquid nitrogen and stored at -80°C. The Animal Care Committee of the University of Washington approved all procedures.
Cell Culture
Culture of coronary plaque smooth muscle cells (SMCs), normal aortic and neonatal medial SMCs, human umbilical endothelial cells and human microvascular endothelial cell-1 cells has been described previously.21 SMCs were grown in Waymouth’s media supplemented with 20% fetal bovine serum (FBS) together with 1x antimycotic and antibiotic agents.
c-FLIP Antibody Preparation and Characterization
A peptide of the N-terminal DED of c-FLIP corresponding to amino
acids 10 to 27 (sequence EEALDTDEKEMLIFLCRD) was used to generate an
affinity-purified rabbit polyclonal antibody, termed MAG1. To examine
the c-FLIP specificity of MAG1, we used an in vitro
transcription and translation (Promega-TNT) system
in conjunction with mammalian cell transfections. Full-length c-FLIP
was cloned into the PCDNA 3.1 vector with an anti-express fusion
epitope and then translated in vitro followed by Western blotting and
detection with either an express antibody (Invitrogen, Carlsbad, CA) or
MAG1 (Figure 1, A and B)
. Baby hamster
kidney-21 cells that had been stably transfected with CrmA were
transiently transfected with a c-FLIP expression vector that encodes
c-FLIPL and c-FLIPS, using Fugene 6 (Roche
Biochemicals, Indianapolis, IN) or Lipofectamine (GIBCO/BRL, Rockville,
MD) as described previously13
(see Figure 1
).
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The primary antibodies used for immunohistochemical analyses
include a polyclonal antibody against c-FLIP (MAG1, 1:100 dilution), a
monoclonal antibody (mAb) recognizing endothelium (CD31; DAKO,
Carpinteria, CA; 1:1000), an mAb-recognizing macrophage (CD68; DAKO;
1:1000), and an mAb recognizing SMC -actin (Boehringer Mannheim,
Indianapolis, IN; 1:2000). All antibodies were diluted in
phosphate-buffered saline (PBS) containing 10% normal goat serum and
1% bovine serum albumin and incubated with the tissue sections for 1
hour at room temperature. Negative controls included substitution of
the primary antibody with nonimmune immunoglobulin G (IgG) of the same
class. In addition, competitive inhibition of the MAG1 antibody with
the immunizing peptide was performed by preincubating the two for 30
minutes to 1 hour at 37°C. All immunohistochemical- and TUNEL-stained
sections were scored by two observers who were blinded for the specimen
identity and the antibody used. The method for TUNEL with alkaline
phosphatase has been described previously.1
TUNEL analyses
were always performed, including human tonsil and rat thymus as
positive controls of human and rat samples, respectively, to provide
internal consistency. Cell proliferation was measured by BrdU infusion
and immunohistochemical detection as previously reported.20
The TUNEL-labeling and -proliferating cells for each high-power field
were shown as a percentage of total cells (i.e., TUNEL- or
BrdU-labeling index = TUNEL- or BrdU-positive cells/total cells
per high-power field x 100 (n = 6) injured
rat common carotid arteries per time point). Nonspecific cytoplasmic
TUNEL staining without nuclear involvement was considered negative. For
the human coronary arteries (n = 21), both the
TUNEL-labeling index and c-FLIP intensity in the intimal region, medial
region, and endothelium were simultaneously examined on double-stained
slides. At least four sections from each specimen were examined. Ten
random fields per section were examined at high magnification (x100).
A total of 200 to 400 cells from each section were counted at high
power (x100). The results of immunohistochemical studies for
c-FLIP expression were assessed by scoring the intensity for a given
antigenic determinant, using a scale of 0 = negative, 1+ = weak,
2+ = moderate, and 3+ = strong. The median was then assigned for all
individual scores.
Double Staining
The first immunostaining using MAG1 antibody was developed with 3,3'-diaminobenzidine (Sigma), which produced a brown reaction product. The second sequence of staining was done on the same sections for TUNEL, with an avidin-alkaline phosphatase-substrate system and Vector Red (Vector ABC Kit, Vector Laboratories), which produced a red reaction product.
Preparation of Riboprobes
Full-length human c-FLIP complementary DNA (cDNA) (1.4 kb) was
subcloned into pBluescript II (Stratagene, La Jolla, CA) as described
previously.13
To generate antisense and sense RNA probes
for in situ hybridization, plasmids were linearized with
XbaI and KpnI, respectively. The plasmids were then in vitro
transcribed with 35S-labeled -thio UTP (New England
Nuclear-Dupont, Boston, MA) by a modified method of Wilcox et
al.22
To improve penetration into sections during
hybridization, transcripts were shortened by alkaline hydrolysis to a
calculated average length of 250 bases.
In Situ Hybridization
In situ hybridizations was performed with 35S-labeled riboprobes on 5-µm-thick, deparaffinized sections of 4% paraformaldehyde-fixed tissue. Riboprobes were separated from unincorporated counts by passage over G-50 NICK columns (Pharmacia, Piscataway, NJ). The peak radioactive fractions were treated with phenol/chloroform, and the aqueous phase was precipitated with ethanol and resuspended to 300,000 cpm/ml in TE buffer (10 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid, pH 7.5). One microliter of the riboprobe was mixed in 50 µl hybridization buffer and applied to each section, and hybridizations were performed at 55°C overnight. Washes included treatment with RNase A (20 mg/ml, Sigma) for 30 minutes at 37°C and a stringent wash in 0.1 x standard saline solution at 55°C for 2 hours. After dehydration in graded alchols and air-drying, slides were dipped in NTB2 emulsion (Eastman Kodak Co., Rochester, NY), exposed in the dark at 4°C for 14 days, and developed as described previously.22 Hematoxylin was used as a counterstain.
Western Blotting
c-FLIP protein was detected by using whole-cell lysates and tissues including human vascular plaque and uninjured or injured rat common carotid arteries at several time points. The samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to poly(vinylidene difluoride) membranes, and processed as described previously.23 The protein concentration of the lysate was determined using the Pierce micro BCA reagents (Rockland, IL). The MAG1 antibody was used at a dilution of 1:100 and a final concentration of 1.4 µg/ml of peptide affinity-purified IgG. The secondary antibody, an anti-rabbit IgG labeled with horseradish peroxidase, was used at a dilution of 1:30,000 (Biorad Laboratories, Hercules, CA). The signals was visualized by enhanced chemiluminescence (ECL; Amersham). Intensity of the signal was measured by densitometric analysis of autoradiograms. X-ray films were scanned with a transmission scanner (UMAX UC1260, UMAX Data System Inc.) using Adobe Photoshop software (version 3.0; Adobe), and the transmission values were converted to values of optimal density by NIH Image software (version 1.59; National Institutes of Health, Bethesda, MD). The profile of each band was plotted by using NIH Image, and the densitometric intensity corresponding to each band was measured as an intensity value. c-FLIP protein intensity was normalized by using calponin expression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although a number of antibodies to c-FLIP are available
commercially, we wished to be sure of the specificity of our MAG1.
Expression data and protein data show that the gene can be transcribed
as at least two forms; c-FLIPS contains only the regions
homologous with the viral protein and c-FLIPL is a
full-length transcript.24
We first tested the specificity
of the MAG1 antibody. This antibody is expected to recognize
c-FLIPL and c-FLIPS proteins, because these
isoforms are identical in their amino acid composition at the
N-terminal death effector domain (DED).13
Using both
in vitro transcription-translation and transfection
analyses, we found that the MAG1 antibody specifically recognized a
54-kD protein (Figure 1, B and D)
but not the LacZ gene product
(Figure 1, A and C)
, representing the correct size of
c-FLIPL in both assays. As a positive control for protein
expression, we used express epitope-tagged LacZ and c-FLIP constructs
in both assays. As shown in Figure 1
(A and C), both LacZ and c-FLIP
proteins were detected by antiexpress antibody. The MAG1 antibody also
recognized c-FLIPS in transfected mammalian cells (Figure 1E)
.
We next used the MAG1 antibody to test the expression of endogenous
c-FLIP in total cell lysates from different SMCs, endothelial cells,
and THP1, a monocytic cell line. As shown in Figure 2A, a
54-kD
c-FLIPL protein was detected by MAG1
antibody in cultured plaque SMCs (lane 1), neonatal VSMCs (lane
2), medial VSMCs (lane 3), human microvascular endothelial cell-1 (lane
6), human umbilical endothelial cells (lane 7), and THP1 (lane 8). On
the other hand, c-FLIP protein was not detectable in astrocyte and
mesangial cells (lanes 4 and 5, respectively). Additionally,
54-kD c-FLIPL protein was also detectable in human coronary
plaque lysates (Figure 2B)
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We next examined c-FLIP expression in a rat vascular balloon
injury model. In uninjured rat carotid arteries, strong
immunoreactivity to c-FLIP protein was detected in the medial SMCs
(Figure 3A)
. However, 48 hours after
balloon injury, c-FLIP immunoreactivity was greatly diminished (Figure 3E)
. At this time point, TUNEL-positive cells were abundant in the
media (Figure 3, F and G)
. Detailed analysis by high-power
magnification of carotid arteries from the 48-hour time point doubly
stained for c-FLIP and TUNEL revealed that TUNEL-positive cells lacked
c-FLIP protein (Figure 3, U–W
, short arrows) and c-FLIP-positive cells
(Figure 3, U–W
, long arrows) also lacked TUNEL positivity. The counts
at 48 hours showed that only 0.8% of cells with c-FLIP were TUNEL
positive in comparison with 39.8% of cells without c-FLIP (Table 1)
. Medial SMCs at day 7 stained weakly
for c-FLIP, and high levels of TUNEL-positive cells could be detected
in the media at this stage (Figure 3, I–K
; Figure 4B
). By day 14,
medial SMCs remained to stain weakly for c-FLIP, although the frequency
of TUNEL positivity is low. When c-FLIP expression was regained in the
media by week 4, no TUNEL-positive cells were found (Figure 3, Q–S)
.
Interestingly, neointima was strongly immunoreactive to c-FLIP at day 7
after the injury and remained strongly immunoreactive until 4 to 6
weeks (Figure 3Q
; results not shown). On day 7, a number of
TUNEL-positive cells could be detected in the intima, and 24.3%
TUNEL-positive cells were also found to be c-FLIP positive (Figure 3, I–K
; Table 1
). However, by day 14 the majority of c-FLIP-positive
cells in the neointima were no longer TUNEL positive (Figure 3, N and Q
; Table 1
). Overall, double staining for c-FLIP and TUNEL in the
injured rat common carotid artery suggested a complex regulation of
c-FLIP in TUNEL-positive and -negative cells. In addition, c-FLIP
expression is down-regulated initially in the media but returns to
normal levels by 28 days after the injury.
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-FLIP protein was down-regulated at 24 and 48 hours after balloon
injury. However, c-FLIP expression was increased at later time points
(from day 4 to 6 weeks; Figure 4A
). These results are in agreement with
immunohistochemical studies of rat carotid arteries after balloon
angioplasty (Figure 3)
. In addition, the results from the Western
analysis confirmed that immunoreactivity observed in the vascular cells
was due to the expression of a 54-kD cFLIPL protein. c-FLIP
protein in the vessel wall after balloon angioplasty was quantified by
densitometric analysis of c-FLIP expression from two separate
experiments, and band intensities were normalized to calponin
expression (Figure 4A)
. These results suggest that c-FLIP is
down-regulated at the 24-h time point after balloon injury and
upregulated in the intima and media by 4 weeks. c-FLIP Expression in Human Coronary Artery and Apoptosis
We next examined the distribution of c-FLIP protein and mRNA in
nonatherosclerotic and atherosclerotic human coronary arteries by
immunohistochemistry and by in situ hybridization. Strong
immunoreactivity to c-FLIP protein (also termed DIT) was observed in
all nonatherosclerotic lesions, also termed. As shown in Figure 5
, strong c-FLIP immunoreactivity was
seen in intimal and medial SMCs (Figure 5, A and H)
and in endothelial
cells (Figure 5, A and D)
. This immunoreactivity was specific because
preincubating the MAG1 antibody with the neutralizing MAG1 peptide
completely abolished the immunoreactivity in human coronary arteries
(Figure 5B)
. Immunostaining serial sections from corresponding human
coronary arteries with smooth muscle -actin antibody and CD31
antibody revealed that these c-FLIP-positive cells are SMCs and
endothelial cells, respectively (Figure 5, C, E and I)
. Similarly,
in situ hybridization with an antisense c-FLIP complementary
RNA (cRNA; Figure 5, F and J
) revealed that c-FLIP messenger RNA (mRNA)
was detectable in SMCs and in endothelial cells. No significant signal
was detected above the background when the control, sense c-FLIP cRNA,
was used to hybridize in nonatherosclerotic coronary arteries (Figure 5, G and K)
. Interestingly, although c-FLIP was widely expressed in all
nonatherosclerotic coronary arteries, TUNEL staining was almost
exclusively not detectable in these regions (Table 2)
.
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, medial smooth
muscle layer (Figure 6K)
, and in plaque macrophages (Figure 6, C and F)
. The second type of atherosclerotic plaque, however, showed weak or
undetectable immunoreactivity to c-FLIP in the luminal endothelium
(Figure 7C)
, in the medial smooth muscle
layer (Figure 7F)
, and in the atherosclerotic plaque SMCs (Figure 7I)
.
c-FLIP immunoreactivity was completely abolished when the neutralizing
peptide was added in all cases (Figure 6, B, D, I, and L
; Figure 7, B, D, G, and J
).
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. As shown in
Figure 8A
, TUNEL-positive endothelial
cells (arrows) did not express detectable c-FLIP immunoreactivity.
Similarly, neither TUNEL-positive macrophages (Figure 8, C and D)
nor
SMCs (Figure 8, F and G)
express detectable c-FLIP. Detailed analysis
of human coronary arteries (atheroma and DIT; n = 21;
Table 2
) for possible correlation between TUNEL positivity and c-FLIP
intensity was further carried out by counting TUNEL-positive cells in
the intima, media, and endothelial lining and simultaneously grading
c-FLIP intensity in the double-stained slides. As shown in Figure 9
and Table 2
, the stronger c-FLIP
intensity, the less TUNEL labeling index in the intima, media, and the
endothelium. The majority of TUNEL-positive cells lacked c-FLIP
staining, whereas cells that stain strongly for c-FLIP showed low or no
TUNEL labeling, although not all of the cells that express low or
undetectable levels of c-FLIP are TUNEL positive (Figure 8, A, C, and F
; Figure 9
). Similar results were found when we counted the number of
c-FLIP-positive and -negative macrophages in TUNEL-positive or
-negative zones (Table 3)
. These results
may indicate that c-FLIP plays a role in the regulation of the death in
human coronary arteries.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is interesting that c-FLIP becomes overexpressed in the neointima at later times in the rat model and is similarly and consistently prominent in nonatherosclerotic human intima. In contrast, in the human advanced atherosclerotic plaques, c-FLIP protein is very variable. In many plaques, but not all, the protein is absent or down-regulated in both endothelial cells and VSMCs. Double labeling with TUNEL and c-FLIP in human coronary arteries indicates that c-FLIP immunoreactivity in general correlates inversely with the frequency of TUNEL positivity. A few TUNEL-positive cells <4% in intima of human coronary arteries) had strong c-FLIP staining. This finding is probably because cells die by multiple pathways. c-FLIP appears to be part of a pathway that should protect against death from Fas, tumor necrosis factor receptor 1 (TNF-R1), or TNF-related apoptosis-inducing ligand (TRAIL), but not death receptor-3 (DR3).23 Taken together, these data suggest that a c-FLIP, like Bcl-2/Ced-9-related proteins, may play a role during vascular injury and in atherosclerotic lesion formation.
It is important that evidence for the c-FLIP function in any system is at least partially inferential from its structure. Studies dependent on overexpression, on the other hand, have both protective and apoptotic effects. For example, the caspase consensus-active site cysteine of the full-length form, c-FLIPL, is absent and is substituted for by a tyrosine residue. Therefore, c-FLIP is proteolytically inactive.16 Consistent with the expected function, expression studies by a number of groups demonstrated a protective effect of c-FLIP on apoptosis induced by the death receptor ligations for TNF-R1, Fas, and TRAIL when expressed at low levels.9,11,14,16 This inhibitory effect coincides with observations made for the viral homologs. Confusingly, other expression studies reported a marked cytotoxic effect of transfected c-FLIP by activation of caspase 8 when expressed at high levels.10,13,15 This effect of high levels of c-FLIP could represent an artificial role of very high levels of the prodomain activity to aggregate and activate caspase 8. A possible mechanism for such an artifact appears in two studies of the overexpressed molecule. Scaffidi et al29 found that inhibition depends on the interaction of the prodomain of c-FLIP with FADD. At normal concentrations, this interaction blocks access of caspase 8 to FADD, thus preventing formation of caspase 8 aggregates required for the activation of this enzyme. In contrast, Shu et al10 suggested that, at high levels, c-FLIP itself might act to aggregate caspase 8. Although the role of c-FLIP in apoptotic signaling is controversial, recent biologic data support the consensus that c-FLIP is protective. v-FLIP acts as a dominant negative for FADD-mediated death, apparently by competitively binding the prodomains of caspase 8 or 10 and thus blocking Fas-mediated apoptosis.8 Similarly, the sensitization of T cells to Fas-mediated apoptosis after T-cell antigen receptor stimulation is mediated by the down-regulation of c-FLIP, and the effect can be restored by providing cytoplasmic c-FLIP to the cells.17 Most relevant to this paper, however, is a recent report that human umbilical vein endothelial apoptosis is dose-dependent and correlates with down-regulation of c-FLIP.30 As with the T-cell study, this study found that the cytotoxity of oxidized lipids was reversed by transfecting endothelial cells with a c-FLIP expression plamid. These findings suggest that c-FLIP competitively inhibits binding of caspase 8 to the Fas receptor complex, thus shutting off the downstream Fas-signaling pathway.
Another candidate for a molecule protecting intimal cells from death is Bcl-XL. Perlman et al noted that induction of apoptosis in vascular media early after balloon injury coincides with down-regulation of Bcl-XL protein, an antiapoptotic molecule, assessed by immunohistochemistry in the arterial media.31 Pollman et al showed that the neointima after balloon injury stained uniformly to Bcl-XL, although some cell death could be observed.32 They also showed that an antisense Bcl-XL construct is able to promote thinning of the neointima after balloon injury. We have shown previously that c-FLIP interacts with Bcl-XL,13 suggesting that these antiapoptotic molecules may interact in controlling cell death in the intima.
It is important that these in vivo studies of antiapoptotic molecules, except for the antisense studies of Bcl-XL, are phenomenological. The absence of c-FLIP in dying cells, indicated by simultaneous staining with TUNEL and an antibody, may be a consequence of protease generated during death rather than a cause. Although we have no direct way of answering this concern, in vitro studies with death receptor-activated death in lymphocytes show that c-FLIP is truncated to a form still recognized by the MAG-1 antibody or antibodies directed at the same peptide sequence.17 A further caution is the use of TUNEL to identify dead cells. TUNEL data clearly depend on the method used. For example, the reported value of TUNEL-labeled cells obtained from atherosclerotic plaques ranged from less than 2% up to 60%.33 In addition, Geng et al suggested that, if the apoptotic bodies are not removed or remain TUNEL positive after engulfment, then high labeling indices may be independent of the time course of cell death itself.2 Thus, TUNEL positivity probably only reflects occurrence of death rather than giving an absolute value.34 To provide experiment-to-experiment consistency in the current study, however, TUNEL analyses were always performed including human tonsil and rat thymus as positive controls of human and rat samples, respectively.
In summary, c-FLIP is widely expressed in the normal vessel wall and down-regulated in the media of the rat common carotid artery after balloon injury and in human atherosclerotic atheroma. The selective expression of c-FLIP in rat neointimal lesions and in diffuse intimal thickening is consistent with previous studies that have demonstrated that intimal cells exhibit a differential pattern of gene expression.35 For human atherosclerotic plaques, the majority of TUNEL-positive cells lack detectable c-FLIP. Loss of c-FLIP in atherosclerotic plaques may be an important step in the ultimate breakdown of the atherosclerotic lesion.
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Footnotes |
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Supported by National Institutes of Health grants HL 03174, HL 61860, and HL 26405.
Accepted for publication September 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and interleukin-1ß. Arterioscler Thromb Vasc Biol 1996, 16:19-27This article has been cited by other articles:
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