© 1999 American Heart Association, Inc.
Original Contribution |
Nuclear Factor-
B Regulates Induction of Apoptosis and Inhibitor of Apoptosis Protein-1 Expression in Vascular Smooth Muscle Cells
From the Cardiovascular Research Unit, Center for Molecular Medicine (W.E., G.K.H.), Karolinska Institute, Stockholm, Sweden; the Vienna International Research Cooperation Center (R.d.M.), Wien, Austria; and the Institut für Prophylaxe der Kreislaufkrankheiten (G.D., K.S.C.W., C.W.), München, Germany.
Correspondence to Dr Wolfgang Erl, Cardiovascular Research Unit, CMM L8:03, Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail wolfgang.erl{at}cmm.ki.se
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Abstract |
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Abstract—Apoptosis is important in normal development as well as in diseases such as atherosclerosis. However, the regulation of apoptosis is still not completely understood. We now show that the transcription factor nuclear factor-
B (NF-B) controls the
induction of apoptosis in human and rat vascular smooth muscle
cells (SMCs). SMCs in high-density culture exhibited a high NF-B
activity and were insensitive to induction of apoptosis.
Inhibition of NF-B by adenovirus-mediated overexpression of its
inhibitor IB
caused a marked increase in cell death
at low but not high cell density. Elevating endogenous
IB levels by inhibiting its degradation with proteasomal
inhibitors resulted in induction of apoptosis in
low-density SMCs, as detected by increased binding of annexin V,
reduced mitochondrial membrane potential, and increased hypodiploid
DNA. In high-density cultures, protection against apoptosis was
associated with the expression of inhibitor of
apoptosis protein-1 (IAP-1). Transfer of IB reduced human
IAP-1 mRNA levels, which suggested that IAP-1 is transcriptionally
regulated by NF-B. This was confirmed through identification of a
motif with NF-B–like binding activity in the human IAP-1 promoter
region. Moreover, antisense inhibition of IAP-1 sensitized high-density
SMCs to the induction of cell death. Together, our data imply that SMCs
at high density are protected by an antiapoptotic mechanism
that involves increased expression of NF-B and IAP-1. Interference
with pathways that control the susceptibility to programmed cell death
may be helpful in the treatment of diseases where dysregulation of
apoptosis is involved, eg, atherosclerosis
and restenosis.
Key Words: apoptosis • nuclear factor-B • restenosis • gene transfer
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dysregulated apoptosis has been implicated as a pathogenetic mechanism in a variety of human diseases. Recently, compelling evidence has demonstrated that apoptotic cell death may play an important role in cardiovascular disorders.1 Apoptosis of vascular smooth muscle cells (SMCs) is critically involved in the progression of atherosclerosis and may contribute to the instability of advanced atherosclerotic plaques.1 2 3 4 After angioplasty, the "response to injury program" involves induction of SMC proliferation, which may result in restenosis of the diseased vessel. In this case, apoptosis may aid the remodeling of the injured artery and prevent the development of restenosis.1 4 5 6
Little is known about the mechanisms that regulate apoptotic
programs in SMCs. However, recent studies on a variety of cell types,
such as B cells, hepatocytes, and
osteoclasts,7 8 9 suggest that the transcription factor
nuclear factor-B (NF-B) plays a crucial role in the regulation of
apoptosis. These cell types share an important feature with
SMCs: a constitutive activity of NF-B.7 8 9 10 11 The
NF-B–like activity in SMCs may be composed of a putative SMC-Rel
protein or p65 and p50/Rel protein heterodimers and may be controlled
by serum components.10 11 In situ detection of nuclear p65
and p50 has revealed NF-B activity within human
atheroma, eg, in intimal SMCs but not in undiseased
arteries.11 12 Moreover, NF-B can be induced in SMCs
after arterial balloon injury.13 This activity
appears to be essential for the proliferation of SMCs, which is
demonstrated by the selective inhibition of SMC growth after
microinjection of the NF-B inhibitor protein IB or
of oligonucleotides that harbor NF-B
elements.14 15
Previous studies have not addressed whether the activity of NF-B may
also affect the viability of SMCs. To test the hypothesis that the
inhibition of NF-B induces apoptosis in SMCs, we used
adenovirus-mediated transfer of IB, which selectively inhibits
NF-B,16 as well as inhibitors of
proteasomal IB degradation.17 18 In this study, we
demonstrate that the inhibition of NF-B induced apoptosis in
human and rat SMCs. However, cells grown at high density were protected
from this lethal effect. These cells not only exhibited higher levels
of NF-B activity but also increased mRNA expression of the recently
identified human inhibitor of apoptosis protein-1
(IAP-1).19 The expression of IAP-1 was under the control
of NF-B, and we discovered an NF-B–like binding element in its
promoter region. Moreover, antisense inhibition of IAP-1 sensitized
high-density SMCs to the induction of cell death. Together, our data
suggest that NF-B–regulated expression of IAP-1 may protect SMCs
from apoptosis.
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Materials and Methods |
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Cell Culture
Rat aortic smooth muscle cells (rSMCs) were harvested from male Sprague-Dawley rats by enzymatic digestion, as described.20 Human umbilical artery smooth muscle cells (hSMCs) were prepared by cutting umbilical arteries into ring segments of 1- to 2-mm length, removing connective tissue, and incubating these rings in T-25 Falcon Primaria flasks (Becton Dickinson) with medium covering the flask bottom. When cells started to grow after 1 to 2 weeks, the rings were removed, and the remaining colonies were trypsinized and reseeded in T-25 flasks. Human aortic SMCs were purchased from Clonetics Corp. Rat SMCs were grown in DMEM/F12 (all media from Life Technologies) with 10% FCS and 50 µg/mL gentamicin, and human SMCs were grown in Waymouth medium with 15% FCS and antibiotics. Cells were passed every 3 to 5 days (rSMCs) or 1 to 2 weeks (hSMCs) and seeded at a density of 1x104 cells/cm2 for propagation in T-75 flasks or at the specified densities 48 hours before the respective treatment in 24-well plates or T-75 or T-25 flasks. Experiments were performed between passages 5 and 10. All other reagents were from Sigma Chemical Co.
Cell Viability Assays
Cell viability was determined by a modified MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
reduction assay.20 Cells were grown in 24-well plates for
48 hours and treated as indicated, washed with PBS, and 300-µL MTT
solution (0.5 mg/mL) in culture medium (0.5% FCS) was added to each
well. After 3 hours of incubation at 37°C, MTT was discarded and the
purple formazan product in the cells solubilized by addition of
acidic isopropanol (0.04 mol/L HCl). After lysing for 10 minutes,
2x100 µL from each well were transferred to a 96-well plate.
Absorbance was measured in an ELISA reader at 570 nm, with the
absorbance at 690 nm to correct for background, and viability was
expressed as the percentage of untreated controls. As an alternative
method to analyze cell viability, trypan blue exclusion assays
were performed after adherent and nonadherent cells were harvested. To
validate the MTT assay as a measure of cell viability, trypan blue
exclusion was performed in some experiments in parallel.
Infection of SMCs and Western Blot Analysis
A recombinant adenovirus that contained the coding sequence of
IB under the CMV promoter (rAd.IB) was constructed
as described.16 rSMCs seeded at
1x104 or 10x104
cells/cm2 and hSMCs seeded at
0.4x104 or 4x104
cells/cm2 for 48 hours were washed with PBS and
incubated at a multiplicity of infection (MOI) of 500 (plaque forming
units, PFU) with rAd.IB or control adenovirus DL-312 in medium
without FCS. After 1 hour at 37°C, the adenovirus was washed off and
fresh growth medium was added. Cells were inspected microscopically
after 24 and 48 hours. Some infected cells were used for extraction of
nuclear protein for electrophoretic mobility shift assay (EMSA) after
24 hours. To analyze the efficiency of transduction, SMCs
seeded at the above densities were also infected with rAd encoding the
green fluorescent protein (rAd.GFP) as a marker vector at an
identical MOI of 500 (PFU). After 48 hours, the fluorescence
intensity was analyzed by flow cytometry, and the percentage of
GFP-positive SMCs was determined as described.21
To assess IB expression, SMCs infected with or without
rAd.IB or treated with N-p-tosyl-L-lysine
chloromethyl ketone (TLCK) were lysed in sample buffer, and whole-cell
lysates were separated by 12.5% SDS-PAGE. The amount of protein loaded
was adjusted to the number of viable cells. Proteins were then
electrophoretically transferred to nitrocellulose membranes. These were
blocked for 1 hour at 25°C, incubated with a monoclonal antibody
(mAb) to IB (Santa Cruz Biotechnology), and reacted with a
peroxidase-labeled sheep anti–mouse Ig mAb. Blots were developed with
chemiluminiscence (ECL, Amersham) and then exposed to X-Omat AR
film.
Detection of Apoptosis by Annexin V Staining
Adherent SMCs infected as above were washed with binding
buffer and incubated with FITC-labeled annexin V in binding buffer
(Annexin V FITC Kit, Immunotech) for 20 minutes at 25°C in the dark.
Cells were washed 3 times, harvested by scraping, resuspended in 1 mL
PBS with 1.5 mmol/L Ca2+ and 1% FCS, and
fixed with 1 mL 2% paraformaldehyde to prevent cell
aggregation. SMCs were analyzed by flow cytometry in a FACScan
with a single cell gate. After appropriate markers for negative and
positive populations were set, the percentage of annexin-V–positive
cells was determined.
Analysis of Mitochondrial Membrane Potential
Identification and quantification of apoptotic and
nonviable cells was achieved by a multiparameter
assay,22 23 which detected a decrease in mitochondrial
membrane potential (
) in combination with staining for propidium
iodide (PI). To measure , cells were incubated with
3,3'-dihexyloxacarbocyanine iodide (DiOC; Molecular Probes) at 37°C
for 15 minutes (16 nmol/L; 525 nm) followed by immediate
analysis of fluorochrome incorporation in a Becton Dickinson
FACS Calibur cytometer. Each sample was incubated with PI (1 µg/mL;
600 nm) 10 seconds before analysis to identify viable and
nonviable cells. An acquisition gate was set to exclude cell debris and
aggregates (R1) and 10,000 cells within this gate were
analyzed. In control experiments (data not shown), cells were
treated with carbonyl cyanide m-chlorophenylhydrazone (50
µmol/L) to induce breakdown.
Isolation of Nuclei and DNA Fragmentation Analysis
To detect DNA fragmentation in nuclei of apoptotic
cells, we used cell cycle analysis, in which apoptotic
cells with a DNA content <2N appear in the sub-G1
region.24 Cells were grown for 48 hours and treated with
the proteasomal inhibitors MG-132 (Z-Leu-Leu-Leu-CHO) or
lactacystin (Biomol) for 24 hours. The IC50 for
inhibition of NF-B activity is 3 µmol/L for MG-132 and
10 µmol/L for lactacystin.18 Cultures were scraped
and adherent and nonadherent cells were pooled, washed, resuspended in
0.5 mL PBS, and fixed with 5 mL of FACS lysing solution (Becton
Dickinson). Cells were then processed with the Cycle Test Plus DNA
reagent kit (Becton Dickinson). After cells were washed with 10 mL PBS
and 2x1-mL citrate buffer, they were lysed in 250 µL buffer A for 10
minutes and 200 µL buffer B was added for 10 minutes. Nuclei were
stained with PI solution for 5 minutes and analyzed in a FACS
Calibur with instrument settings from the DNA quality control kit
(Becton Dickinson), activated doublet discrimination mode, and
a single nuclei gate. Typical histograms were analyzed by
manual gating and setting a marker (M1) on the sub-G1 peak.
Preparation of Nuclear Extracts and EMSA
EMSA was performed as described.25 Nonadherent
cells were removed and adherent cells were rinsed with PBS and
harvested in 1 mL of 20 mmol/L KCl buffer (20 mmol/L HEPES,
22% glycerol, 20 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT,
1 mmol/L PMSF, 1 µg/mL leupeptin, and 15 µg/mL aprotinin).
Isolated nuclei were resuspended in 41 µL of 20 mmol/L KCl
buffer, and 39 µL of 0.6 mol/L KCl buffer was added. Nuclear proteins
were extracted by incubation on ice for 30 minutes. After cells were
centrifuged for 15 minutes at 8000g, supernatants
with nuclear proteins were transferred into precooled tubes and protein
concentrations were determined spectrophotometrically. Nuclear protein
(10 µg) was mixed with double-stranded
oligonucleotides that corresponded to the NF-B
binding motifs 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Santa
Cruz) and 5'-AGCAGAGCT-TTCCC-3' (human
IAP-1),19 which were labeled with
[32P]-dATP with T4 polynucleotide
kinase. A 25-fold excess of unlabeled oligonucleotide
was used for competition to identify specific binding, whereas
competition with unlabeled oligonucleotide with a
mutated sequence was ineffective. After the reaction was allowed to
bind for 15 minutes, samples were separated on nondenaturating 4%
polyacrylamide gels and exposed to x-ray films. To obtain
comparable intensities, EMSA with the IAP-1 motif required longer
exposure. The absorbance of NF-B–specific bands was
analyzed by laser densitometry and expressed as optical density
(OD)xmm.
Reverse Transcription–Polymerase Chain Reaction
Reverse transcription–polymerase chain reaction (RT-PCR) was
essentially performed as described.26 27 RNA was isolated
from cells grown in 10-cm dishes by lysis with 7.5 mol/L guanidine-HCl,
3.5 mmol/L N-lauroyl-sarcosine, adjusted to pH 5.2 with
1 mol/L acetic acid, by shearing chromosomal DNA in a needle, followed
by phenol and diethylether extraction, and finally by ethanol
precipitation. cDNA was produced from total RNA (0.3 µg) by M-MLV
reverse transcriptase (Life Technologies). PCR was performed with
random hexamers and primers specific for human IAP-1 (sense:
5'-CAGTGGATATTTCCGTGGCT-3', antisense: 5'-TTTCATCTCCTGTG TCT-3')
and ß-actin selected from regions with minimal homology to yield
products of 676 and 540 bp, respectively.19 24 cDNA
was amplified by 32 cycles with Taq DNA polymerase, 48 pmol
of human IAP-1 primer, and 36 pmol ß-actin primer in a thermocycler
480 (Perkin-Elmer Cetus) set to the cycle 95°C for 30 seconds, 58°C
for 60 seconds, and 72°C for 60 seconds. PCR products were
analyzed with 1% agarose gel electrophoresis and were
quantified after HPLC separation on a nonporous diethyl-aminoethyl
column with a 0.3 to 0.6 mol/L NaCl gradient buffered at pH 9.0 and UV
detection at 260 nm (Abimed-Gilson) as
described.26 27 The respective peaks appeared at
retention times predicted by weight standards, and areas under peaks
were integrated. Human IAP-1 mRNA expression was normalized to levels
of ß-actin to compensate for variations in mRNA extraction.
Antisense Inhibition of IAP-1
Several phosphorothioate antisense 19-mer
oligonucleotides directed against various regions of
human IAP-1 mRNA19 were designed according to a recent
report28 and suggestions from recent
reviews.29 30 Among these, an antisense
oligonucleotide directed against a sequence that
encompassed the initiation site of human IAP-1 mRNA (5'-GTTCATAAT
GAAATGAATG-3') revealed the most obvious effects and was called
IAP-1.3, whereas an inversed sequence with corresponding
oligonucleotide composition called IAP-1.s3 (5'-GTA
AGTAAAGTAATACTTG-3') was used as a control. Human aortic SMCs
seeded at 0.4x104
cells/cm2 or 4x104
cells/cm2 were grown for 48 hours and transfected
with oligonucleotides (1 µg DNA) in 6-µL Tfx-50
reagent (Promega), with an optimized protocol for human aortic SMCs
(Promega notes). Cells were incubated with Tfx/DNA for 1 hour at 37°C
in Opti-MEM medium, before normal growth medium (2:1) was added. Cells
were then grown for 24 hours and treated with MG-132 (1 µmol/L)
in medium with 0.5% FCS. After 24 hours, cells were harvested, and the
percentage of dead cells was determined by trypan blue exclusion.
Statistical Analysis
Data are expressed as mean±SD unless otherwise stated and were
statistically analyzed by use of Student t test with
appropriate Bonferoni corrections, as indicated. Differences with
P<0.05 were considered significant.
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Results |
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Density-Dependent Sensitivity to Apoptosis in SMCs
The radical scavenger pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-
B activation,25 31 has been
shown to induce apoptosis in SMCs.32
Interestingly, we found that SMCs seeded at low or high density and
grown for 48 hours showed profoundly different sensitivity to induction
of cell death by PDTC, as determined by MTT (Figure 1
). Treatment with PDTC (100
µmol/L) for 24 hours reduced the viability of rSMCs seeded at
1x104 cells/cm2 by 60%
and that of human aortic SMCs at 0.4x104
cells/cm2 by 50%. In contrast, rSMCs seeded at
10x104 cells/cm2 or human
aortic SMCs at 4x104
cells/cm2 showed no decrease in MTT activity in
response to PDTC (Figure 1). Thus, SMCs at low but not high
density were sensitive to induction of cell death with PDTC, which
implied a role of cell density and NF-B inhibition in susceptibility
to apoptosis.
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Overexpression of IB Induces Apoptosis in SMCs at Low
Cell Density
To directly investigate the involvement of NF-B in
apoptosis of SMCs, cells were seeded at defined densities and
subsequently infected with a recombinant adenovirus that expressed
IB. The expression of IB in rSMCs (seeded at
1x104 or 10x104
cells/cm2) and hSMCs (seeded at
0.4x104 or 4x104
cells/cm2) was analyzed by Western blot
analysis(Figure 2A).
Expression of endogenous IB was not detectable in
uninfected cells by use of a detection system with moderate
sensitivity. Overexpression of IB was achieved by
adenovirus-mediated transfer of IB (Figure 2A). The level
of IB expression was similar in lysates from equivalent numbers
of infected SMCs and was therefore independent of species or cell
density (Figure 2A).
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After adenoviral infection, rSMCs seeded at low density
(1x104 cells/cm2) revealed
little microscopic evidence of cell death after 24 hours of culture
(not shown) but were clearly found to undergo cell death after 48 hours
of culture when overexpressing IB (Figure 2B
). In
contrast, rSMCs seeded at high density (10x104
cells/cm2) appeared to be resistant to
induction of cell death by overexpression of IB (Figure 2B). Similarly, microscopic examination (Figure 2B)
revealed that hSMCs seeded at a low density
(0.4x104 cells/cm2) were
considerably more susceptible to induction of apoptosis than
hSMCs seeded at a high density (4x104
cells/cm2). Infection with control adenovirus had
no effect on viability at any cell density examined (not shown). To
obtain a more quantitative measure of cell death, we harvested hSMCs
treated as above after 48 hours of culture; differences in cell
viability were analyzed by trypan blue exclusion.
Adenovirus-mediated overexpression of IB induced a marked
reduction in the viability of rSMCs and hSMCs seeded at low density,
whereas SMCs at high density showed no significant decrease in
viability on overexpression of IB (Figure 3A and 3B). In contrast, infection with control
adenovirus did not affect viability of SMCs at any density (Figure 3A and 3B). The effects were sustained after prolonged periods
of time, ie, 72 hours (data not shown).
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To confirm that the reduction of viability was due to
apoptosis, we used FITC-conjugated annexin V, which binds to
phosphatidylserine that is translocated from the
inner to the outer leaflet of the cell membrane and therefore exposed
when a cell enters apoptosis.22 33
Consistent with reduced viability, the percentage of annexin
V–positive cells was increased by overexpression of IB in rat
and human SMCs seeded at low density, which reflected induction of
apoptosis. However, it was not altered in SMCs seeded at high
density or by infection with control adenovirus (Figure 3C and 3D).
To rule out that the observed effects on viability and
apoptosis were due to differences in susceptibility to
infection, we also infected SMCs at a different density with a
recombinant adenovirus expressing GFP (Figure 3E through 3H). At
an equivalent moi, the percentage of GFP-positive cells after 48 hours
was comparable or even higher in high-density (76±2% in rat, 70±5%
in human, mean±SD, n=3) compared with low-density (56±3% in rat,
58±2% in human) SMCs. This clearly indicates that high-density cells
show similar or better transduction efficiency than subconfluent cells
and excludes that the effects we observed were due to differences in
susceptibility to infection.
Inhibition of IB Degradation Induces Apoptotic Death
in Low-Density SMCs
To determine whether cell death induced by IB-mediated
NF-B inhibition was due to the induction of apoptosis in
SMCs, we analyzed sequential alterations in and in
plasma membrane integrity during early stages of
apoptosis.22 23 Because the onset of the effects
on SMC viability induced by virally transferred IB could not be
precisely timed, we studied SMC apoptosis after treatment with
TLCK, an inhibitor of proteolytic IB
degradation.17 TLCK treatment resulted in induction of
apoptosis and cell death in rSMCs seeded at low density, as
indicated by the percentage of cells with decreased
(DiOClow) or PI+ staining (Figure 4A). These effects were more
marked in the presence of 0.5% FCS than 10% FCS, whereas induction of
cell death could not be detected in rSMCs seeded at high density (data
not shown).
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To confirm that these signs of apoptosis were followed by
a reduction in SMC viability after treatment with TLCK, we measured MTT
activity. Indeed, we found a dose-dependent decrease in viability
induced by TLCK in rSMCs seeded at low but not high density (Figure 4B). Similar results were obtained with trypan blue exclusion
(data not shown) and in MTT assays after treatment with another
proteasome inhibitor, MG-132 (Figure 5). This indicates that inhibition
of NF-B was associated with an induction of apoptosis in
SMCs growing at low but not high density and supports the notion that
NF-B plays a role in protecting SMCs against apoptosis.
Western blot analysis revealed that TLCK treatment of
low-density SMCs resulted in a similar increase in IB levels as that
achieved by adenoviral IB transfer (Figure 4C).
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Cell cycle analysis with DNA staining of isolated nuclei was
used as an additional, specific parameter that detected
apoptotic cells by their hypodiploid DNA.24
Treatment of low-density SMCs with the proteasome
inhibitors MG-132 and lactacystin resulted in an increased
percentage of apoptotic cells with a DNA content <2N, as
evident by the appearance of a marked peak in the sub-G1 region (Figure 5A).
A dose-dependent induction of cell death in low but not
high-density SMCs by MG-132 treatment was confirmed by parallel MTT
assays (Figure 5B).
Lower NF-B Activity in Apoptosis-Prone Low-Density
SMCs
To further elucidate a potential involvement of NF-B in
the differential susceptibility of SMCs to the induction of
apoptosis, EMSA was performed to compare the interactions of an
oligonucleotide that contained a classic NF-B
binding motif with nuclear protein extracts isolated from low- or
high-density rSMCs. High-density SMCs exhibited a higher NF-B
binding activity than low-density cells (Figure 6A and 6B). Specific binding of
NF-B was identified by inhibition with an unlabeled competitor
oligonucleotide, whereas competition with a mutated
oligonucleotide had no effect (Figure 6A and 6B). In low-density SMCs, treatment with TLCK resulted in a substantial
(50%) reduction in NF-B activity (Figure 6A and 6B), which
was also inhibited by overexpression of IB (not shown). Although
high-density rSMCs showed a reduction (40%) in NF-B activity on
TLCK treatment or IB transfer, the level of NF-B activity
remained comparable to that of untreated low-density SMCs (Figure 6A and 6B; not shown). These results suggest that a high
constitutive NF-B activity may be associated with protection against
apoptosis in high-density SMCs, whereas inhibition of NF-B
may be responsible for the induction of apoptosis in
low-density SMCs.
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Increased NF-B–Dependent IAP-1 Levels in
Apoptosis-Resistant High-Density SMCs
To investigate the mechanisms for protection against
apoptosis in high-density SMCs, we analyzed the
expression of two important inhibitors of
apoptosis: bcl-234 and IAP-1,
a member of another protein family that suppresses
apoptosis.19 Although bcl-2 protein was
not detectable (not shown), RT-PCR showed that human IAP-1 mRNA was
expressed in hSMCs (Figure 7A and 7B).
The level of IAP-1 mRNA was considerably higher in SMCs growing at high
density than at low density (Figure 7A and 7B). Because this
pattern paralleled that observed for NF-B activity, we
analyzed the effect of IB overexpression on IAP-1.
Adenovirally mediated overexpression of IB virtually extinguished
expression of IAP-1 mRNA in low-density SMCs, whereas IAP-1 mRNA
remained detectable in high-density SMCs (Figure 7A and 7B).
This was consistent with differential effects on
apoptosis and NF-B activity. Inhibition of IAP-1 expression
by IB suggested that IAP-1 may be regulated through the NF-B
pathway. Therefore, we analyzed the published IAP-1
sequence19 and identified a NF-B-like motif
(5'-AGCAGAGCTTTCCC-3') in the promoter region at -433 to
-424 bp upstream of the transcription initiation site. EMSA with this
sequence revealed a NF-B–like binding activity, which indicated an
unorthodox NF-B motif (Figure 7C). Thus, the data imply that
IAP-1 transcription is promoted by NF-B-like elements and that
protection against apoptosis in high-density SMCs may involve
increased levels of NF-B activity and IAP-1 expression.
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Antisense Inhibition of IAP-1 Sensitizes High-Density SMCs to Cell
Death Induction
Finally, to support that NF-B–regulated IAP-1 expression
may causally contribute to the rescue of high-density SMCs from cell
death, we performed experiments with an antisense
oligonucleotide (IAP-1.3) directed against the
initiation site of IAP-1. Transfection of low- or high-density SMCs
with either antisense or control oligonucleotides
hardly affected viability after 24 hours (not shown). As expected,
MG-132 induced cell death in low- but not high-density SMCs (Figure 8). Transfection of low-density
SMCs with antisense or control oligonucleotides did not
affect the induction of cell death by NF-B inhibition (Figure 8). However, the antisense oligonucleotide
IAP-1.3 sensitized apoptosis-resistant high-density
SMCs to the induction of cell death, whereas the control
oligonucleotide IAP-1.s3 with inversed sequence and
corresponding oligonucleotide composition had no effect
(Figure 8). Consistent with the results based on trypan
blue exclusion, transfection of high-density SMCs with IAP-1.3 resulted
in a 2-fold increase in the percentage of annexin V-positive cells
after treatment with 1 µmol/L MG-132 for 24 hours (data not
shown). This demonstrates that IAP-1 can confer protection against cell
death in high-density SMCs with high NF-B activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SMCs exhibit a constitutive activity of NF-
B,10 11 which has been shown to promote their
proliferation.14 15 However, the mechanism by which
NF-B controls the expansion of SMC populations remains unclear. We
now show that (1) induction of apoptosis depends on the density
of SMCs; (2) high-density SMCs are resistant to
apoptosis and express a high constitutive NF-B activity,
whereas low-density SMCs are prone to undergo apoptosis and
exhibit less NF-B activity, (3) inhibition of NF-B by
overexpressing its inhibitor IB or by preventing
IB degradation results in apoptosis in low-density SMCs;
(4) overrepression of IB prevents expression of
antiapoptotic IAP-1; (5) NF-B is thus required for
expression of IAP-1, which contains a NF-B–like motif in its
promoter region; and (6) protection of high-density SMCs from the
induction of cell death is mediated by IAP-1. Together, these data
indicate that NF-B may promote SMC growth by modulating
apoptosis, that the antiapoptotic machinery develops in
parallel with increasing SMC density, and that NF-B–regulated
expression of IAP-1 is an important component of this machinery in
SMCs.
Inhibition of NF-B by overexpression of IB resulted in a
selective induction of apoptotic cell death in rat and human
SMCs, as shown by trypan blue exclusion and annexin V binding, which
was dependent on cell density, ie, restricted to low density. In fact,
SMCs seeded at high density appeared to be protected from the induction
of apoptosis, despite equivalent levels of IB expression
after adenoviral transfer at both densities. Infection with rAd.GFP
indicated that these effects were not due to differences in
transduction efficiency between high- and low-density cells. This
extends previous findings that inhibition of NF-B can induce
apoptosis or cell death in different cell
types.7 8 9 35 36 In contrast to drug-mediated NF-B
inhibition or microinjection of IB,7 8 9
adenovirus-mediated gene transfer of IB enabled us to inhibit
NF-B–like activities specifically and effectively in a large
proportion of the cell population. Our data imply that a constitutive
NF-B activity protects against apoptosis in high-density
SMCs. A sufficient NF-B activity may thus be crucial to maintain and
support both SMC proliferation and survival.
To detect the effects of NF-B inhibition on SMC viability, we
analyzed trypan blue exclusion, which is widely used in studies
of apoptosis.34 Mitochondrial dysfunction is an
initial step in the death program, and a reduction in is a
specific marker to detect early apoptotic
changes.22 23 Because the characteristic cleavage of DNA
into nucleosome-sized fragments, considered as a hallmark of
apoptosis, is a late event in the apoptotic process and
does not occur in all cell types,37 was measured in
rSMCs. In this setting, we used TLCK, an inhibitor of
NF-B activity that prevents IB degradation and thus increases
cytoplasmic IB levels more rapidly than adenovirus-mediated
transfer of IB, which required more than 24 hours for the onset
of detectable effects in human and rat SMCs. Western blot and trypan
blue exclusion analysis after adenoviral IB transfer or
TLCK treatment revealed similar efficacy in increasing IB levels
and inducing cell death in low-density SMCs. Treatment of SMCs with
TLCK resulted in sequential alterations of and plasma membrane
integrity, ie, a reduced staining with DiOC accompanied by an increase
in PI+ cells. Consistent with results after IB
overexpression, TLCK treatment did not reduce viability in high-density
SMCs. With the use of fluorescence staining of isolated nuclei
to detect DNA degradation in apoptotic cells, we confirmed that
MG-132 and lactacystin, which are more potent and selective
inhibitors of proteasomal IB degradation than TLCK,
induce apoptosis in low-density SMCs.
Viability depends on the balance between apoptosis and proliferation. Because apoptosis is a kinetic and transient process that ultimately leads to cell death, a moderate increase in the percentage of apoptotic cells at a given time (as evident in the apoptosis assays used) can cumulatively result in a 50% reduction of viability, as seen in MTT assays over 24 hours.
The lower susceptibility to apoptosis in high-density
cells was associated with a higher constitutive NF-B–like activity,
which provides further evidence that NF-B is involved in the
regulation of programmed cell death. Moreover, this higher NF-B was
less clearly inhibited by increasing levels of IB in high-density
cells. This supports the notion that a high constitutive NF-B
activity may protect against apoptosis. Several groups have
shown an essential role for NF-B, in particular its p65 subunit, in
the prevention of tumor necrosis factor- (TNF-)–induced
apoptosis.38 39 40 This infers a negative feedback
loop by which NF-B activation that results from TNF- signaling
may counteract TNF--induced cell death programs and clearly
separates the apoptosis-inducing potential of TNF- from
NF-B activation. We now demonstrate that a constitutive NF-B
activity, which may be independent of agonist or
cytokine-induced death signals, favors survival programs in
SMCs. The inhibition by IB indicates that p65 and
c-Rel rather than p50 are candidates likely to mediate this
activity. This is supported by the ineffectiveness of the p50-specific
inhibitor SN50 to induce
apoptosis (W.E. et al., unpublished data). It is also possible
that specific factors of the Rel/NF-B family are involved in
mediating different pathways. In fact, such a distinction has recently
been reported in B lymphocytes.41
To elucidate potential mechanisms of protection, we studied mRNA
expression of human IAP-1, a member of a novel class of molecules, the
inhibitor of apoptosis proteins of nonviral
origin,42 which can protect against apoptosis by
inhibiting cell-death caspases.43 44 45 High-density hSMCs
revealed an increased expression of IAP-1 mRNA, which could be
responsible for protection against apoptosis in our study.
Similarly, increased expression of IAP-2 and X chromosome–linked IAP
has been found in high- versus low-density SMCs (W.E. et al,
unpublished data, 1998). In contrast, we did not detect expression of
anti–apoptotic bcl-2 protein in SMCs at any
density, although the related bcl-xL
has been shown to be present in neointima, and its
inhibition induced apoptosis and regression of vascular
disease.46 This complies with results that IAPs and
bcl-2 confer protection in separate but overlapping
pathways.19 Overexpression of IB blocked IAP-1
mRNA expression in low-density SMCs, but reduced its expression only in
high-density cells. This parallels effects of IB overexpression
on SMC viability and further infers a protective role of IAP-1. Our
data are consistent with recent findings in
endothelial cells in which cytokine-induced
IAP expression is dependent on NF-B and overexpression of X
chromosome–linked IAP protected against TNF-–induced
apoptosis.24 The conclusion that IAP-1 expression
is regulated by NF-B in SMCs is supported by a report that shows
that IAP-1 mediates the antiapoptotic activity of
v-Rel.47 Notably, we found a NF-B–like
binding activity to a newly identified motif within the human IAP-1
promoter region in high-density hSMCs. Other studies have found IAP-2
but not IAP-1 to be under the control of NF-B,48 which
indicates that IAP genes may be differentially regulated by NF-B in
distinct cellular systems. Our present results suggest that the
expression of IAP-1 may be regulated by engagement of a NF-B–like
promoter element. This supports recent findings that induction of IAP-1
and IAP-2 serves as an important mechanism by which NF-B prevents
apoptosis.45 In addition to the transfer of IAP of
viral or bacterial origin, such NF-B–dependent mechanisms may
essentially contribute to accomplish host cell survival during
bacterial or viral infections.42 49 We now directly
demonstrate by antisense inhibition that IAP-1 confers protection
against cell death in high-density SMCs with high NF-B activity.
This suggests that IAP-1 plays a crucial role in controlling
apoptosis in these cells. Recent reports have demonstrated that
expression of IAP-1 or IAP-2 alone was not sufficient to rescue cells
with inactive NF-B from TNF-induced
apoptosis,45 48 which suggests that multiple
antiapoptotic gene products that are activated by
NF-B function cooperatively to achieve optimal protection against
apoptosis. Our findings in hSMCs that the suppression of IAP-1
results in a remarkable sensitization to induction of cell death
support a prominent role of IAP-1 among the components of this
antiapoptotic machinery and may indicate that a disruption of
the protective network can affect resistance to apoptosis.
The finding that inhibition of constitutive NF-B induces
apoptosis in low-density SMCs suggests that the death program
is a default pathway that is modulated by cellular NF-B activity. A
high frequency of apoptosis may be important to permit high
cell turnover and tissue remodeling in growing cell populations. These
situations may be mimicked in a low density culture, whereas a
high-density culture may reflect a more stable situation in normal,
adult tissue. NF-B may be a signal linking cell density to
apoptosis and thus to cell turnover. Pathological tissue growth
can be controlled by inducing apoptosis, and transfer of
IB may therefore be a promising way to prevent restenosis
caused by excessive SMC proliferation after angioplasty and
arterial injury. Also, evidence for activated
NF-B has been provided in intimal SMCs of human atheroma
but not nonatherosclerotic arteries.11 12 The activation
of NF-B in SMCs after balloon injury has been associated with
intimal lesion formation and inflammatory responses.13
Adenovirus-mediated gene transfer by pressure-assisted delivery of
constitutive endothelial nitric oxide synthase in
balloon-injured arteries has been shown to allow virus penetration
through the elastic laminae to deeper layers and to reduce SMC
proliferation and neointima formation.50
Recently, adenoviral gene transfer of fas ligand to the
vessel wall has been shown to induce apoptosis in
fas-bearing SMCs, thereby inhibiting balloon injury-induced
neointima formation in rat carotid
arteries.51 Since nitric oxide can induce
apoptosis in SMCs and has been shown to inhibit NF-B
mobilization in vascular cells,52 53 54 it may induce SMC
apoptosis by inhibiting NF-B activity. The feasibility of
gene transfer to adult human vessels has recently been
demonstrated;55 however, high titers of infection may
cause activation of vascular
endothelium.56 Hence, adenoviral IB
overexpression and antiinflammatory and antioxidative agents that
inhibit NF-B mobilization may act in concert to induce SMC
apoptosis and to inhibit endothelial
activation.16 25 26 57 This may attenuate inflammatory
reactions after angioplasty or adenoviral gene transfer, which improves
the prevention of neointima formation and contributes to
the benefits of NF-B inhibition.
In conclusion, the sensitivity to induction of apoptosis in
exponentially proliferating low-density SMCs contrasts strongly with
the resistance to apoptosis of high-density SMCs with a
"resting" phenotype. Such differences in the regulation of
cell death and turnover could have important implications for the
prevention of restenosis in diseased vessels. Considering the
limited success of preventing restenosis in humans by
inhibiting SMC proliferation, the key role of NF-B and IAP-1 in the
regulation of SMC apoptosis demonstrated herein offers an
attractive new target for the therapeutic control of
neointima formation.
|
Acknowledgments |
|---|
This study was supported by the Swedish MRC (grant 6816), Heart-Lung Foundation and Cancer Research, Deutsche Forschungs-gemeinschaft (grant Er 270/1-1 to W.E. and We 1913/2-1 to C.W.), August-Lenz Stiftung (K.S.C.W.), and an exchange program of Deutscher Akademischer Austauschdienst. We thank A. Olsson for performing EMSA.
Received August 10, 1998; accepted January 7, 1999.
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References |
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 J. Vaage and G. Valen Preconditioning and cardiac surgery Ann. Thorac. Surg., February 1, 2003; 75(2): S709 - 714. [Abstract] [Full Text] [PDF]
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 V. Poulaki, C. S. Mitsiades, A. M. Joussen, A. Lappas, B. Kirchhof, and N. Mitsiades Constitutive Nuclear Factor-{kappa}B Activity Is Crucial for Human Retinoblastoma Cell Viability Am. J. Pathol., December 1, 2002; 161(6): 2229 - 2240. [Abstract] [Full Text] [PDF]
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W. Martinet, D. M. Schrijvers, G. R.Y. De Meyer, J. Thielemans, M. W.M. Knaapen, A. G. Herman, and M. M. Kockx Gene Expression Profiling of Apoptosis-Related Genes in Human Atherosclerosis: Upregulation of Death-Associated Protein Kinase Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2023 - 2029. [Abstract] [Full Text] [PDF]
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M. Aragno, R. Mastrocola, E. Brignardello, M. Catalano, G. Robino, R. Manti, M. Parola, O. Danni, and G. Boccuzzi Dehydroepiandrosterone Modulates Nuclear Factor-{kappa}B Activation in Hippocampus of Diabetic Rats Endocrinology, September 1, 2002; 143(9): 3250 - 3258. [Abstract] [Full Text] [PDF]
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Y. Wang, S. Chan, and B. K. Tsang Involvement of Inhibitory Nuclear Factor-{kappa}B (NF{kappa}B)-Independent NF{kappa}B Activation in the Gonadotropic Regulation of X-Linked Inhibitor of Apoptosis Expression during Ovarian Follicular Development in Vitro Endocrinology, July 1, 2002; 143(7): 2732 - 2740. [Abstract] [Full Text] [PDF]
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 B. Tian, Y. Zhang, B. A. Luxon, R. P. Garofalo, A. Casola, M. Sinha, and A. R. Brasier Identification of NF-{kappa}B-Dependent Gene Networks in Respiratory Syncytial Virus-Infected Cells J. Virol., June 5, 2002; 76(13): 6800 - 6814. [Abstract] [Full Text] [PDF]
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 M. Cejna, J. M. Breuss, H. Bergmeister, R. de Martin, Z. Xu, M. Grgurin, U. Losert, H. Plenk Jr, B. R. Binder, and J. Lammer Inhibition of Neointimal Formation after Stent Placement with Adenovirus-mediated Gene Transfer of I{kappa}B{alpha} in the Hypercholesterolemic Rabbit Model: Initial Results Radiology, June 1, 2002; 223(3): 702 - 708. [Abstract] [Full Text] [PDF]
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A. J. Chase, M. Bond, M. F. Crook, and A. C. Newby Role of Nuclear Factor-{kappa}B Activation in Metalloproteinase-1, -3, and -9 Secretion by Human Macrophages In Vitro and Rabbit Foam Cells Produced In Vivo Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 765 - 771. [Abstract] [Full Text] [PDF]
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 T. Weber, M. Lu, L. Andera, H. Lahm, N. Gellert, M. W. Fariss, V. Korinek, W. Sattler, D. S. Ucker, A. Terman, et al. Vitamin E Succinate Is a Potent Novel Antineoplastic Agent with High Selectivity and Cooperativity with Tumor Necrosis Factor-related Apoptosis-inducing Ligand (Apo2 Ligand) in Vivo Clin. Cancer Res., March 1, 2002; 8(3): 863 - 869. [Abstract] [Full Text] [PDF]
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J.M. Breuss, M. Cejna, H. Bergmeister, A. Kadl, G. Baumgartl, S. Steurer, Z. Xu, Y. Koshelnick, J. Lipp, R. De Martin, et al. Activation of Nuclear Factor-{kappa}B Significantly Contributes to Lumen Loss in a Rabbit Iliac Artery Balloon Angioplasty Model Circulation, February 5, 2002; 105(5): 633 - 638. [Abstract] [Full Text] [PDF]
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S. Meiners, M. Laule, W. Rother, C. Guenther, I. Prauka, P. Muschick, G. Baumann, P.-M. Kloetzel, and K. Stangl Ubiquitin-Proteasome Pathway as a New Target for the Prevention of Restenosis Circulation, January 29, 2002; 105(4): 483 - 489. [Abstract] [Full Text] [PDF]
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 D.L. MANN The Yin/Yang of Innate Stress Responses in the Heart Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 363 - 370. [Abstract] [PDF]
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M. D. Southall, J. S. Isenberg, H. Nakshatri, Q. Yi, Y. Pei, D. F. Spandau, and J. B. Travers The Platelet-activating Factor Receptor Protects Epidermal Cells from Tumor Necrosis Factor (TNF) alpha and TNF-related Apoptosis-inducing Ligand-induced Apoptosis through an NF-kappa B-dependent Process J. Biol. Chem., November 30, 2001; 276(49): 45548 - 45554. [Abstract] [Full Text] [PDF]
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 R. V. Sharma, M. V. Gurjar, and R. C. Bhalla Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappa B activation in VSM cells from female rats J Appl Physiol, November 1, 2001; 91(5): 2400 - 2406. [Abstract] [Full Text] [PDF]
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 C. Wahl, F. Oswald, U. Simnacher, S. Weiss, R. Marre, and A. Essig Survival of Chlamydia pneumoniae-Infected Mono Mac 6 Cells Is Dependent on NF-kappa B Binding Activity Infect. Immun., November 1, 2001; 69(11): 7039 - 7045. [Abstract] [Full Text] [PDF]
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T. Watanabe, M. Yoshizumi, M. Akishita, M. Eto, K. Toba, M. Hashimoto, K. Nagano, Y.-Q. Liang, Y. Ohike, K. Iijima, et al. Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1738 - 1744. [Abstract] [Full Text] [PDF]
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W. Erl and W. Siess Sphingosine-1-Phosphate and the Leading Edg-1 of Vascular Smooth Muscle Cells Circ. Res., September 14, 2001; 89(6): 474 - 476. [Full Text] [PDF]
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 G. Valen, Z.-q. Yan, and G.o. K. Hansson Nuclear factor kappa-B and the heart J. Am. Coll. Cardiol., August 1, 2001; 38(2): 307 - 314. [Abstract] [Full Text] [PDF]
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A. Orlandi, A. Francesconi, D. Cocchia, A. Corsini, and L. G. Spagnoli Phenotypic Heterogeneity Influences Apoptotic Susceptibility to Retinoic Acid and cis-Platinum of Rat Arterial Smooth Muscle Cells In Vitro : Implications for the Evolution of Experimental Intimal Thickening Arterioscler Thromb Vasc Biol, July 1, 2001; 21(7): 1118 - 1123. [Abstract] [Full Text] [PDF]
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 M. MANDERSCHEID, U. K. MEßMER, R. FRANZEN, and J. PFEILSCHIFTER Regulation of Inhibitor of Apoptosis Expression by Nitric Oxide and Cytokines: Relation to Apoptosis Induction in Rat Mesangial Cells and RAW 264.7 Macrophages J. Am. Soc. Nephrol., June 1, 2001; 12(6): 1151 - 1163. [Abstract] [Full Text]
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 G.K. Hansson The stabilized plaque: will the dream come true? Eur. Heart J. Suppl., June 1, 2001; 3(suppl_C): C69 - C75. [PDF]
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 T.-S. Lee and L.-Y. Chau Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells Am J Physiol Cell Physiol, March 1, 2001; 280(3): C709 - C718. [Abstract] [Full Text] [PDF]
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C. W. Xiao, K. Ash, and B. K. Tsang Nuclear Factor-{{kappa}}B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor {{alpha}}-Induced Apoptosis Endocrinology, February 1, 2001; 142(2): 557 - 563. [Abstract] [Full Text] [PDF]
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 J. NEUZIL, T. WEBER, A. SCHRODER, M. LU, G. OSTERMANN, N. GELLERT, G. C. MAYNE, B. OLEJNICKA, A. NEGRE-SALVAYRE, M. STICHA, et al. Induction of cancer cell apoptosis by {alpha}-tocopheryl succinate: molecular pathways and structural requirements FASEB J, February 1, 2001; 15(2): 403 - 415. [Abstract] [Full Text] [PDF]
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Rainer de Martin, M. Hoeth, R. Hofer-Warbinek, and J. A. Schmid The Transcription Factor NF-{kappa}B and the Regulation of Vascular Cell Function Arterioscler Thromb Vasc Biol, November 1, 2000; 20 (11): e83 - e88. [Abstract] [Full Text] [PDF]
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A. M. Schmidt and D. M. Stern Hyperinsulinemia and Vascular Dysfunction: The Role of Nuclear Factor-{kappa}B, Yet Again Circ. Res., October 27, 2000; 87(9): 722 - 724. [Full Text] [PDF]
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H. Obara, A. Takayanagi, J. Hirahashi, K. Tanaka, G. Wakabayashi, K. Matsumoto, M. Shimazu, N. Shimizu, and M. Kitajima Overexpression of Truncated I{kappa}B{alpha} Induces TNF-{alpha}-Dependent Apoptosis in Human Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, October 1, 2000; 20(10): 2198 - 2204. [Abstract] [Full Text] [PDF]
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J. Sadoshima Cytokine Actions of Angiotensin II Circ. Res., June 23, 2000; 86(12): 1187 - 1189. [Full Text] [PDF]
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W. Erl, C. Weber, and G. K. Hansson Pyrrolidine dithiocarbamate-induced apoptosis depends on cell type, density, and the presence of Cu2+ and Zn2+ Am J Physiol Cell Physiol, June 1, 2000; 278(6): C1116 - C1125. [Abstract] [Full Text] [PDF]
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N. J. McCarthy and M. Bennett The regulation of vascular smooth muscle cell apoptosis Cardiovasc Res, February 1, 2000; 45(3): 747 - 755. [Abstract] [Full Text] [PDF]
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K. Walsh and J. M. Isner Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall Cardiovasc Res, February 1, 2000; 45(3): 756 - 765. [Abstract] [Full Text] [PDF]
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F. Chen, L. M. Demers, V. Vallyathan, Y. Lu, V. Castranova, and X. Shi Involvement of 5'-Flanking kappa B-like Sites within bcl-x Gene in Silica-induced Bcl-x Expression J. Biol. Chem., December 10, 1999; 274(50): 35591 - 35595. [Abstract] [Full Text] [PDF]
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S.-Y. Hong, W.-H. Yoon, J.-H. Park, S.-G. Kang, J.-H. Ahn, and T. H. Lee Involvement of Two NF-kappa B Binding Elements in Tumor Necrosis Factor alpha -, CD40-, and Epstein-Barr Virus Latent Membrane Protein 1-mediated Induction of the Cellular Inhibitor of Apoptosis Protein 2 Gene J. Biol. Chem., June 9, 2000; 275(24): 18022 - 18028. [Abstract] [Full Text] [PDF]
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C. J. M. de Vries, T. A. E. van Achterberg, A. J. G. Horrevoets, J. W. ten Cate, and H. Pannekoek Differential Display Identification of 40 Genes with Altered Expression in Activated Human Smooth Muscle Cells. LOCAL EXPRESSION IN ATHEROSCLEROTIC LESIONS OF smags, SMOOTH MUSCLE ACTIVATION-SPECIFIC GENES J. Biol. Chem., July 28, 2000; 275(31): 23939 - 23947. [Abstract] [Full Text] [PDF]
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