© 1996 American Heart Association, Inc.
Articles |
Autocrine Induction of DNA Synthesis by Mechanical Injury of Cultured Smooth Muscle Cells
Potential Role of FGF and PDGF
From the Division of Cardiology, Cedars-Sinai Medical Center, Los Angeles, Calif, and the Department of Cell and Molecular Biology (A.H.-N.), Karolinska Institute, and the Departments of Surgery (U.H.) and Medicine (J.N.), Karolinska Hospital, Stockholm, Sweden.
Correspondence to Jan Nilsson, King Gustaf Vth Research Institute, Karolinska Hospital, 171 76 Stockholm, Sweden.
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Abstract |
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Abstract To determine whether replication of arterial smooth muscle cells (SMCs) in response to mechanical injury would occur in the absence of serum and other cells, we created an in vitro model in which confluent, growth-arrested cultures of rat SMCs were injured by gentle pressure of a soft plastic tube and then kept in serum-free medium for up to 4 days. Replication of SMCs in and around the injury, as measured by tritiated thymidine incorporation, was noted within 24 hours and peaked at 48 hours after injury, whereas noninjured cells remained quiescent. An increased expression of platelet-derived growth factor (PDGF) A mRNA, noted 6 hours after injury, was followed by an increased PDGF AA immunoreactivity in SMCs in and around the zone of injury at 24 and 48 hours after injury. A PDGF A chain antisense oligonucleotide inhibited 87.0±4.0% (P<.005) of SMC replication in the injury zone, whereas the corresponding sense oligonucleotide reduced SMC replication by only 37.2%. An antibody to fibroblast growth factor (FGF) almost completely inhibited SMC replication in the injured zone, whereas an antibody to PDGF AA was without effect. Incubation of SMCs with FGF increased PDGF A mRNA levels in SMCs, and 5 µmol/L PDGF A antisense oligonucleotides reduced FGF-induced SMC replication by 62%. Taken together, these results demonstrate that injured rat SMCs in culture release FGF that activates DNA synthesis of neighboring SMCs both by a direct mechanism and by stimulating the production of PDGF AA.
Key Words: injury • growth factors • smooth muscle cells • antisense
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Mechanical injury of arteries results in a healing response that is characterized by activation of SMC migration and the growth and formation of extracellular matrix, leading to the formation of a thickened neointima.1 A similar process is believed to play an important role in the development of restenosis after percutaneous transluminal coronary angioplasty.
Several growth factors are capable of stimulating the growth of SMCs in culture. These include PDGF,2 FGF,3 epidermal growth factor,4 insulin-like growth factor,5 and neuropeptides.6 SMCs can be induced to synthesize several of these mitogens, which suggests the possibility of autocrine mechanisms as one possible way to induce SMC growth.7 8 9 Accordingly, angiotensin II,10 interleukin-1ß,11 and transforming growth factor–ß12 have been shown to stimulate SMC growth by induction of PDGF AA expression.
The in vivo activation of SMC proliferation has been extensively
investigated in balloon-injured
deendothelialized rat carotid arteries. In this
model, intimal SMC hyperplasia is initiated only if the injury is deep
enough to result in necrosis of medial SMCs,13 suggesting
the possible involvement of factors released from injured SMCs rather
than mitogens released from the aggregating platelets that cover
the denuded intima. Consequently, considerable interest has focused on
local expression and release of growth factors in injured arteries. In
balloon-injured rat carotid arteries, medial SMC replication
commences within 1 to 3 days of injury and is followed by migration of
SMCs into the intima and a phase of rapid intimal SMC
proliferation.1 Northern blot analysis of these
arteries demonstrates an increased expression of PDGF A mRNA 6 hours
after injury and an increased expression of PDGF 
and ß receptors
24 hours after injury.14 Infusion of PDGF into rats
subjected to carotid injury results in increased SMC migration and
intimal proliferation.15 Although PDGF antibodies reduce
neointimal thickness, they have no effect on the rate of
SMC proliferation,16 suggesting that the primary effect is
on PDGF-induced migration. Another mitogen that has received
considerable interest in this context is FGF, which is present in
vascular SMCs and endothelial cells; its main pathway
for secretion is believed to be from injured cells.3 FGF
antibodies inhibit the initial activation of medial SMC DNA synthesis
and migration,17 18 but they have no effect on the
subsequent intimal SMC hyperplasia.8
Although the rat carotid balloon-injury model has been useful in characterizing SMC response, it has not been possible to determine if the activation of SMC growth requires the presence of other cell types and/or serum factors. Therefore, in the present study we established a cell-culture model to determine SMC response to cell damage under precisely controlled and defined conditions. The results demonstrate that activation of SMC growth in response to cell damage involves paracrine or autocrine stimulation by both FGF and PDGF A.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Materials
F-12/DMEM medium was purchased from GIBCO BRL, NCS from Gemini Bio-Products, and [3H]thymidine from DuPont NEN. Antibodies against SMC
-actin (HHF 35), PDGF A, and PDGF B used
for immunohistochemistry were kindly provided by Dr C. Harts,
University of Washington, Seattle. PDGF ß and receptor antibodies
for immunohistochemistry were kindly provided by Dr C.-H. Heldin,
University of Uppsala, Sweden. Antibodies against basic FGF for
immunohistochemistry as well as blocking antibodies against FGF and
PDGF AA were from R&D Systems. Immunopure ABC kits were purchased from
Pierce. The PDGF A chain cDNA probe was kindly provided by Dr C.
Betsholz, University of Gothenburg, Sweden.
Establishment of SMC Injury Model
SMCs were isolated by
collagenase digestion of rat
aorta.19 The cells were cultivated in F-12/DMEM medium
containing 10% NCS and 50 µg/mL gentamicin at 37°C in an
atmosphere of 5% CO2 in air. The purity of the SMC
populations was determined by the presence of muscle-specific
-actin immunoreactivity by using the HHF 35 antibody. Cells were
trypsinized and seeded on 22x22-mm glass coverslips placed in
six-well plates. They were allowed to grow to confluence in the
presence of 10% NCS and subsequently growth arrested by incubation in
serum-free F-12/DMEM medium containing 0.1% endotoxin-free BSA
for 48 hours. Before injury the cells were given fresh serum-free
medium with 0.1% BSA. The cultures were then injured by gentle
pressure with a 2-mm-wide soft plastic tube for 10 seconds.
Analysis of DNA Synthesis
[3H]thymidine
(final concentration, 2 µCi/mL)
was added to each well within 1 hour of injury. The cells were
incubated in this medium at 37°C for 24, 48, 72, or 96 hours and
fixed in 1% buffered paraformaldehyde. Alternatively,
the cells were given [3H]thymidine at 24, 48, or 72 hours
after injury and incubated in this medium for 24 hours before fixation.
The specimens were then dehydrated in ethanol and mounted on glass
slides. The slides were dipped in Kodak NTB2 emulsion, air-dried,
exposed at 4°C for 3 days, developed in Kodak D-19, and stained with
methylene blue. The fraction of labeled nuclei was determined by
counting at least 300 randomly selected cells on each coverslip.
Synthesis of Oligonucleotides
Phosphorothioate-modified
oligonucleotides
were synthesized by using an Applied Biosystems 394 DNA/RNA
synthesizer. The synthesized oligonucleotides were
freeze-dried, redissolved in water, and desalted by using Pharmacia
PD-10 columns. Concentrations were determined by using
spectrophotometric methods. The PDGF A sense
oligonucleotide sequence comprised the first six codons
of human PDGF A mRNA (5'-ATG AGG ACC TTG GCT TGC-3'), and the PDGF
A
antisense oligonucleotide comprised the complementary
sequence 5'-GCA AGC CAA GGT CCT CAT-3'. An antisense
oligonucleotide against c-fms mRNA (5'-TCA
AGC CCA GGT CCT CAG-3') with an 86% homology to the PDGF A antisense
oligonucleotide was used as an additional control.
Northern Blot Analysis
For Northern blot analysis, SMCs were
cultured in
100-mm dishes and serum starved as described above. They were then
injured by pressing an 8-mm elastic plastic tube on the cells. At the
indicated times the cells were scraped off the dishes with a rubber
policeman and suspended in 5 mL ice-cold sodium Tris EDTA buffer
(0.1 mol/L NaCl, 20 mmol/L Tris, and 10 mmol/L EDTA, pH 7.4). After
addition of proteinase K (0.3 mg/mL), the cells were
homogenized and incubated at 37°C for 30 minutes. Gel
electrophoresis of 20 µg total RNA was performed on 1.1% agarose
gels containing 2.2 mol/L formaldehyde. The filters were hybridized in
a buffer containing 50% formamide, 5x saline–sodium citrate
(43.8 g/L NaCl and 22 g/L sodium citrate), 5x Denhardt's solution
(1
g/L each polyvinylpyrrolidone, BSA, and Ficoll 400), 0.1% sodium
dodecyl sulfate, 100 mg/mL salmon sperm DNA, and 10% dextran
sulfate for 20 hours at 42°C with 32P-labeled DNA probes.
Probes were labeled with [-32P]dCTP by using the
random-primer technique according to the protocol of the
manufacturer (Stratagene, Inc). After hybridization, the filters were
washed twice for 30 minutes each time at 55°C in 0.1x
saline–sodium citrate/0.5% sodium dodecyl sulfate and
exposed to x-ray film.
Immunohistochemistry
Cultures were fixed in 3% buffered
paraformaldehyde at 2, 24, 48, 72, and 96 hours after
injury. They were then washed three times for 5 minutes each in PBS,
permeabilized by transfer to PBS/0.2% Triton X-100 for
5 minutes, rinsed in PBS, and incubated with PBS/1.5% blocking serum
(rabbit or horse, depending on the secondary antibody used) for 30
minutes. Excess blocking serum was blotted from the coverslips, and the
cells were then exposed to primary antibodies against -actin,
basic FGF, PDGF A, PDGF B, and PDGF and ß receptors for 18 hours
at room temperature in a humidifying chamber. Controls included use of
nonimmune sera instead of primary antibody as well as omission of the
primary antibody. After washing in PBS three times for 5 minutes each
time, the cells were incubated with biotinylated anti-rabbit or
anti-mouse IgG for 60 minutes, washed three times (5 minutes each
time) in PBS, and stained with avidin–biotinylated alkaline
phosphatase for 60 minutes. The cells were finally incubated with
alkaline phosphatase substrate solution for 30 minutes in darkness and
counterstained with hematoxylin.
Statistical Methods
Data are expressed as mean±SD and
were evaluated by unpaired
Student's t tests. A probability value of less than .05 was
taken as significant.
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Results |
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Morphology of the Injury
Injury resulted in formation of a well-defined, approximately 1- to 2-mm-wide longitudinal zone of injury in the cell culture. Light microscopic analysis of the injured zone immediately after injury showed areas of necrotic cells along the margin of the injury and in the injury itself as well as areas with complete denudation (Fig 1A
). Uptake of trypan blue was observed
in cells still remaining in the zone of injury as well as in cells
along the border region of the injury. At 24 hours cells could be
observed migrating into the zone of injury, and an increasing number of
viable cells accumulated in the injured area during the subsequent days
(Fig 1B).
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Activation of DNA Synthesis
The basal rate of DNA synthesis
in noninjured, confluent culture
serum starved for 48 hours was 0.4±0.1%. To assess the kinetics of
DNA replication, cultures were given a 24-hour pulse of serum-free
medium containing [3H]thymidine and 0.1% BSA at 0, 24,
48, and 72 hours after injury. Before exposure to thymidine cells were
kept in serum-free medium with 0.1% BSA alone. During the first 24
hours after injury, 10.7% of the cells in and around the zone of
injury had taken up [3H]thymidine, whereas only 0.3% of
the cells in uninjured areas were thymidine labeled (Fig 2). A
peak in DNA replicating activity was observed
between 24 and 48 hours after injury, with a thymidine labeling index
of 45.7% in cells in the zone of injury (Figs 1B
and
2). DNA
replication in the zone of injury subsequently declined, whereas a
minor increase in DNA replication was observed in noninjured areas
between 48 and 96 hours after injury.
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) and
noninjured (
) areas was determined by
autoradiography. Each value represents
mean±SD of triplicate determinations.
To study whether activation of
SMC DNA replication in injured SMC
cultures was due to loss of contact inhibition, DNA synthesis was
monitored in parallel nonconfluent cultures. In these experiments the
cells were serum starved for 48 hours and then incubated with fresh
serum-free medium. The thymidine labeling index in serum-free
sparse cultures never exceeded 6% during the 96 hours analyzed
(Fig 3). Experiments in which serum-starved cells
were exposed to medium containing 20% serum at 0, 24, 48, and 72 hours
suggested that the ability of nonconfluent cells to respond to mitogen
stimulation was unchanged throughout the entire experiment (Fig
3).
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) or 20% NCS (
DNA replication of cells in and around the wound in
injured SMC
cultures began to decline 3 days after injury. To study whether this
phenomenon was explained by an unresponsiveness of the cells due to the
prolonged exposure to serum-free medium, these cultures were
exposed to medium containing 20% NCS at 0, 24, 48, and 72 hours after
injury. The results showed that cells in the injury zone remained
responsive to serum mitogens at all times, whereas the response of
cells in noninjured areas decreased with time (Fig 4).
The mechanism responsible for this difference remains to be clarified,
but it may involve an increased sensitivity to contact inhibition with
prolonged serum starvation.
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) was determined by
autoradiography. Before the pulse the cells were
kept in serum-free medium containing 0.1% BSA. Each value
represents mean±SD of triplicate determinations.
Expression of PDGF A Immunoreactivity and mRNA in Injured
Cultures
To analyze if injury affected the expression of growth
factors and growth factor receptors, immunohistochemical studies using
polyclonal antibodies against human FGF, PDGF AA and BB, and PDGF
and ß receptors were used. All antibodies are known to
cross-react with the respective rat protein. A nonspecific binding
of antibodies to injured cells was seen in the wound area during the
first 2 hours after injury, making the immunohistochemical
analysis during this period inconclusive. There was no clear
difference in -actin expression between cells in and around the
wound and cells in noninjured areas of the cultures. A marked increase
in PDGF A chain immunoreactivity was observed in cells present in
and around the wound at 24 and 48 hours after injury (Fig 1C).
At this
time there was also a weak increase in FGF immunoreactivity in the
wound area (Fig 1D), but this was frequently associated with
damaged
cells, and the specificity of this FGF immunoreactivity was uncertain.
All cultures showed diffuse immune staining for PDGF and ß
receptors. The staining was similar in injured and noninjured
areas.
Uninjured SMCs had a low but clearly detectable expression of
PDGF A
chain mRNA. An eightfold increase in PDGF A chain mRNA (as assessed by
autoradiographic scanning) could be detected at 6 hours
after injury. At 24 hours after injury the PDGF A chain mRNA levels had
returned to baseline levels (Fig 5).
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Antibody and Antisense Blocking Experiments
The finding that
injury of growth-arrested SMC cultures
induced activation of DNA synthesis under serum-free conditions
indicates involvement of autocrine or paracrine mitogens. To
analyze the possible role of FGF released from dying cells,
injuries were performed in the presence of 40 µg/mL of a blocking FGF
antibody. Experiments in subconfluent cultures of SMCs demonstrated
that this antibody concentration completely inhibited the DNA synthesis
induced by 10 ng/mL recombinant basic FGF. The presence of this
antibody reduced the fraction of cells in the wound area replicating
DNA during the first 48 hours following injury from 28.3±6.0% to
7.0±2.6% (P<.005; Fig 6, left).
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The
finding of increased PDGF A mRNA levels and PDGF A immunoreactivity
in cells surrounding the wound suggested that injury-induced
activation of DNA synthesis also may involve autocrine stimulation by
PDGF A. However, incubation with PDGF A antibodies in a concentration
sufficient to completely inhibit the mitogenic activity of
10 ng/mL recombinant PDGF A did not affect the DNA synthesis
activated by injury (Fig 6, right). Since the PDGF A encoded by
some splicing variants (ie, the full-length transcript, which
includes exon 6) is retained within the cell,20 the lack
of inhibitory effect of PDGF-blocking antibodies could be
due to an inability of the antibody to reach and react with the
protein. To investigate this possibility, an antisense
oligonucleotide hybridizing a 15-bp sequence
surrounding the initiation codon of PDGF A mRNA was used. At a
concentration of 5 µmol/L, this antisense
oligonucleotide inhibited 62% of injury-induced
activation of DNA synthesis (Fig 7). Incubation with the
corresponding sense oligonucleotide caused a 29%
reduction in injury-induced DNA synthesis, indicating that part of
the effect of the antisense oligonucleotide was due to
general cytotoxicity. The same level of nonspecific inhibition was also
observed in the presence of an oligonucleotide
containing a 2-bp mismatch of the antisense sequence and an antisense
oligonucleotide against the c-fms mRNA
molecule, which shows an 83% sequence homology in this region.
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Effect of FGF on PDGF AA Expression
The observation that both
FGF antibodies and PDGF A antisense
oligonucleotides appeared to inhibit more than 50% of
injury-induced activation of DNA synthesis indicates that in this
system the mitogenic mechanisms of FGF and PDGF are not
independent of each other. To investigate this possibility, we
analyzed the effect of FGF on PDGF A mRNA accumulation by
Northern blotting. Exposure of lesion SMCs to 10 ng/mL recombinant
basic FGF for 6 hours resulted in a twofold increase in PDGF A mRNA
levels (Fig 8). Moreover, addition of PDGF A antisense
oligonucleotides inhibited almost 50% of FGF-induced
DNA synthesis in subconfluent lesion SMC cultures, whereas the sense
oligonucleotide inhibited FGF-induced DNA synthesis by
about 10% (Fig 9).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Damage to the medial SMC layer is a prerequisite for activation of intimal SMC proliferation in the intact blood vessel.13 This process is associated with increased expression of a number of growth factor genes in the injured artery.14 It has been difficult to determine which of these factors are of biological importance. Inhibition of balloon injury–induced activation of SMC DNA synthesis and replication by blocking antibodies indicate that FGF plays an important role during the first days after injury,17 whereas PDGF antibodies have no significant effect on cell proliferation.16 However, these studies are complicated by the difficulty of determining whether sufficient antibody concentrations reached the cellular level. Our results suggest that injury of confluent cultures of human SMCs may be a useful model for study of the mechanisms involved in injury-induced activation of SMC replication. By injuring cells through static pressure rather than removing the cells by scraping with a rubber policeman, the effects of growth factors released from dying cells still remaining in the wound can be assessed. This allows analysis of the SMC response to injury under defined serum-free conditions as well as the application of sufficient concentrations of blocking antibodies and antisense oligonucleotides. Injury-induced DNA synthesis in growth-arrested SMCs under serum-free conditions clearly indicates that growth of SMCs can be stimulated by factors released from injured or dying cells and that this process does not require the presence of any other cell type.
Several observations argue against the possibility that the activation of cell growth in the injured area is due to loss of contact inhibition. The fraction of [3H]thymidine-labeled cells in parallel, nonconfluent, and noninjured SMC cultures never exceeded 6% during the entire experiment. Nonconfluent cells are of course not an ideal control for the growth response of injured confluent cells. It is possible that the actual mechanical release from contact inhibition will allow cells arrested at a late stage of the G1 phase of the cell cycle to continue into S phase and divide for a single cell cycle. However, this would not explain continued DNA replication for up to 4 days. Moreover, although most cells present in the border region of the wound were completely surrounded by other cells, their thymidine labeling index was increased more than tenfold compared with cells in noninjured areas. Finally, the observation that FGF antibodies inhibit injury-induced DNA synthesis strongly argues against the possibility that this phenomenon is due to loss of contact inhibition.
The restriction of replicative activity to a few cell layers closest to disintegrated cells in the wound border and the inhibition of 75% of the DNA synthesis by FGF antibodies during the first 48 hours following injury suggest that FGF released from damaged cells plays an important role in the early activation of human SMC growth in response to injury. FGF is responsible for the early activation of SMC DNA synthesis in balloon-injured rat carotid arteries. The role of FGF in activation of SMC growth after balloon angioplasty in humans remains speculative. However, FGF immunoreactivity has been demonstrated both immediately after angioplasty as well as in manifest restenosis lesions.21
PDGF A chain mRNA expression is increased in balloon-injured rat carotid arteries within 6 hours of injury.14 SMCs isolated from the neointima that develops in these arteries in response to balloon injury produce a PDGF-like mitogen.22 These observations suggest autocrine stimulation of SMC growth by PDGF AA after injury. However, infusion of antibodies against PDGF did not reduce the rate of SMC DNA replication. The interpretation of these results are complicated by the difficulty of determining whether sufficient amounts of antibody penetrated the vascular wall. In our study we found increased expression of PDGF A mRNA levels after 6 hours and an increased PDGF A immunoreactivity in the wound region after 24 hours, but PDGF antibodies were without effect on DNA replication. This latter finding suggests that the inability of PDGF AA antibodies to inhibit SMC replication in vivo is not due to an insufficient penetration of the antibody into the vessel wall.
The inability of PDGF A antibodies to interfere with initiation of DNA synthesis after injury could be due to an interaction between PDGF AA and its receptor already inside the cell, ie, intracrine stimulation. We studied this possibility by using antisense oligonucleotides. Indeed, exposure of the cells to PDGF A chain antisense oligonucleotide almost completely inhibited injury-induced activation of DNA synthesis. This type of intracrine activation is responsible for stimulating the growth of v-sis–transformed fibroblasts23 (v-sis is the viral oncogene homologue of PDGF B). Interestingly, Behl and coworkers24 used the same antisense oligonucleotide to PDGF A to study autoinduction of growth by PDGF A in a melanoma cell line and found an almost identical level of inhibition as that observed in the present study. Several earlier studies have also demonstrated the existence of PDGF intracrine or autocrine loops in SMCs. The transition of rat SMCs from the contractile to the synthetic phenotype, which occurs early in primary culture as well as in balloon-injured arteries, is associated with the development of a transient capacity to grow in serum-free medium and the production of PDGF A.7 Similarly, neonatal rat SMCs demonstrate a high level of DNA synthesis under serum-free conditions and a continuous production of a PDGF-like mitogen.25 The stimulatory effect of interleukin-111 and transforming growth factor–ß12 on SMC DNA synthesis is due to activation of the endogenous production of PDGF A in the cells. Wilson and coworkers26 have demonstrated that activation of SMC DNA synthesis in response to mechanical strain is mediated by autocrine action of PDGF AA. Moreover, the growth-stimulatory effect of oxidatively modified LDL has also been attributed to an increased cellular expression of PDGF A.27 PDGF AA is a markedly less potent mitogen for SMCs than PDGF BB. Indeed, Inui et al28 have shown that in SMCs isolated from the aorta of spontaneously hypertensive rats PDGF AA increases protein synthesis but does not affect the rate of DNA synthesis. However, in SMCs isolated from the aorta of normotensive rats, PDGF AA clearly acts as a mitogen.29
The specificity of the biological effects of antisense oligonucleotides remains a matter of controversy. Several investigators have demonstrated the antiproliferative effects of antisense oligonucleotides against c-myb and c-myc on SMCs both in vitro and in vivo.30 31 However, recent evidence has shown that this effect is due to a nonspecific effect of a 4G sequence in the oligonucleotide rather than by inhibition of c-myb and c-myc production.32 Although no 4G sequence was present in the PDGF A antisense oligonucleotide used in our study, the results should still be interpreted with some caution. The specificity controls used in the present study included the sense oligonucleotide and an oligonucleotide containing a 2-bp mismatch. Both gave rise to a limited reduction in DNA synthesis.
Blocking antibodies to FGF and PDGF A antisense oligonucleotides inhibit injury-induced activation of DNA synthesis by more than 50%, which suggests that the two growth factors may not act independently. This notion is supported by the observations that FGF increases PDGF A mRNA expression with similar kinetics as mechanical injury and that addition of PDGF A antisense oligonucleotides inhibits part of the mitogenic effect of recombinant FGF. Thus, the growth-stimulatory effects of FGF may include both a direct, immediate activation of the mitogenic signal cascade as well as an indirect, sustained effect mediated by stimulation of PDGF A production.
In summary, the present findings demonstrate that cultured human SMCs may be used for studies of the mechanisms involved in injury-induced activation of SMC proliferation. They further suggest that SMC proliferation in and around the zone of injury is caused by the concerted action of FGF released from damaged cells and an FGF-induced intracrine PDGF A loop.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the American Heart Association, the Swedish Medical Research Council, the Swedish Heart and Lung Foundation, the Grand Foundation, the Tore Nilssons fund, the Swedish Society of Medicine, and the King Gustaf Vth 80 Birthday Fund.
Received August 14, 1995; accepted October 23, 1995.
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