© 1999 American Heart Association, Inc.
Original Contribution |
Tumor Necrosis Factor-
Upregulates Angiotensin II Type 1 Receptors on Cardiac Fibroblasts
From the Department of Medicine, Division of Cardiology, University of California, San Diego Medical Center, San Diego, Calif.
Correspondence to Barry Greenberg, MD, Department of Medicine/Cardiology, UCSD Medical Center, 200 W Arbor Dr, San Diego, CA 92103-8411. E-mail bgreenberg{at}ucsd.edu
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Abstract |
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Abstract—Angiotensin II (Ang II) plays an important role in post–myocardial infarction (MI) remodeling. Most Ang II effects related to remodeling involve activation of the type 1 receptor (AT1). Although the AT1 receptor is upregulated on cardiac fibroblasts post-MI, little is known about the mechanisms involved in the process. Consequently, we tested whether growth factors known to be present in the remodeling heart increased AT1 mRNA levels. Using quantitative competitive reverse transcription–polymerase chain reaction, we found that norepinephrine, endothelin, atrial natriuretic peptide, and bradykinin had no significant effect on AT1 mRNA levels. Ang II, transforming growth factor-ß1, and basic fibroblast growth factor reduced AT1 mRNA levels (P<0.02). Tumor necrosis factor-
(TNF-), however, produced a marked increase in
AT1 mRNA. After 24 hours of TNF- incubation,
AT1 mRNA increased by 5-fold above control levels
(P<0.01). The EC50 for the TNF- effect
was 4.6 ng/mL (0.2 nmol/L). Interleukin (IL)-1ß caused a 2.4-fold
increase, whereas IL-2 and IL-6 had no significant effect. Studies of
TNF- enhancement of AT1 mRNA levels demonstrate that the
increase was not due to a change in transcript stability. TNF-
treatment for 48 hours also resulted in a 3-fold increase in
AT1 surface receptor and a 2-fold increase in Ang
II–induced production of inositol phosphates. The present
findings provide evidence for TNF- regulation of AT1
receptor density on cardiac fibroblasts. Because TNF- concentration
and AT1 receptor density increase in the
myocardium after MI, these results raise the possibility
that TNF- modulates post-MI remodeling by enhancing Ang II effects
on cardiac fibroblasts.
Key Words: AT1 • cardiac fibroblast • tumor necrosis factor- • post–myocardial infarction remodeling
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Introduction |
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There is considerable evidence that the renin-angiotensin system plays an important role in post–myocardial infarction (MI) cardiac remodeling. After MI, the renin-angiotensin system is activated both systemically1 and locally within the heart.2 Angiotensin II (Ang II) is a growth factor that can stimulate processes known to be associated with cardiac remodeling, such as myocyte growth (hypertrophy) and extracellular matrix (ECM) protein synthesis (fibrosis3–7). Moreover, the administration of angiotensin-converting enzyme inhibitors8–11 and Ang II receptor blockers12 13 inhibits post-MI remodeling8 9 12 13 and reduces mortality10 11 in experimental animal models. Angiotensin-converting enzyme inhibitors also reduce mortality in MI survivors.14 15
Cardiac fibroblasts are involved in post-MI remodeling through the generation of replacement scar tissue in the infarct zone and the production of fibrosis in noninfarcted segments of myocardium.2 Ang II stimulation of cardiac fibroblasts increases cell division16 and enhances production of ECM proteins such as fibronectin and collagens.4 5 Ang II also induces cardiac fibroblasts to secrete a paracrine growth factor(s) that stimulates hypertrophy of cardiac myocytes.6 These growth-promoting effects of Ang II are mediated through the Ang II type 1 receptor, AT1. Stimulation of this G protein–coupled receptor results in the activation of phospholipase C, the production of inositol phosphates (IP), and a rise in intracellular calcium.7 17 There is evidence that AT1 mRNA levels and receptor density are increased after MI18 19 and that these changes occur predominantly in cardiac fibroblasts.19 20 Although upregulation of the AT1 receptor would be expected to enhance fibroblast activities involved in post-MI remodeling, little is known about the mechanism(s) responsible for this increase in receptor density.
A variety of growth factors, including neurotransmitters, hormones, and
cytokines, are increased systemically and/or locally in the
heart after MI. Many of these agents are known to modulate fibroblast
activities such as cell proliferation and ECM synthesis.21
Thus, we hypothesized that some of these factors may be involved in the
post-MI regulation of the AT1 receptor density on
cardiac fibroblasts. In initial experiments, we assessed the effects of
selected candidate agents on AT1 mRNA levels in
neonatal rat cardiac fibroblasts. Results derived from the testing of
various humoral candidates indicate that tumor necrosis factor-
(TNF-) has a unique capacity to substantially increase
AT1 mRNA levels.
TNF- is a pleiotropic cytokine that plays an important role
in the response to tissue injury and wound healing.22
Increased amounts of this cytokine have been detected in
regions of the infarcted heart23 24 25 26 where
AT1 upregulation is known to occur. Consequently,
we proceeded to characterize TNF- effects on
AT1 mRNA levels. In this study, we demonstrate
that TNF- enhancement of AT1 mRNA levels is
not due to a change in transcript stability. Increases in mRNA levels
are associated with increases in receptor density and with the enhanced
production of IP in response to Ang II treatment. Thus, these
findings provide evidence that TNF- increases the density of
functional AT1 receptors on cultured cardiac
fibroblasts and suggest a potential important in vivo role for this
cytokine in the setting of MI.
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Materials and Methods |
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Cell Cultures
Neonatal rat cardiac fibroblasts were prepared from hearts of 1- to 2-day-old Sprague Dawley rats as described by Kim et al.6 For each experiment, cells were plated from frozen stock (passage 0) in medium (DMEM high glucose; Gibco-BRL) containing 10% FBS. At 90% confluency, medium was replaced with serum-free medium for 24 hours, and cultures were treated according to the experimental design. Fibroblasts were exposed to norepinephrine (NE, Sigma), Ang II (Sigma), human basic fibroblast growth factor (bFGF; R&D Systems), human transforming growth factor-ß1 (TGF-ß1; R&D systems), atrial natriuretic peptide (ANP; Peninsula Laboratories), endothelin (ET; Peninsula Laboratories), bradykinin (BK; Sigma), rat recombinant interleukin (IL)-1ß, human recombinant IL-6, and rat recombinant IL-2 (all from R&D Systems) in serum-free medium for 24 hours. (See Figure 2
for doses used.) Recombinant rat
TNF- (Biosource International) was used for all experiments and
administered according to the experimental protocol. Actinomycin D was
obtained from Calbiochem. Rabbit polyclonal anti-TNF- antibody
was purchased from Genzyme Diagnostics and Biosource
International.
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Isolation of Total RNA and Competitive Quantitative Reverse
Transcription–Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using the Qiagen RNeasy kit.
Amplification of AT1 mRNA was performed using the
Titan 1-tube RT-PCR system (Roche). Reverse transcription was carried
out for 45 minutes at 50°C and PCR for 26 cycles each of 94°C for 1
minute, 42°C for 1 minute, and 68°C for 1.5 minutes. To exclude the
possibility that contaminating genomic DNA may be amplified, control
experiments were carried out in the absence of the reverse
transcriptase.
The AT1 PCR forward and reverse primers have been
previously described, and the reverse primer served as the reverse
transcription primer as well.27 The quantification of
RT-PCR products is illustrated in Figure 1. In these experiments, the DNA fragment
of the target AT1 gene was amplified from 3
quantities of RNA obtained from the same sample. Amplification was done
in the presence of a constant copy number of synthetic deletion mutant
cRNA of an AT1A gene clone from which 63 bp were
removed (bases 502 to 564, kindly provided by Dr Eric Clauser,
Collège de France, Paris, France).27 The
primers used in these experiments amplify mRNA from both
AT1A28 and AT1B29
subtypes. The DNA products can be distinguished from each other by
the presence of a unique EcoRI restriction site on
the AT1A subtype. Restriction digests with
EcoRI revealed that AT1 PCR fragments
from both the control and TNF-–treated rat fibroblasts were of the
AT1A type. AT1B PCR
fragments were not detected in these experiments.
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, untreated) is plotted against log RNA
concentration. The amount of RNA containing the same number of
AT1 mRNA molecules as the mutant is denoted with an arrow
and represents equal amplification.
The ethidium bromide gels depicted in Figure 1A
illustrate the
competition created between the amplification of the wild-type (target)
and the deletion mutant PCR products. The amplification
products of the target and mutant mRNA were equal when the input
RNA concentrations of the target AT1 mRNA matched
that of the cRNA mutant. This point was derived by extrapolation, as
illustrated in Figure 1B. To obtain the numerical value of
AT1 mRNA levels, the intensity of the bands was
determined from digitized images of gel. The values for the
mutant-derived bands were corrected for the difference in fragment size
due to the deletion, and the values for the log target/mutant (band
intensity) were plotted against log RNA concentration. The points were
then fitted with a linear line, and the value of x when
y=0 was considered the value of the RNA concentration that
contains the same number of AT1 mRNA molecules as
the mutant cRNA. AT1 mRNA molecules per nanogram
of total RNA was derived from that number. When
AT1 mRNA was induced after TNF- treatment, the
amount of RNA used in the assay was reduced accordingly, as shown in
Figure 1A.
Receptor Binding Assays
Binding of Ang II was performed on intact adherent cells plated
in multiwell plates, which had been treated with 100 ng/mL TNF- for
48 hours. The procedure used was described by Villarreal et
al,4 with modifications according to Widdowson et
al.30 The modification included the use of varying
concentrations of 0.1 to 10 nmol/L [3H]Ang II
(Amersham Life Science) for total binding, incubation of the cells with
ligand for 2 hours at 4°C, and protein determination from an aliquot
of each culture dish well. On the basis of specific activity,
[3H]Ang II counts were converted to fmol Ang II
bound and normalized per mg of protein. Nonspecific binding was
determined in the presence of "cold" Ang II, and competition for
binding was assessed in the presence of losartan and PD123319
(RBI). Maximal binding, Bmax, and the
dissociation constant, Kd, were derived in
2 ways. Specific binding was plotted against
[3H]Ang II concentration and fitted to a
hyperbolic curve according to the equation
B=Bmaxx[[3H]Ang
II]/(Kd+[[3H]Ang
II]), where B is amount of [3H]Ang
II bound and Bmax and
Kd are derived (Figure 5) using the
Prism program (GraphPad). Bmax and
Kd were also determined by means of
Scatchard plot analysis.
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Cell Counts
Cells were plated and treated in a fashion identical to that for
binding studies. Medium was removed and saved. After trypsinization,
cell suspension in a known volume was pooled with the original cell
medium. Cells were counted in the presence of trypan blue to identify
nonviable cells.
Production of Inositol Phosphates
For the determination of Ang II–induced IP
production, fibroblasts were plated and treated for 48 hours
with TNF- (100 ng/mL) in inositol-free medium. During the last 24
hours of TNF- treatment, cells were loaded with
[3H]myo-inositol (NEN). Isolation of
IP was performed according to a previously described
procedure.31 IP production in response to Ang
II exposure over the range of 10-9 to
10-6 mol/L were measured at 45 minutes in
the presence of LiCl (10 mmol/L). To determine the effect of Ang
II receptor antagonists, cells were incubated with either
losartan (AT1 antagonist) or
PD123319 (AT2 antagonist). Because
cell counts indicated minimal variations in cell number among wells,
data were expressed as counts per well.
Data Analysis
Data are presented as mean±SEM. Significant differences
were determined by t test or ANOVA. Curve fits were
generated using the Prism computer program (GraphPad). A P
value <0.05 was considered statistically significant.
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Results |
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Modulation of AT1 mRNA Levels
The possibility that growth factors present in the remodeling heart might be playing a role in the regulation of AT1 receptor density was initially assessed by measuring the effect of these agents on AT1 mRNA levels. Control AT1 mRNA levels in the cultures of untreated neonatal cardiac fibroblasts ranged from 3000 to 8000 molecules per nanogram RNA with an average of 5667±536 (n=14). The effects of the various agents on AT1 mRNA levels were expressed as change from control levels within each experiment (Figure 2
). NE, ET, ANP, and BK
did not significantly affect AT1 mRNA levels. Ang
II, TGF-ß1, and bFGF each significantly reduced
AT1 mRNA levels to 38%, 24%, and 44% of
control levels, respectively (all P<0.02). Exposure to
TNF-, however, increased AT1 mRNA nearly
5-fold above control levels (P<0.01). IL-1ß increased
AT1 mRNA levels by 2.4-fold (P<0.04),
whereas IL-6 and IL-2 had no significant effects on
AT1 mRNA levels.
TNF- Effects on AT1 mRNA Are Time and Dose
Dependent
To characterize the time dependence of the TNF- effect, total
RNA was extracted from cardiac fibroblasts at 2, 6, 12, 24, and 48
hours after exposure to serum-free medium containing TNF- (10 ng/mL,
0.57 nmol/L) and from control fibroblasts exposed to serum-free medium
in the absence of TNF- (Figure 3A).
TNF- treatment resulted in an increase in AT1
mRNA as early as 6 hours after exposure. AT1 mRNA
levels reached a 5-fold (P<0.01) increase above control
levels by 24 hours, and this effect was maintained at 48 hours.
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Dose dependency of TNF- on AT1 mRNA levels was
determined at 24 hours of exposure. TNF- concentrations used for the
dose response determination ranged from 0.1 to 500 ng/mL (6 pmol/L to 3
nmol/L, Figure 3B). The TNF- effective dose for 50%
AT1 mRNA upregulation was found to be 4.6 ng/mL
(0.26 nmol/L).
In the above experiments, cardiac fibroblasts were exposed to a single
application of TNF- for up to 48 hours. The effects of prolonged
exposure to TNF- were determined by applying 100 ng/mL TNF- to
fibroblasts every 48 hours for up to 6 days. The results showed that
AT1 mRNA levels increased to 8.4-fold of basal
levels after 4 days and were 7-fold higher after 6 days. Removal of
TNF- after 48 hours of exposure resulted in the return of
AT1 mRNA to basal levels within 3 days.
To exclude the possibility that impurities in the TNF- or the
release of autocrine factors from fibroblasts were responsible for the
upregulation of the AT1, experiments were
performed in the presence of neutralizing antibodies to TNF-. The
use of TNF- antibodies either concomitantly with TNF- (n=2) or
with conditioned medium from fibroblasts pretreated with TNF- (n=1)
suppressed the induction of AT1 mRNA.
TNF- Does Not Increase AT1 mRNA Levels by Enhancing
Message Stability
Previous studies had demonstrated that selected humoral agents can
increase AT1 receptor density by enhancing
transcript stability (eg, insulin treatment of rat vascular smooth
muscle cells32 ). Thus, the effect of TNF- on
AT1 mRNA stability was investigated. After
induction of cardiac fibroblast AT1 mRNA levels
by TNF- (50 ng/mL) for 24 hours, cultures were treated with the
transcription inhibitor actinomycin D (5 µg/mL) in the
continuing presence of TNF-. Total RNA was then extracted from
fibroblasts at various defined intervals. AT1
mRNA levels were quantified by RT-PCR as described above. Figure 4 depicts the fraction of
AT1 mRNA levels relative to the levels at the
beginning of actinomycin D treatment (time=0) for untreated and
TNF-–treated cultures (mean±SEM, n=3). Although TNF- enhanced
AT1 mRNA levels in a manner depicted in Figure 3, it did not alter the rate of mRNA degradation in the presence
of actinomycin D, which indicates that it did not increase the
stability of the AT1 transcripts.
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TNF- Enhances Density of AT1 Cell Surface
Receptor
Binding studies were performed to determine whether TNF-
increased cell surface AT1 receptor density.
Figure 5A illustrates that total binding
levels for [3H]Ang II (10 nmol/L) were
approximately doubled at 24 hours and further increased to 3.8 above
control levels by 48 hours, whereas nonspecific binding in the presence
of excess unlabeled Ang II (10-6 mol/L) remained
essentially unchanged. Addition of the selective
AT1 antagonist losartan
(10-5 mol/L) resulted in nearly complete
displacement of bound radioligand, whereas addition of the
AT2 antagonist PD123319
(10-5 mol/L) had no significant effect, which
demonstrates that an increase in AT1 receptor
density was the primary cause for the increase in Ang II binding to
cultured fibroblasts.
Saturation binding experiments were performed to obtain the
dissociation constant (Kd) and receptor
density (Bmax) for control and TNF-–treated
cardiac fibroblasts. Although the dissociation constant for binding was
not significantly different in control fibroblasts (4.12±0.58 nmol/L,
n=3) and TNF-–treated fibroblasts (4.53±0.47 nmol/L, n=3),
Bmax in treated cells (1313±124 fmol/mg protein)
was 2.8-fold higher (P<0.01, n=3) than
Bmax in control cells (466±70 fmol/mg protein).
Data from a representative experiment are illustrated
in Figure 5B and 5C.
The effect of TNF- on cell proliferation was assessed to
determine whether increases observed in surface receptor density were
due to an increase in cell number. No significant differences in the
number of viable or dead cells between control and TNF-–treated
fibroblasts was noted (25 wells counted per each cell group in 4
individual experiments). On the basis of the number of cells per well,
total protein level per well, and level of Ang II binding, the number
of AT1 receptors was estimated to be
105 per cell.
Upregulation of AT1 Receptor by TNF- Results in
Enhancement of Ang II–Induced IP Production
Figure 6 illustrates levels of Ang
II (10-9 to 10-6
mol/L)–induced IP production in untreated fibroblasts or
fibroblasts pretreated for 2 days with TNF- (100 ng/mL). Control
levels used for normalization of the data in each experiment were
derived from fibroblasts treated with neither TNF- nor Ang II
(Figure 6, control [0 mol/L Ang II]). In untreated
fibroblasts, Ang II stimulated similar levels of IP production
throughout a concentration range of 10-9 to
10-6 mol/L. Pretreatment of the cells with
TNF-, however, altered the profile of IP production. Basal
levels (0 mol/L Ang II) were reduced to one third of the corresponding
levels of untreated cells (P<0.001, n=3). Increasing
concentration of Ang II progressively increased IP production
in TNF-–treated cells, reaching maximal levels at
10-7 to 10-6 mol/L Ang
II, with a significant 2-fold increase observed at
10-6 mol/L Ang II relative to untreated cells
(Figure 6, P<0.003, n=3). PD123319
(10-5 mol/L), the AT2
antagonist, did not significantly affect IP
production, whereas losartan
(10-5 mol/L), the AT1
antagonist, produced essentially complete blockade when
used in 100-fold excess.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ang II activation of cardiac fibroblasts plays an important role in post-MI cardiac remodeling. Most of the known effects of Ang II on cardiac fibroblasts are mediated through the AT1 receptor, and there is evidence that this receptor is upregulated after MI. The present study shows that TNF-
increases
AT1 mRNA levels in neonatal rat cardiac
fibroblasts. The increase in AT1 mRNA levels is
associated with an increase in membrane receptor density and enhanced
production of Ang II–stimulated second messengers within the
cell. Because TNF- has been identified in the post-MI heart, these
findings raise the possibility that TNF- may play an important role
in modulating post-MI remodeling through its effects on cardiac
fibroblast AT1 receptors.
Potential Neurohormonal Modulators of the AT1
Receptor
A variety of agents that could potentially alter cardiac
fibroblast function have been identified in the heart after MI or
during the development of heart failure. To examine whether one or more
of these agents may be involved in the regulation of the
AT1 receptor, cultured neonatal fibroblasts were
exposed to a group of preselected candidate neurotransmitters, growth
factors, and cytokines. In vitro experiments are advantageous
for this purpose in that a selected agent can be examined in the
absence of systemic effects. The results demonstrate that TNF-
markedly increased AT1 mRNA levels. IL-1ß
produced a smaller increase. A previous study done using cultured rat
vascular smooth muscle cells showed that IL-1ß produced an increase
in AT1 mRNA of magnitude similar to that observed
in cardiac fibroblasts.33 Interestingly, in that study,
TNF- had no significant effect on AT1 mRNA
levels, which suggests that the upregulation seen in cardiac
fibroblasts in the present study is cell or tissue specific.
The reduction by Ang II and lack of any significant effect with ET seen in these experiments are consistent with reported observations in cardiac fibroblasts.34 However, our results with NE treatment contrast with those previously reported, which noted a modest 60% upregulation of AT1 mRNA levels after a 24-hour treatment of neonatal cardiac fibroblasts with NE.34 TGF-ß1, the secretion of which from cultured cardiac fibroblasts is stimulated by Ang II,21 decreased AT1 mRNA to 24% of control levels. A similar reduction in AT1 mRNA levels was seen with bFGF. A decrease in AT1 mRNA levels induced by bFGF has been seen in vascular smooth muscle cells and has been attributed to a decrease in AT1 gene transcription rate and destabilization of the AT1 message.35
Induction of AT1 mRNA Levels
The increase in AT1 mRNA with TNF-
is seen as early as 6 hours after exposure to the cytokine and
peaks at 24 hours after treatment. Maximal effect of a single dose led
to a 5-fold increase in mRNA levels, whereas continued application of
TNF- over 6 days resulted in a progressive increase in mRNA levels.
Removal of the cytokine was associated with a return to basal
levels within 3 days. These observations may have implications relevant
to the in vivo setting after MI. AT1 is
upregulated after MI at the peri-infarction zone19 13
predominantly on cardiac fibroblasts.20 Investigators
studying the post-MI heart have reported that macrophages
infiltrating the necrotic region and its border zones, including cells
surrounding the vasculature, appear to be involved in
production of TNF-.23 26 This
production of TNF- is sustained over an extended period in
both the border zone and remote segments of the
myocardium.26 Thus, the continued presence of
high levels of TNF- in the border zone and noninfarcted regions of
the myocardium is consistent with the possibility
that TNF- may be responsible for the regulation of
AT1 seen in these regions.19 20
Increased AT1 mRNA stability and/or
enhancement of rate of AT1 gene transcription
could account for enhancement of AT1 mRNA levels.
However, the degradation rate of AT1 mRNA was
unaffected by TNF- treatment, which indicates a lack of effect on
message stability. Future work should address the possibility of
enhancement of transcription and the identification of gene enhancer
sequences that are responsive to TNF-. The promoter for
AT1A has been isolated, and putative response
elements have been identified on the basis of sequence
analysis.36 Although the presence of putative
response elements to TNF-, such as activator protein-1
and nuclear factor-
B,37 38 have been identified on the
AT1 gene promoter, a systematic analysis
of their activity in the cardiac fibroblasts is required.
Induction of Density of Functional AT1
Receptors
The increase in mRNA was followed by an increase in
AT1 surface receptor density without change in
receptor affinity. Our results also showed that upregulation of the
AT1 mRNA and receptor density occurs on
individual cells rather than as a result of TNF-–induced cell
proliferation. Ang II–induced synthesis of IP has been previously
described and is associated with AT1
activation.7 Our data demonstrate that increased
AT1 receptor density in response to TNF-
results in the enhanced production of second-messenger IP by
Ang II.
Potential Role of Fibroblast AT1 Receptors in
Cardiac Remodeling
This study provides evidence that TNF- is involved in the
upregulation of AT1 receptor density on cardiac
fibroblasts in the post-MI rat heart. The significance of this
observation is related to the role that Ang II activation of the
AT1 receptor on cardiac fibroblasts plays in
post-MI cardiac remodeling. Previous studies have shown that
AT1 receptors are substantially more abundant on
cardiac fibroblasts than on cardiac myocytes.6 39 Exposure
of cardiac fibroblasts to Ang II leads to an increase in cell
number.16 Ang II also stimulates production of the
ECM proteins4 5 collagens I and III and fibronectin and
other substances, such as
TGF-ß1,40 which are related to the
deposition of interstitial matrix and scar formation. Ang
II also induces cardiac fibroblasts to produce a paracrine factor(s)
that stimulates myocyte hypertrophy.6 All of
these effects of Ang II on cardiac fibroblasts are mediated through the
AT1 receptor. Increased density of
AT1 receptors on cardiac fibroblast after MI
would be expected to enhance Ang II–mediated effects on the remodeling
process. The Ang II type 2 receptor, AT2, has
been reported to mediate antigrowth and antiproliferation
functions.41 However, we found no evidence of
AT2 upregulation in binding studies.
In summary, the results of this study demonstrate that TNF-
increases the density of functional AT1 receptors
on cardiac fibroblasts. These findings identify a previously
unrecognized association that could link the effects of disparate
systems that are believed to be involved in post-MI remodeling.
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Acknowledgments |
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This study was supported in part by the National Heart, Lung, and Blood Institute (Grant NL-03160, to F.J.V.). We thank Dr W. H. Dillmann for his helpful suggestions and Juan Alvergue for technical assistance.
Received December 22, 1998; accepted May 25, 1999.
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