Vascular Biology |
From the Division of Biomedical Sciences (N.W., L.V., Y.Z., M.B.S.), University of California, Riverside; Chiron Corp (S.H.), Emerville; and Elan Pharmaceuticals (J.F.), Menlo Park, Calif.
Correspondence to M.B. Stemerman, MD, Division of Biomedical Sciences, University of California, Riverside, CA 92521. E-mail michael.stemerman{at}ucr.edu
| Abstract |
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B. Because the induced expression of ICAM-1 and MCP-1 in ECs
has been implicated in endothelial activation and a
number of important vascular disorders, it is suggested that AP-1
activation may play an important role in the pathogeneses of
inflammation, angiogenesis, and
atherogenesis.
Key Words: endothelial activation AP-1 adenovirus adhesion molecules chemokines
| Introduction |
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B (NF-
B) has
been shown to play a pivotal role in EC activation,2 3 4
increasing evidence indicates the importance of the transcription
factor activator protein-1 (AP-1) in
endothelial homeostasis and dysfunction. AP-1 is a
regulatory protein complex composed of members of the Jun and Fos
families. These trans-acting elements interact with AP-1
binding sites in target genes to mediate cellular responses to
environmental stimuli.5 In ECs, AP-1 activity can be
induced by a variety of factors, such as
cytokines,6 bacterial endotoxin,
antioxidants,7 hypoxia,8 fluid shear
stress,9 10 11 and LDL.12 Sequence
analysis indicates that AP-1 binding sites are recurrent
elements in the promoters of genes encoding adhesion molecules and
chemokines. However, the regulatory effects of AP-1 proteins in human
ECs remain unclear. It is not known whether AP-1 proteins are
sufficient to induce phenotypic changes or whether they must act in
concert with other effectors. Using an adenovirus-mediated gene
transfer protocol, we found that the AP-1 proteins c-Fos and c-Jun
directly cause phenotypic activation and induction of intercellular
adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1
(MCP-1) in human ECs. The coinduction of ICAM-1 and MCP-1 may
represent coordinated events in endothelial
activation processes, such as inflammation, angiogenesis, and
atherogenesis. | Methods |
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5). An intermolecular recombination of the shuttle
vector and
5 then creates a new virus that carries the
c-jun or c-fos cDNA under the control of a
tetracycline-responsive transactivator
(tTA).14 A recombinant adenovirus expressing tTA (AdtTA)
was constructed with the tTA cDNA and upstream cytomegalovirus promoter
from pUHD15-1.14 An adenovirus expressing the
ß-galactosidase gene (Adß-gal) was constructed from pUHC13-3 as
described previously.15 The recombinant adenovirus
carrying the gene for the inhibitor of NF-
B
(rAdI
B
, provided by Dr F.H. Bach, Harvard Medical School, Boston,
Mass) was constructed to express the porcine I
B
gene
(ECI-6).16 The adenoviruses were plaque-purified,
amplified, and titered in 293 cells.17 The functional
titers as determined by plaque assays on 293 cells were
4x1011 plaque-forming units per milliliter (AdtTA),
1.6x1011 pfu/mL (Adß-gal), 1.6x1011 pfu/mL
(AdJun), 1.2x1011 pfu/mL (AdFos), and
1.3x1011 pfu/mL (rAdI
B
).
For adenoviral infection, confluent human umbilical vein
endothelial cells (HUVECs) were exposed to adenoviral
vectors at a multiplicity of infection (MOI) of 100 for 2 hours. Unless
specified otherwise, AdtTA was added with AdJun, AdFos, or Adß-gal to
induce transgene expression (with a combined MOI of
100). The cells
were incubated for the indicated time courses after viruses were washed
off.
Cell Culture and Reagents
HUVECs were harvested by collagenase treatment of
umbilical cords and cultured on plates coated with collagen. Cells were
maintained in medium 199 supplemented with 20% FBS, 20 mmol/L
HEPES (pH 7.4), 1 ng/mL recombinant human fibroblast growth factor, 90
µg/mL heparin, and antibiotics.12 Cells within 3
passages were used in these experiments. Phorbol 12-myristate
13-acetate (PMA) was purchased from Sigma Chemical Co, human
recombinant tumor necrosis factor-
(TNF-
) from Knoll
Pharmaceutical, and carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG-132)
from Biomol.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated by using Trizol reagent (GIBCO/BRL),
fractionated on formaldehyde/agarose gels, transferred to a nylon
membrane, and hybridized to random-primed cDNA probes for human
c-jun, c-fos, ICAM-1, MCP-1, and von
Willebrand factor.
Protein Extraction and Western Blot Analysis
Nuclear proteins were extracted from HUVECs as previously
described.12 Protein concentration was measured with the
bicinchoninic acid protein assay kit (Pierce). Twenty micrograms of
protein per sample was resolved by SDSpolyacrylamide gel
electrophoresis (PAGE), transferred onto an Immobilon-P membrane
(Millipore), and analyzed with primary rabbit polyclonal
antibodies to c-Jun (1:2000), c-Fos (1:1000), I
B
(1:1000)
(all from Santa Cruz), and a horseradish peroxidaseconjugated
secondary antibody (sheep anti-rabbit, 1:5000; Sigma), followed by
enhanced chemiluminescence detection (Amersham).
Electrophoretic Mobility Shift Assay (EMSA)
Six micrograms of nuclear extract was mixed with 1 µg of
poly(dI-dC) and DNA binding buffer and incubated at room temperature
for 10 minutes. 32P-labeled
oligonucleotides were then added to the reaction and
incubated for 20 minutes at room temperature. The
oligonucleotides were end-labeled by using T4
polynucleotide kinase and [
-32P]ATP (ICN).
Additionally, a 100-fold molar excess of unlabeled competing
oligonucleotides was used in some experiments to test
the specificity of binding. The sequences of
oligonucleotides are as follows: (1) consensus AP-1,
5'-GCT TGA TGA GTC AGC CGG AA-3'; (2) NF-
B, 5'-AGT TGA GGG GAC TTT
CCC AGG C-3'; (3) SP1, 5'-ATT CGA TCG GGG CGG GGC GAG C-3'; (4) GATA,
5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3'; (4) ICAMTPA-responsive
element (TRE) (-321), 5'-TAG ACC GTG ATT CAA GCT TAG-3'; (5)
ICAMNF-
B (-223), 5'-TTT AGC TTG GAA ATT CCG GAG CT-3'; (6)
ICAMAP-1/Ets (-940), 5'-GCT GCT GCC TCA GTT TCC CAG-3'; and (7)
ICAMTRE/AP-1 (-1290), 5'-TGG CCA GTG ACT CGC AGC CCC-'3.
Reporter Gene Constructs and Transactivation Assays
Construction of promoter-luciferase plasmids pGLAP-1Luc and
pGL2-Luc was previously described.12 In brief,
pGLAP-1Luc contains 3 consensus AP-1 binding sites upstream from an
HSV-TK TATA box. As a control reporter plasmid, pGL2-Luc contains only
the TATA box in a pGL2 basic vector (Promega). Plasmids pRSVß-gal
and pGL1.3 were provided by Dr T. Parks (Boehringer Ingelheim
Pharmaceuticals Inc, Ridgefield, Conn). pGL1.3 contains 1344 bp of the
human ICAM-1 upstream region fused to the luciferase reporter
gene.18 For the transactivation assay, HUVECs grown in
6-well plates were first transfected with 6 µg of plasmid DNA,
consisting of a mixture of 5 µg of a promoter-luciferase construct
and 1 µg of pRSVß-gal plasmid, by the calcium phosphate
precipitation method. After 6 hours, the cells were exposed to AdtTA
with AdJun and/or AdFos for 2 hours. AdtTA alone was used as the
control. Forty-eight hours after the transfection, cell lysates were
harvested to measure luciferase activity. The ß-gal activity was also
measured to normalize transfection efficiency.
Statistical Analysis
Quantitative data were expressed as mean±SEM. Statistical
analysis was performed using the Student's t test.
Differences were considered significant when probability values were
<0.05. For nonquantitative data, the results were expressed as
representatives of at least 3 independent
experiments.
| Results |
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100% of HUVECs. The protein expression of transgenes in HUVECs can
be detected as early as 16 hours after infection, reaching a peak at 24
hours. At the titers tested (up to 500 MOI), infection with Adß-gal
and AdtTA did not cause either cytopathological changes or the
nonspecific induction of endogenous genes, such as
c-jun, c-fos, and ICAM-1 in HUVECs. Similarly,
the adenoviruses expressing c-jun and c-fos were
generated. After infection of HUVECs with AdFos or AdJun, total RNA and
nuclear protein were extracted to examine the expression of
c-fos and c-jun mRNA and protein. As shown in
Figure 1C
B probes could completely compete
for the binding induced by overexpression of c-jun and
c-fos. In addition, DNA binding activity to other sequences,
including NF-
B, SP-1, and GATA, was not affected by overexpression
of c-fos and c-jun. For a functional
analysis, a promoter transactivation assay was performed with a
chimeric promoter-luciferase reporter, pGLAP-1Luc, which
contains 3 copies of the consensus AP-1 motifs. Expression of this
AP-1dependent gene was enhanced 3-fold in HUVECs coinfected with
AdJun and AdFos (Figure 2B
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Induction of Coordinated Expression of ICAM-1 and MCP-1
To elucidate the consequences of AP-1 activation, we further
examined its effect on the transcriptional induction of genes involved
in phenotypic modulation of ECs. ICAM-1 and MCP-1 genes were induced in
HUVECs earlier than 24 hours after infection with AdFos and AdJun
(Figure 3A
). Peak levels of steady-state
mRNA for ICAM-1 and MCP-1 were found 24 to 48 hours after coinfection.
Infection with Adß-gal and AdtTA did not induce ICAM-1 or MCP-1
expression. Therefore, coinduction of ICAM-1 and MCP-1 genes appeared
to be the specific result of increased levels of c-Jun and c-Fos
expression. Coexpression of c-Fos and c-Jun did not regulate gene
expression of von Willebrand factor, which is considered an
EC-specific molecular marker. None of these genes was found to be
induced by infection with Adß-gal and AdtTA as the controls for
adenoviral vectors. In addition, overexpression of c-jun and
c-fos markedly potentiated ICAM-1 and MCP-1 induction by PMA
(10 ng/mL) (Figure 3B
). These findings indicate that AP-1 is an
important signaling pathway for PMA induction of ICAM-1.
|
Many vascular adhesion molecule genes (including those for ICAM-1)
contain functional NF-
B binding sites in their promoter
regions,21 which have been suggested as the critical
cis-elements for cytokine-induced
transcriptional induction. In addition, interaction between AP-1 and
NF-
B has recently been reported to play a role in the activation of
another vascular adhesion molecule, VCAM-1.22 Therefore,
to determine whether the ICAM-1 induction and the EC activation induced
by the AP-1 activation process were dependent on an NF-
B pathway, we
examined the induction of ICAM-1 in the absence of NF-
B activation.
MG-132, an aldehyde peptide, is an inhibitor of
proteasomes23 and an effective antagonist of
NF-
B activation.24 Pretreatment of HUVECs with MG-132
can block NF-
B activation and largely abolish TNF-
induced
ICAM-1 activation (Figure 4A
). As a more
specific NF-
B antagonist, rAdI
B
was used to
overexpress I
B
in HUVECs (Figure 4B
), which was previously
demonstrated to specifically inhibit NF-
B activation in
ECs.16 As a result, TNF-
induction of ICAM-1 was
similarly inhibited in the rAdI
B
-infected HUVECs. However, ICAM-1
induction by AP-1 overexpression was not blocked by either MG-132 or
I
B
(Figure 4C
). These data strongly suggest that AP-1induced
ICAM-1 activation can occur independently of the NF-
B pathway.
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Enhancement of Promoter Activity and Binding to AP-1 Sites in the
ICAM-1 Promoter
Activation of ICAM-1 gene expression is known to occur at the
level of transcription.25 To further elucidate the
mechanism by which overexpression of AP-1 proteins activates
ECs, a transactivation assay with the use of a 1.3-kb ICAM-1
promoterdriven luciferase reporter construct (ICAM-Luc) was first
used to test whether adenovirally mediated AP-1 (c-Jun/c-Fos)
overexpression could enhance ICAM-1 promoter activity. As shown in
Figure 5B
, expression of the ICAM-Luc
reporter (pGL1.3) was enhanced
4-fold by coinfection with AdJun and
AdFos but not with control virus AdtTA. Because this 1.3-kb region of
the ICAM-1 promoter contains multiple putative AP-1like sequences and
NF-
B motifs (Figure 5A
), EMSA with the
oligonucleotides corresponding to these motifs was
performed to precisely localize the sequences that are functionally
involved in AP-1 induction of ICAM-1 gene expression. As shown in
Figure 5C
, individual overexpression of c-Jun or c-Fos induced little
binding activity to any of the sequences, whereas coexpression of c-Jun
and c-Fos resulted in strong binding activity to the proximal AP-1/TRE
site, moderate binding to the distal AP-1, but no binding to the
AP-1/Ets sequence. This result suggests that the proximal AP-1/TRE site
may function as the cis-element for AP-1 (likely,
c-Jun:c-Fos heterodimers) to activate ICAM-1 transcription.
DNA binding activity at the ICAM-1 NF-
B sequence, on which
cytokine induction critically depends,26 was not
found to be induced by AdFos and/or AdJun. This result,
consistent with the antagonist experiment,
indicates that NF-
B, either as the
trans-activator or the cis-element,
is not indispensable for ICAM-1 gene activation when the AP-1 pathway
is activated. AP-1 proteins may act through an independent
regulatory mechanism to cause EC phenotypic activation by using
specific recognition sites in the target genes.
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| Discussion |
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B pathway.
AP-1 transcription factors are formed through dimerization between the
members of the Fos and Jun families.27 Recent studies have
suggested AP-1 to be an important regulator in
endothelial function and pathological processes. First,
the AP-1 binding motif has been identified as a recurrent sequence in
the promoters of many genes biologically significant in the conversion
of ECs into a proinflammatory or procoagulant status, such as ICAM-1,
VCAM-1,21 E-selectin,28 MCP-1,29
and tissue factor.30 Second, AP-1 proteins and activity in
ECs can be increased by a variety of acute and chronic vascular
injuries, such as inflammatory cytokines,6 fluid
shear stress,9 hypoxia,8 and a high
level of LDL.12 Because these stimuli have pleiotropic
effects on various transcription pathways and cellular functions,
overexpression of AP-1 proteins has been used as a specific approach to
dissect the regulatory effect of AP-1 on endothelial
function.22 31 However, owing to the refractory response
of human primary ECs to many transfection methods,32 33
such an approach usually must be performed in either transformed cell
lines or nonhuman cells and is therefore limited in its
physiological relevance to human disease. To assay
the regulatory effect of AP-1 proteins on the expression of
endogenous genes and to allow biochemical analysis
in human ECs, it is necessary to include a highly efficient
transfection system. Considering the advantages of a recombinant
adenovirus as an effective gene delivery vector into various
transfection-resistant cell types,34 we
constructed replication-deficient adenoviruses expressing
c-jun and c-fos genes to establish AP-1
activation in HUVECs. For adenovirus-mediated gene expression, however,
the potential effects due to the vectors themselves rather than
specific transgenes must be ruled out. In this study, the viruses
encoding tTA protein and ß-gal were used as the controls for each
experiment. As a result, the adenoviruses were found to not induce
AP-1, ICAM-1, or MCP-1. As an additional indicator of specificity, we
found that infection with adenoviruses encoding c-jun and
c-fos could selectively activate AP-1 but not other
transcription factors such as NF-
B, SP-1, and GATA. Thus,
adenovirus-mediated gene transfer of c-jun and
c-fos can effectively and specifically establish an
experimental model for AP-1 activation in human ECs. Although infection
with AdJun can increase DNA binding to consensus AP-1 sequences and
AP-1 promoter activity, coinfection with both AdJun and AdFos (with the
same combined MOI) elicited the more significant AP-1 activity (Figure 2
). This result is in agreement with previous studies in other cell
types, in which cotransfection of c-Jun and c-Fos expression vectors
resulted in more potent transactivation of AP-1dependent indicator
genes than what is typically achieved by using c-Jun expression
alone.20 Likely, the c-Fosmediated increase in AP-1
activity is due to formation of c-Jun:c-Fos heterodimers, which are
more efficient in DNA binding and transcriptional regulation than the
c-Jun homodimer.35 36 Thus, the interaction between c-Jun
and c-Fos may play an important role in cellular regulation. In
addition, induction of endogenous c-Jun has been observed
to follow the expression of adenovirus-encoded c-jun in ECs.
This may represent the positive regulatory loop of c-Jun
activation, which is thought to occur through interaction between the
c-Jun:ATF-2 heterodimer and the cAMP-responsive element binding
protein in the c-jun promoter.37 38
With successful adenovirus-mediated gene transfer of c-jun
and c-fos in HUVECs, we asked whether AP-1 activation could
cause EC phenotypic modulation. A pronounced induction of ICAM-1 and
MCP-1 gene expression was observed after overexpression of
c-jun and c-fos. This is the first report of a
direct link between AP-1 activation and the induction of both a
chemokine and an adhesion molecule in ECs, which is particularly
significant in endothelial biology. ICAM-1 and MCP-1
are important cell adhesion and chemokine mediators of leukocyte and EC
interactions. MCP-1 is primarily chemotactic for
monocytes,39 whereas ICAM-1 is important in mediating the
adhesion and extravasation of circulating leukocytes.25
Therefore, coinduction of ICAM-1 and MCP-1 may represent
coordinated events in endothelial activation
processes.40 In particular, both ICAM-1 and MCP-1 have
been identified as being involved in mononuclear cell infiltration in
experimental models of atherosclerosis and in lesions
from human subjects.41 42 43 Atherogenic factors such as LDL
have been reported to induce both AP-112 and
ICAM-1.44 This observation will further our understanding
of AP-1 as an important regulator in the pathobiological activation of
the vascular endothelium. On the basis of the
observation that c-jun and c-fos coexpression
appeared to be more efficient for induction of ICAM-1/MCP-1 expression
(Figure 3
), which is consistent with the ICAM-1AP-1/TRE
binding assay and ICAM-1 promoter transactivation assay (Figure 4
), we
hypothesize that Jun:Fos heterodimers might be a functional form of
the AP-1 complex in inducing ICAM-1 and MCP-1 expression. In the
future, studies using c-fos and c-jun ribozymes
or adenoviruses expressing antisense c-fos and
c-jun will be helpful to detail the role of c-Fos and c-Jun
in mediating EC activation.
As transcription factors, AP-1 proteins may activate gene
expression through their binding to specific cis-elements
within the promoters. Alternatively, AP-1 may exert its regulatory
effects through complex interaction with other regulatory proteins such
as NF-
B.45 In fact, many vascular adhesion molecule
genes have both AP-1 and NF-
B elements in their promoters. Recently,
NF-
B has been shown to be a pivotal player in transcriptional
regulation of EC activation.21 However, among the various
stimuli, some, such as proinflammatory cytokines, are
activators of both AP-1 and NF-
B, while others, such as
LDL,12 are AP-1 but not NF-
B inducers. Furthermore,
some excitants, such as the antioxidant pyrrolidine
dithiocarbamate,46 are activators of AP-1 but
inhibitors of NF-
B. Therefore, it would be intriguing to
block NF-
B to test whether this AP-1mediated effect occurred
independently or was dependent on an interaction with NF-
B.
Typically, NF-
B (a p50/p65 heterodimer) is retained in the cytoplasm
by association with I
B
protein in quiescent ECs. After
stimulation, I
B is phosphorylated by I
B
kinase,47 which action causes I
B
degradation and
release of NF-
B to the nucleus.2 In the current study,
2 strategies were used to block NF-
B activation in ECs. The
proteasome inhibitor MG-132 has been widely used because it
can reduce nuclear translocation of p50/p65 and block TNF-
induced
activation of NF-
Bdependent-promoters.24 A further
specific blockade, the adenovirus rAdI
B
, can effectively block
TNF-
induced (Figure 4
) and even basal levels of NF-
B activation
in ECs.16 As a result, TNF-
stimulated ICAM-1 can be
largely blocked by treatment with NF-
B antagonists.
However, neither of the NF-
B blockers could reduce AP-1mediated
ICAM-1 induction, suggesting that this induction is indeed an
NF-
Bindependent event. This evidence supports our hypothesis that
AP-1 activation can circumvent the NF-
B pathway and independently
induce certain types of gene expression in ECs. In light of the fact
that certain chronic vascular stimulating agents such as LDL
(antioxidants as well) selectively induce AP-1 activation, this
AP-1specific event may reflect a gene activation pathway distinct
from that used by many acute vascular stimuli, such as proinflammatory
cytokines and bacterial endotoxin.
The 5'-flanking region of the human ICAM-1 gene contains a variety of
putative regulatory enhancer elements, including multiple AP-1like
sites and NF-
B sites.18 Our EMSA data have revealed
that infection with AdJun and AdFos induced the most pronounced binding
to the proximal AP-1/TRE site and, to a lesser extent, to the distal
AP-1 site. This finding is in accordance with a previous report that
transcriptional induction of ICAM-1 by tissue plasminogen
activator in human neuroblastoma cells involves Jun- and
Fos-containing complexes binding to this AP-1/TRE site.48
It was also described that in human ECs, the antioxidant pyrrolidine
dithiocarbamate can induce binding to the AP-1 site of the ICAM-1
promoter by complexes composed of Fos and Jun proteins.46
Moreover, promoter deletion analysis has indicated that the
inducibility of ICAM-1 by LDL largely depends on the presence of this
proximal AP-1/TRE element (Y.Z. et al, unpublished data, 1999).
Although the responsiveness of the MCP-1 promoter and the functionality
of the putative AP-1 site have not been examined in the present
study, an AP-1/TRE site was similarly found to be responsible for the
inducibility of a 550-bp MCP-1 promoter by fluid shear
stress.49 Taken together, our data indicate that AP-1
regulatory proteins may mediate EC activation through the cognate
sequence in the promoter regions of the target genes. Future studies
with site-directed mutagenesis experiments will be helpful to explore
whether ICAM-1 induction depends on the proximal AP-1/TRE site and the
potential roles of its flanking sequences and interaction with other
sites.
In conclusion, we have presented a unique model system in which
AP-1 complexes are overexpressed by adenoviral vectors in primary human
ECs. Our study demonstrates that AP-1 overexpression is itself
sufficient to induce adhesion molecule and chemokine genes in ECs
through an NF-
Bindependent pathway. These results provide the
important insight that AP-1 plays a key regulatory role, whereby a
variety of stimuli activate ECs in a specific pattern of gene
expression and subsequently contribute to the development of vascular
pathological processes.
| Acknowledgments |
|---|
B
construct and Dr H.L. Liao and O. Friedli for their
assistance in the experiments. Received November 3, 1998; accepted January 27, 1999.
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N.-H. Cho, S.-Y. Seong, M.-S. Huh, N.-H. Kim, M.-s. Choi, and I.-s. Kim Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1 Infect. Immun., September 1, 2002; 70(9): 4841 - 4850. [Abstract] [Full Text] [PDF] |
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A. Masamune, K. Kikuta, M. Satoh, A. Satoh, and T. Shimosegawa Alcohol Activates Activator Protein-1 and Mitogen-Activated Protein Kinases in Rat Pancreatic Stellate Cells J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 36 - 42. [Abstract] [Full Text] [PDF] |
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