1 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA
2 Division of Orthopedic Rheumatology, Department of Orthopedic Surgery,
University of Erlangen-Nuremberg, 91054, Germany
3 Molecular Biology Section, Division of Biology, University of San Diego, San
Diego, CA 92093, USA
4 Department of Cell Biology, Harvard Medical School, Boston, MA 02215,
USA
5 Program in Cancer Biology, Department of Radiation Oncology Stanford
University, Stanford, CA 94305, USA
6 Department of Medicine, University of Pennsylvania School of Medicine,
Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: schipani{at}helix.mgh.harvard.edu)
Accepted 19 February 2004
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SUMMARY |
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Key words: von Hippel-Lindau tumor suppressor protein, Cartilage development, HIF1
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Introduction |
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The fetal growth plate is a constitutively avascular tissue, and we have
recently demonstrated that it is hypoxic, with a typical outside-inside
gradient of oxygenation (Schipani et al.,
2001). Adaptation of cells and tissues to hypoxic
microenvironments requires the presence of hypoxia-inducible factor 1
(HIF1
), which is the key molecule in hypoxic response, regulating the
expression of glycolytic enzymes and several growth factors, including VEGF
(Semenza, 2000
). This
transcription factor is ubiquitously expressed, but under normoxic conditions
it is hydroxylated on specific proline residues by a recently discovered
family of prolyl-hydroxylases (Jaakkola et
al., 2001
). The von Hippel Lindau tumor suppressor protein (pVHL),
which is a recently identified novel E3 ubiquitin ligase
(Iliopoulos et al., 1995
),
recognizes the proline-hydroxylated form of HIF1
, and targets it for
polyubiquitination and degradation by the proteasome. Conversely, under
hypoxic conditions, oxygen-sensitive prolyl-hydroxylase activity is reduced,
and HIF1
translocates into the nucleus. Within the nucleus it binds to
its putative partner HIF1ß, also termed ARNT (aryl hydrogen receptor
nuclear translocator). This complex binds to specific hypoxic responsive
elements (HRE), thereby initiating the transcription of specific genes
(Forsythe et al., 1996
).
pVHL consists of 213 amino acids, and is expressed in most tissues and cell
types (Iliopoulos et al.,
1995). Heterozygous missense mutations of the VHL gene
have been identified as the likely cause of the von Hippel Lindau syndrome
(Latif et al., 1993
). This
syndrome is characterized by a dominantly inherited predisposition to develop
pheochromocytomas, and highly vascular tumors of the kidney, the central
nervous system and the retina (Maher and
Kaelin, 1997
). Tumorigenesis is associated with either the loss or
inactivation of the wild-type allele, following Knudson's two hit hypothesis
(Knudson and Meadows, 1980
).
pVHL has been shown to form a stable multiprotein complex which contains
Elongin B (TCEB2), Elongin C (TCEB1), CUL2 and RBX1
(Kibel et al., 1995
;
Ohh et al., 2000
). As
mentioned above, this multiprotein complex has E3 ubiquitin-ligase activity,
and one of its main targets is HIF1
(Lisztwan et al., 1999
;
Iwai et al., 1999
). The
importance of pVHL for proteolysis of HIF1
is further underlined by the
finding that cells lacking a functional pVHL are unable to degrade this
transcription factor, which ultimately results in an accumulation of
HIF1
. Homozygous disruption of Vhlh in mice results in early
embryonic lethality caused by abnormalities of placental vasculogenesis
(Gnarra et al., 1997
). Thus,
little is known about the physiological role of pVHL during fetal development
and postnatal life.
We have recently reported that HIF1 is essential for cell growth and
survival of murine growth plate chondrocytes in vivo
(Schipani et al., 2001
).
Chondrocytes lacking functional HIF1
undergo massive cell death in the
center of the growth plate (Schipani et
al., 2001
). Interestingly, viable chondrocytes at the periphery
display a significant increased rate of actively proliferating cells.
Furthermore, we have provided clear evidence that HIF1
is necessary for
type II collagen accumulation by hypoxic growth plate chondrocytes in vitro
(Pfander et al., 2003
). Based
on these findings, we hypothesized pVHL, a key molecule in the degradation
pathway of HIF1
, to be an important modulator of long bone development,
possibly through the regulation of HIF1
stability. The aims of this
study were to explore the role of pVHL in endochondral bone development, and
to investigate whether the lack of pVHL might affect expression of HIF1
target genes in growth plate chondrocytes.
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Materials and methods |
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Alizarin Red S staining, histological and in situ hybridization analyses, Tunel-assay
Alizarin Red S staining was performed as described previously
(Schipani et al., 1997).
For light microscopy, tissues from E14.5, E15.5, E16.5, E17.5 and E18.5
embryos (delivered by caesarean section), from newborns, and from 5- and
30-day-old mice, were fixed in 10% formalin/PBS (pH 7.4) and stored in
fixative at 4°C. Paraffin blocks were prepared by standard histological
procedures. Sections (5-6 µm in thickness) were cut from several levels of
the block, and stained with Hematoxylin (H) and Eosin (E). In situ
hybridization was performed using complementary 35S-labeled
riboprobes as described previously (Lee et
al., 1996).
For Tunel assay, paraffin sections from hindlimbs of newborn mice were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Tunel assay was performed using the In Situ Cell Death Detection Kit (Roche, Germany), according to the manufacturer's instructions.
Bromodeoyxyuridine (BrdU) labeling
E14.5, E15.5 and E16.5 pregnant mice were injected intraperitoneally with
100 µg BrdU/12 µg FdU per gram body weight, 2 hours prior to sacrifice.
After sacrifice, embryo hindlimbs were dissected, fixed and embedded in
paraffin, and longitudinal sections across the tibia and femur were obtained.
To identify actively proliferating cells, nuclei that had incorporated BrdU
were detected using a Zymed BrdU immunostaining kit (Zymed Laboratories, San
Francisco, USA).
HIF1 immunohistochemistry
For HIF1 detection, fresh frozen sections from newborn mice were
fixed in acetone for 20 minutes at -20°C and then permeabilized with 0.1%
Triton X-100 in 0.1% sodium citrate. After blocking, sections were incubated
with the commercially available antibody C-19 that specifically recognizes an
epitope in the C-terminal portion of the HIF1
protein (Santa Cruz
Biotech, CA, USA), at a dilution of 1:100. Detection of binding was by the
Streptdavidin-HRP system provided by the TSA kit (Perkin Elmer-Life Sciences,
MA, USA), according to the manufacturer's conditions.
Chondrocyte isolation, western blot analysis, real-time PCR, Elisa
Chondrocytes were isolated from newborn
Vhlh+f//+f mice as described
(Pfander et al., 2003). In
brief, forelimbs and hindlimbs were dissected, and distal epiphyses of radius,
ulna and tibia were isolated in HBSS (Gibco BRL, MD, USA). Epiphyses were
digested in 0.25% trypsin/EDTA for 30 minutes at 37°C, and in 195 U/ml
collagenase type II in HBSS (Worthington, NJ, USA). Chondrocytes were plated
at a density of 4x105 cells per well of a six-well plate, and
grown in monolayer cultures in high glucose DMEM (Gibco BRL, MD, USA)
supplemented with 10% FBS (Hyclone, UT, USA) and 1% penicillin/streptomycin.
On day 1 post-plating, adherent chondrocytes were infected with adenovirus
containing either ß-galactosidase or Cre recombinase (generously supplied
by Frank J. Giordano, Yale University, New Haven, CT, USA) to create wild-type
chondrocytes or Vhlh null cells. Deletion of Vhlh was
confirmed by semi-quantitative PCR analysis across the 3' loxP site. For
immunoblotting, cells were lyzed, and detection of HIF1
hydroxylated at
Pro564 was performed as previously described
(Chan et al., 2002
). Detection
of
-tubulin with a specific antibody (Research Diagnostics, NJ, USA)
was used as control of equal loading.
Total RNA was isolated from wild-type chondrocytes or Vhlh null
cells as previously described (Pfander et
al., 2003), and then transcribed into single cDNA using AMV
reverse transcriptase (Boehringer Mannheim, Germany) and random hexamer
primers, according to the manufacturer's instructions. For real-time PCR
analyses, cDNA was diluted to a final concentration of 10 ng/µl. For PCR
reactions, SYBR-Green Mastermix (Applied Biosytems) was used. Total cDNA (50
ng) was used as template to determine the relative amount of mRNA by real-time
PCR (ABI Prism 7700 sequence detection system) using specific primers. The
reaction was conducted as follows: 95°C for 4 minutes, and then 40 cycles
of 15 seconds 95°C and 1 minute 60°C. ß-actin was amplified as an
internal control. Cycle threshold (Ct) values were measured and calculated by
the Sequence detector software. Relative amounts of mRNA were normalized to
ß-actin and calculated using the software program Microsoft Excel.
Relative mRNA contents were calculated as x=2-
Ct, in
which
Ct=
E-
C,
E=CtVHL
null-Ctß-actin and
C=CtWT-Ctß-actin (wild-type expression was
taken as 1). Specific primers for VEGF, phosphoglycerokinase 1 (PGK1) and type
II collagen mRNA were designed as described
(Pfander et al., 2003
). For
ß-actin the following sequences were used: ß-actin forward, AGG CCC
AGA GCA AGA GAG G; and ß-actin reverse, TAC ATG GCT GGG GTG TTG AA.
Protein concentrations of soluble VEGF isoforms were determined using the
DuoSet Elisa Kit for mouse VEGF (R&D Systems, MN, USA) as previously
described (Pfander et al.,
2003). Briefly, cell culture supernatants (wild-type chondrocytes
or Vhlh null cells) were harvested and stored at -20°C. VEGF
Elisa was conducted according to the manufacturer's instructions.
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Results |
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Lack of functional pVHL has been linked to both sporadic and familial cases of renal clear cell carcinomas, which are known to derive their histological appearance from accumulation of glycogen and lipids. We thus explored the possibility that the atypical chondrocytes of the Vhlh null epiphysis could have a `clear cell' phenotype. Notably, PAS or Oil Red staining did not reveal any increased accumulation of glycogen or lipids, respectively, in the Vhlh null chondrocytes in comparison with control cells (data not shown).
To test whether the increased cell size of resting chondrocytes in Vhlh null animals might be the result of an ectopic or premature process of hypertrophic differentiation, type X collagen expression was investigated by in situ hybridization on histological sections of newborn mice. In both wild-type and mutant epiphyses, type X collagen mRNA expression was spatially restricted to the hypertrophic zone (Fig. 3C,G). Furthermore, a similar expression pattern of type II collagen mRNA was present in both Vhlh null and control growth plates (Fig. 3A,B,E,F). Consistent with these findings, spatial distribution of mRNA encoding the PTH/PTHrP receptor (PPR) and Indian Hedgehog (IHH), which are mainly expressed at the transition between proliferation and hypertrophy, was also similar in null and control specimens (Fig. 3D,H; and data not shown). Lastly, no differences in number and distribution of apoptotic chondrocytes were detected between mutant and control growth plates using the Tunel assay (data not shown). Taken together, our data indicate that the phenotypic alterations of resting chondrocytes in the Vhlh null growth plate were not the result of ectopic or premature hypertrophy.
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Expression of HIF1-dependent genes is upregulated in the Vhlh null growth plate
We next investigated whether the transcription factor HIF1 is
stabilized and accumulates in the absence pVHL. In the Vhlh null
growth plate, the intensity and number of chondrocytes that stained positive
for HIF1
protein were clearly increased, demonstrating that deletion of
Vhlh indeed leads to an accumulation of HIF1
(Fig. 6A,B). Western blot
analysis of protein lysates of primary chondrocytes grown in normoxic
conditions confirmed a dramatic accumulation of HIF1
hydroxylated at
Pro564 in cells lacking Vhlh, when compared with the control
(Fig. 7A).
|
In situ hybridization analysis of newborn wild-type epiphysis revealed that
PGK transcripts were expressed in the center of both the proliferative and
prehypertrophic, with a typical outside-inside pattern that resembled the
pattern of oxygenation of the growth plate
(Fig. 6C)
(Schipani et al., 2001). By
contrast, in the newborn mutant growth plate, the outside-inside pattern of
expression of PGK mRNA was perturbed; PGK mRNA was detectable throughout the
whole epiphysis, including in a few scattered hypertrophic chondrocytes
(Fig. 6D). Furthermore, resting
chondrocytes located in close proximity to the articular surface showed an
increased expression of PGK mRNA (Fig.
6D).
We then analyzed VEGF mRNA expression by in situ hybridization. Our in situ hybridization analysis showed that in the newborn wild-type growth plate, VEGF mRNA was present not only in hypertrophic chondrocytes, but also, although to a lesser extent, in the center of the proliferative zone (Fig. 6E). In the newborn mutant growth plate, similar to PGK transcripts, VEGF mRNA was detectable in all layers. In addition, VEGF mRNA expression appeared to be granular and was upregulated in chondrocytes located next to the articular cap (Fig. 6F). Similar mRNA expression patterns of VEGF and PGK were observed in postnatal life (data not shown).
Consistent with these findings, measurements of soluble VEGF in the supernatant of primary chondrocytes grown in normoxic conditions, and real-time PCR of total RNA isolated from the same cells confirmed a dramatic accumulation of both VEGF protein and mRNA in cells lacking Vhlh when compared with controls (Fig. 7B,C). Furthermore, PGK mRNA levels were elevated in Vhlh null chondrocytes when compared with controls, even if to a lesser degree than VEGF mRNA (Fig. 7C).
Taken together, these findings strongly support the hypothesis that lack of
pVHL in chondrocytes leads to an accumulation of HIF1 protein and to an
increased expression of its target genes.
Growth plates lacking both Vhlh and Hif1a display the Hif1a null phenotype
In the final set of our experiments, we investigated whether the unique
phenotype in mice lacking functional pVHL resulted from accumulation of the
transcription factor HIF1 and through activation of HIF1
target
genes. To address this question, we created double mutant mice lacking both
the transcription factor HIF1
and pVHL in cartilage
(Vhlh/Hif1a null). These mice died within the first hours
after birth, like the mice that lack only HIF1
in cartilage
(Hif1a null). The limbs of Vhl/Hif1a null newborns
were essentially identical to the limbs isolated from Hif1a null
newborns (data not shown). Both types of mutant limbs were much shorter and
were deformed compared with wild-type controls
(Fig. 8H-J). Furthermore, as in
the Hif1a null growth plate, the center of the Vhl/Hif1a
null growth plate was remarkably hypocellular as result of massive cell
death, which generated a spatially localized defect extending from the joint
space to the primary spongiosa (Fig.
8H-J). Lastly, similar to the Hif1a null growth plate,
the proliferation index of the viable chondrocytes surrounding the central
area of cell death was significantly increased in the
Vhl/Hif1a null growth plate in comparison with controls
(Fig. 8D-G). Both mutant
phenotypes were extremely severe at birth, and were already clearly evident at
E.14.5 (Fig. 8A-C).
|
![]() |
Discussion |
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The VHL tumor suppressor protein is required to regulate HIF1, and
inactivation of VHL has been linked to the development of a variety of highly
angiogenic tumors, including hemangioblastoma of the retina and central
nervous system, clear cell carcinoma of the kidney, and pheochromocytoma
(Maher and Kaelin, 1997
). Loss
of pVHL function leads to an increased stability of HIF1
and
overexpression of proteins encoded by HIF1
target genes, such as VEGF
and PGK (Gnarra et al., 1996
;
Cramer et al., 2003
). However,
although HIF1
is the best characterized pathway that is affected by
pVHL loss of function, pVHL has been reported to have a range of effects
within the cells that are not clearly related to HIF-1
activation.
Identification of HIF1
-dependent and -independent effects of pVHL
action is thus an open field of intense investigation.
In this study, we report the novel and intriguing finding that the tumor
suppressor protein pVHL is necessary for normal chondrocyte proliferation in
vivo. Vhlh null growth plates show a dramatic decrease in chondrocyte
proliferation at different stages of fetal development, as determined by BrdU
labeling. The role of pVHL in regulating cell proliferation is not fully
elucidated (Kondo and Kaelin,
2001). Our data differ from previous studies that have shown that
lack of functional pVHL impairs cell cycle exit under certain experimental
conditions (Bindra et al.,
2002
; de Paulsen et al.,
2001
; Pause et al.,
1998
). However, our findings are consistent with the recent report
that lack of pVHL inhibits cell proliferation in a teratocarcinoma model
(Mack et al., 2003
). It is
highly possible that pVHL actions on the cell cycle may indeed vary in
different cell types. This hypothesis would be in agreement with the
observation that, despite the ubiquitous pattern of VHL expression,
only a restricted subset of tumors is observed in patients suffering from the
von Hippel Lindau syndrome (Kaelin and
Maher, 1998
).
Conditional inactivation of HIF1 in chondrocytes leads to the
increased proliferation rate of viable chondrocytes at the periphery of the
growth plate, while central cells undergo massive cell death
(Schipani et al., 2001
). In
addition, a concomitant decrease in the mRNA expression of the CDK-inhibitor
p57kip2 is also detectable in murine growth plates lacking
HIF1
. p57kip2 is apparently required for exit of epihyseal
chondrocytes from the cell cycle (Nagahama
et al., 2001
; Yan et al.,
1997
). Upregulation of p57kip2 has been identified by
microarray analysis in renal epithelial cells exposed to hypoxia
(Leonard et al., 2003
).
Consistent with these findings, Vhlh deficient growth plates
displayed a mild but consistent increase in p57kip2 mRNA
expression, as shown by in situ hybridization analysis. This increased
expression of p57kip2 transcripts may be partly responsible for the
hypocellularity and the decreased proliferation rate observed in Vhlh
null growth plates. However, chondrocyte proliferation is regulated at
multiple levels by numerous factors, such as FGFs, BMPs, PTHrP, IHH, cell-cell
and cell-matrix adhesion, and biomechanical signals (Shum et al., 2002). It is
therefore likely that regulation of chondrocyte proliferation by pVHL does not
involve only modulation of p57kip2 expression. It will be now
interesting to study whether and how the pVHL/HIF1
system interacts and
cooperates with all the different pathways that regulate cell proliferation in
the developing growth plate.
The resting zone of the Vhlh null growth plate was mainly occupied
by enlarged chondrocytes with a high cytoplasm to nucleus ratio, with
similarities to pre-hypertrophic or hypertrophic chondrocytes. However, no
evidence of ectopic hypertrophic differentiation could be found by analyses of
type X collagen, PPR or IHH mRNA expression. It is possible that the decreased
mitotic activity is the pathogenic event that leads to both the
hypocellularity and the appearance of atypical cells observed in the
Vhlh null growth plate. However, to our knowledge this unique
cartilage phenotype characterized by increased cell size and reduced
proliferation has not been described in other knockout models in which
chondrocyte proliferation has been reported to be severely impaired, such as
the Ihh knockout (St-Jacques et
al., 1999). Therefore, the increased cell size observed in the
Vhlh null growth plate probably is not the necessary or direct
consequence of the decreased cell proliferation per se. It is possible that
lack of pVHL in chondrocytes uncouples cell size and cell proliferation by
regulating a still unknown molecular mechanism. Alternatively, pVHL may
regulate chondrocyte size and shape through pathways that are distinct from
those involved in chondrocyte proliferation. In this regard, it has been
recently reported that pVHL is a microtubule-associated protein that can
protect microtubules from depolymerization in vivo
(Hergovich et al., 2003
), and
this function appears to be independent of its ability to engage in E3 ligase
complex formation.
It is well known that chondrocyte proliferation and differentiation require
their attachment to the matrix (Terpstra
et al., 2003). In our in vivo model, the lack of Vhlh in
cartilage leads not only to decreased proliferation, but also to increased
matrix between cells, as shown by histological analysis. Hypoxia increases
type II collagen accumulation in vitro in a HIF1
-dependent manner
(Kurz et al., 2001
;
Pfander et al., 2003
).
Furthermore, recently it has been shown that hypoxia increases a group of
procollagen hydroxylases that are indispensable for collagen fiber formation
through stabilization of the transcription factor HIF1
(Hofbauer et al., 2003
). It is
thus highly possible that the increased matrix deposition observed in
Vhlh null growth plates might result from accumulation of HIF1
in the mutant chondrocytes, leading to both an increased expression of type II
collagen mRNA and to an enhanced synthesis of procollagen hydroxylases that
are critically required for collagen triple helix formation. However, at this
stage of investigation, a HIF1
-independent role of pVHL in regulating
matrix accumulation cannot be excluded. Notably, it has been shown that pVHL
can bind directly to fibronectin, a very important matrix protein
(Ohh et al., 1998
). A specific
role of pVHL in extracellular matrix formation is also suggested by the
finding that pVHL regulates metalloproteinases production and activity
(Koochekpour et al., 1999
).
Further investigation will be needed in order to establish the role of pVHL in
the regulation of cartilage matrix and, more generally, in chondrogenesis. It
will be then crucial to identify whether this role is indeed dependent on
HIF1
transcriptional activity.
VEGF is one of the best indicators of HIF1 transcriptional activity;
consistent with this finding is the observation that tumors caused by
Vhlh inactivation are highly angiogenic and accumulate HIF1
.
Blood vessel invasion of the epiphysis, a crucial step in endochondral bone
development, has been shown to be regulated in part by VEGF activity
(Gerber et al., 1999
;
Horner et al., 1999
;
Maes et al., 2002
;
Zelzer et al., 2002
;
Ortega et al., 2003
;
Haigh et al., 2000
;
Carlevaro et al., 2000
).
Several groups have reported that VEGF mRNA expression is mainly restricted to
the hypertrophic zone (Gerber et al.,
1999
; Horner et al.,
1999
). Our data show that VEGF mRNA is also produced by
proliferating chondrocytes, suggesting a broader role of VEGF in chondrocyte
biology. In agreement with this hypothesis, more recent reports indicate that
VEGF is an important modulator of chondrocyte differentiation, and it is also
a critical survival factor for chondrocytes
(Zelzer et al., 2002
;
Maes et al., 2004
;
Zelzer et al., 2004
).
VEGF expression in chondrocytes is in part regulated by
HIF1-dependent mechanisms (Schipani
et al., 2001
; Pfander et al.,
2003
). Consistent with this notion, in the present study we
detected a change in the expression pattern of VEGF mRNA in the resting and
proliferating zones of Vhlh null growth plates, probably caused by
the stabilization of HIF1
. Interestingly, despite increased levels of
VEGF expression in Vhlh null resting-zone chondrocytes, no ectopic
blood vessel invasion was observed at this site. Furthermore, a delay of
angiogenesis at the secondary ossification site was clearly noticeable in the
Vhlh null specimens. Taken together, these findings suggest that
several other factors may be involved in regulating angiogenesis during
endochondral bone development (Ivkovic et
al., 2003
), particularly at the secondary ossification center.
In our final experiment we investigated whether the morphological
alterations, the decreased mitotic activity and the increased deposition of
extracellular matrix molecules within the Vhlh null growth plate
might result from the accumulation of HIF1 and the de-regulation of
HIF1
target genes. To address this crucial question we used conditional
inactivation of both Vhlh and Hif1a in all cartilaginous
elements. The growth plate phenotype of Hif1a/Vhlh null mice
was virtually indistinguishable from that observed in Hif1a null
animals. This result is consistent with the idea that the altered endochondral
ossification process observed in Vhlh null mice could result, at
least in part, from increased activity of HIF1
, and, consequently, from
de-regulation of HIF1
target gene expression. Further investigations
will be needed to establish whether HIF1
is the only target of pVHL
activity in growth plate chondrocytes.
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ACKNOWLEDGMENTS |
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