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(Circulation. 1999;99:2750-2756.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Dilated Cardiomyopathy Is Associated With Significant Changes in Collagen Type I/III ratio
From the Medical Clinic II, University Hospital Benjamin Franklin, Freie Universität Berlin, Germany.
Correspondence to Matthias Pauschinger, MD, Medical Clinic II, University Hospital Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany.
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Abstract |
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Background—It is controversial whether myocardial fibrosis in end-stage dilated cardiomyopathy (DCM) is associated with altered collagen type I/type III (Col I/Col III) ratio.
Methods and Results—Patients with DCM (ejection fraction [EF] <50%, n=12) and with mild global left ventricular dysfunction (EF >50%, n=18) were examined. Col I, Col III, and transforming growth factors-ß1 (TGF-ß1) and -ß2 (TGF-ß2) gene expression in endomyocardial biopsies was evaluated by quantitative competitive reverse transcriptase–polymerase chain reaction (qRT-PCR). Collagen content was quantified after picrosirius red and immunohistological staining and by hydroxyproline assay. In patients with EF <50%, there was a pronounced 2- to 6-fold increase of myocardial Col I mRNA abundance (P<0.01), with a corresponding 1.6-fold increase at the protein level versus that found in patients with EF >50%. The Col III mRNA abundance showed a 2.0-fold increase (P<0.04). There was a relevant shift in the Col I/Col III mRNA ratio for DCM patients (Col I/Col III, 8.2) compared with patients with an EF >50% (Col I/Col III, 6.4). In addition, total collagen content was increased in patients with EF <50% (n=3) (4.3±0.1%) compared with patients with EF >50% (n=8) (2.7±0.9%) (P<0.004). The biochemically determined ratio of hydroxyproline/total protein (n=12) was correlated to the Col I mRNA abundance (P<0.05, r=0.77). TGF-ß1 and TGF-ß2 showed elevated myocardial mRNA abundances (1- to 7-fold and 4- to 5-fold, respectively) in DCM patients.
Conclusions—Differential increase of Col I and Col III leads to an increased Col I/Col III ratio in DCM myocardium. Because Col I provides substantial tensile strength and stiffness, this may contribute to systolic and in particular diastolic dysfunction in DCM.
Key Words: cardiomyopathy • collagen • growth substances • remodeling • polymerase chain reaction
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The myocardial extracellular matrix is composed of a complex network of structural protein, mainly collagen type I (Col I) and collagen type III (Col III), which provide architectural support for the muscle cells and also play an important role in myocardial function.1 2 Several studies of dilated cardiomyopathy (DCM) have demonstrated changes in collagen content at the protein level, characterized primarily by an accumulation of Col I.3 4 5 However, whether there is also an increase in Col III remains controversial. The ratio of collagen types within the heart is of significance because of their different mechanical properties.6 For example, tissues with predominance of Col I (such as tendon) are characterized by strength and stiffness, whereas tissues containing large amounts of Col III (such as skin) are characterized by greater elasticity. Yoshikane et al5 documented an increase in the number and thickness of collagen fibers in end-stage DCM by electron microscopic and immunohistological methods. A biochemical analysis by Bishop and colleagues3 showed an increase in the absolute amounts of both Col I and Col III. In contrast, Weber et al7 demonstrated a pronounced increase in predominantly thin collagen fibers and a decrease in thick collagen fibers in explanted hearts from patients with end-stage DCM compared with normal myocardium. The apparent discrepancy of these studies may be explained by differences in methodology; however, other factors, such as collagen cross-linking, may have influenced the results.
Our aim was to examine changes in messenger RNA (mRNA) abundance of collagen subtypes in endomyocardial biopsy samples from patients with and without diagnosis of DCM. Furthermore, we also sought to determine whether there were changes in transforming growth factors-ß1 (TGF-ß1) and -ß2 (TGF-ß2), cytokines known to play a crucial role in mediating collagen synthesis.8 9 10 Furthermore, the small amount of tissue available from endomyocardial biopsy samples required that we establish quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) assays to conduct the analysis of multiple genes.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Patients
Thirty patients with the presumptive clinical diagnosis of DCM were analyzed in this study. All patients underwent cardiac catheterization for further evaluation of impaired global or regional left ventricular function initially revealed by echocardiography and by reduced exercise tolerance with a minimum duration of 6 months. Right and left heart catheterization, including standard hemodynamic measurements, was carried out in all patients in whom coronary, hypertensive, and valvular heart disease as well as restrictive or constrictive heart disease had been excluded before an endomyocardial biopsy was obtained. In addition, none of the analyzed patients had decompensated heart failure. Because of the limited number of available biopsies, a subgroup analysis for total collagen content by picrosirius red staining was performed in a subgroup of 11 patients (n=3, EF <50%, and n=8, EF >50%) from the total study group of 30. In addition, in another subgroup of 12 patients (mean EF, 49±11%) from the total study group, the ratio of hydroxyproline concentration/total protein concentration (both µg/mL) was correlated with the collagen mRNA abundance.
All procedures were performed in accordance with ethical standards and with the Helsinki Declaration of 1975. In addition, all patients gave informed consent for all of the invasive studies performed.
Biopsy
Endomyocardial biopsies were taken from the
right ventricular septum in all patients with a Cordis
bioptome. From these small biopsy samples, we measured (1) Col I and
Col III mRNA abundance, (2) mRNA abundance for
TGF-ß1 and TGF-ß2, (3)
mRNA abundance for GAPDH, (4) total collagen content
histologically by picrosirius red staining, (5) Col I
and Col III expression immunohistologically on the
protein level, (6) hydroxyproline concentration biochemically, and (7)
CD3 T lymphocytes as a marker of inflammation.
RNA Extraction
Total RNA was isolated from all 30 samples by a modification of
the lithium chloride method according to Auffray et
al.11
Reverse Transcription
For the PCR, 300 ng total RNA of each biopsy was transcribed
into cDNA by use of avian mycloblastosis virus reverse transcriptase
(AMV RT) and random primer in accordance with the supplier's
protocol (Promega).
qRT-PCR
The qRT-PCR was based on the coamplification of a specific
internal standard with the cDNA to be examined. Because of the
identical primer sequences of the internal standard and cDNA, both
molecules compete for the primers of the PCR mixture in the phase of
primer annealing. A decreasing concentration of internal standard
accounts for an increased amplification of examined cDNA (Figure 1
).
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The internal standard was constructed by use of the PCR mimic–constructing kit (Clontech Laboratories, Inc) with linker primers for Col I, Col III, TGF-ß1, TGF-ß2, and GAPDH. The standard differed in size from the amplified cDNA to distinguish between both PCR products on agarose gels. The difference in size between standard and endogenous cDNA was in the range of 10% to 20% of base length. The exogenous DNA standard was applied in qRT-PCR in 5 dilutions ranging from 0.1 to 0.16 attomol, calculated by an estimated length of 500 bp while a constant concentration of cDNA was added. The amounts of cDNA were 1.5 ng for Col I, 3 ng for Col III, 2 ng for TGF-ß1, 6 ng for TGF-ß2, and 0.6 ng for GAPDH.
The qRT-PCR program consisted of a denaturing phase at 94°C for 1 minute, primer annealing at 60°C for Col III, TGF-ß1, TGF-ß2, and GAPDH and at 72°C for Col I, each for 90 seconds, and an extension temperature of 72°C for 2 minutes. The PCR ran for 35 cycles and was still in the linear phase.
The PCR reacting volume of 25 µL consisted of 0.4 µmol/L of each primer, 0.2 mmol/L dNTPs, 2.0 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl, and 4 U of Taq polymerase (Perkin-Elmer).
Oligonucleotides Used for qRT-PCR
Primers were selected on 2 different exons, separated by an
intron to differentiate between amplified DNA and amplified
reverse-transcribed RNA. The primer sequences are listed in Table 1.
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Densitometric Analyses of qRT-PCR Products
Each qRT-PCR product was loaded on an ethidium
bromide–stained agarose gel (1.5%). For analysis of bands,
the gel was photographed under 254-nm UV light with a photo documentary
system (Ultra Violet Products Gel Documentation System Image Store
5000) and quantified with the NIH image program (Ultra-Violet
Products Limited). All values were measured twice to avoid
inaccuracy. The abundances of all different mRNA species were related
to GAPDH mRNA to increase accuracy.
Histology
Picrosirius red staining was used for the
histological assessment of the total collagen content
by use of computer-assisted morphometry (Lucia software program).
Formalin-fixed tissue was processed for paraffin embedding, sectioned
at a thickness of 5 µm, and stained with picrosirius red
(Polyscience, Inc). The collagen content of the sections was measured
when viewed with circularly polarized light according to previously
published methods.12 For each sample, a total of 10 fields
were analyzed with a x20 objective lens.
Histological analyses of
endomyocardial biopsies for myocardial inflammation
were performed in a standard manner and evaluated according to the
Dallas classification.13
Immunohistology
Endomyocardial biopsies were directly
embedded in OCT (Miles Laboratories, Inc) and frozen at -70°C. With
a cryostat, the biopsies were cut into 5-µm thin sections, mounted on
a slide, and fixed in acetone for 10 minutes.
Antibodies
Biopsies were incubated with monoclonal mouse anti-human
antibodies against Col I (Oncogene Research Products) and Col III
(Sigma Chemical Co), with a working solution for Col I of 1:300 and Col
III of 1:150. Monoclonal antibodies directed against CD3 T lymphocytes
were used according to Kühl et al.14
Immunohistological Staining
Each primary antibody was added to a section of 6 slices and
incubated for 45 minutes at room temperature in a humidified chamber.
After incubation, unbound antibodies were removed by 2 washing steps
with PBS for 5 minutes. Then, peroxidase-conjugated rabbit anti-mouse
antibody (Dianova GmbH), diluted at 1:200 in PBS containing 10% FCS,
was added and likewise incubated for 45 minutes at room temperature.
After another 2 washing steps with PBS, the specifically bound
antibodies were detected by a stain reaction with carbazole for 12
minutes in the dark. For quantification of stained areas, the sections
were viewed with a light microscope at a magnification of x40 and
digitized. With the Lucia software program (Nikon), the relation
between stained and unstained areas was calculated and the Col I and
Col III protein was measured as percentage proportion. For each biopsy
and antibody, 6 sections were measured and averaged. The quantification
of CD3 T lymphocyte content was performed at a magnification of x400
according to Kühl et al.14
Hydroxyproline Determination
The endomyocardial biopsies were
homogenized in 330 µL of 6 mol/L hydrochloric acid for 15
seconds. Of this homogenate, 30 µL was used for the
determination of the total protein content in the analyzed
tissue sample. Afterward, 300 µL of the 6 mol/L hydrochloric acid
homogenate was heated at 120°C overnight to hydrolyze
collagen into its component amino acids. Hydroxyproline concentration
was measured in an amino acid analyzer in standard fashion. In
addition, the total protein concentration of the analyzed
biopsy was measured with the bicinchoninic acid protein quantification
kit (Pierce). The concentration of tissue hydroxyproline was expressed
as the ratio of hydroxyproline concentration/total protein
concentration (both µg/mL) for each biopsy. The hydroxyproline
concentration was applied to the total protein content, because with
the very small size of endomyocardial biopsies, a
weight-adjusted standardization of the hydroxyproline content would be
not accurate enough.
Statistics
For statistical analysis, the SPSS statistical software
package was used. Statistical analyses were done with the
Mann-Whitney U test, and the mRNA values of each group are
shown with their respective medians and the corresponding interquartile
distances. Statistical significance was accepted at the level of
P
0.05. The other values are expressed as
mean±SD.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Patients and Hemodynamic Parameters
The patients were assigned to 2 groups on the basis of cardiac function. Twelve patients (3 women, 9 men) 34 to 60 years old (mean, 47±11 years) had global left ventricular dysfunction (EF <50%) and were classified as having DCM. Eighteen patients (7 women, 11 men) 17 to 71 years old (mean, 40±14 years) had only mild or regional left ventricular dysfunction (EF >50%) and were classified as having "latent" DCM. The other hemodynamic and clinical data, including the medications, are listed in Table 2
.
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Collagen Quantification
Gene Expression Studies
The mRNA abundance of Col I, measured as Col I/GAPDH mRNA ratio
(0.45±0.49) in the group of patients with an EF <50%, was increased
significantly, by 2.6-fold (P<0.01), versus patients with
an EF >50% (0.172±0.23) (Figure 2).
The mRNA ratio of Col III/GAPDH increased significantly, by 2.0-fold
(P<0.04), from 0.027±0.026 in patients with mild regional
dysfunction and EF >50% versus 0.055±0.066 in patients with global
dysfunction and EF <50% (Figure 2). These changes resulted in
a shift in the ratio between Col I and Col III at the mRNA level for
patients with DCM (ratio, 8.2) compared with the gene expression ratio
of Col I/Col III in the group with an EF >50% (ratio, 6.4).
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The growth factors TGF-ß1 and TGF-ß2 showed a trend toward an increased mRNA abundance in the group with EF <50% (TGF-ß1/GAPDH, 0.048±0.027; TGF-ß2/GAPDH, 0.018±0.014) versus the group of patients with EF >50% (TGF-ß1/GAPDH, 0.029±0.041; TGF-ß2/GAPDH, 0.004±0.014).
Histology and Immunohistology of Collagen
The results of gene expression for Col I were confirmed on the
protein level. A trend toward a greater accumulation of the percentage
proportion of stained and unstained areas of Col I was detected in
patients with DCM (Col I, 20±11%) compared with patients with a
normal EF (Col I, 12±8%). However, a trend toward a decrease of Col
III was found (patients with EF <50%, 30±13%; patients with EF
>50%, 36±11%). The histological analysis of
the total collagen content by picrosirius red staining visualized in
circularly polarized light also showed a significant increase in
fibrosis in patients (n=3) with EF <50% (4.3±0.1%) compared with
patients (n=8) with EF >50% (2.7±0.9%) (P<0.0035; see
Figure 3). This increase in collagen
content appeared to be uniform throughout the biopsy samples.
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Hydroxyproline Content
Because of the limited availability of
endomyocardial biopsies, these biochemical
analyses were performed in a subgroup of 12 patients (EF
<50%, n=6; EF >50%, n=6; mean EF, 49±11%). The ratio of
hydroxyproline concentration/total protein concentration (both µg/mL)
was significantly correlated with the mRNA abundance of Col I
(r=0.77; P<0.05) (see Figure 4) but not with Col III.
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Histology and Immunohistology of Myocardial Inflammation
Active myocarditis according to the Dallas classification was
excluded in all patients. The distribution of CD3 T lymphocytes
differed between groups; patients with global dysfunction and EF <50%
showed a significant (P0.05) increase of CD3 T lymphocytes
of 1.8±0.7 lymphocytes per visual field, compared with the group of
patients with EF >50%, who showed a value of 1.3±0.6 CD3 T
lymphocytes per visual field.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The study revealed that myocardium obtained from patients with a diagnosis of DCM and an EF <50% had an increase in the ratio of Col I/Col III mRNA abundance versus that found in patients with an EF >50%. The total collagen content quantified by computer-assisted morphometry of picrosirius red–stained histological slices in circularly polarized light showed a significant increase in patients with EF <50% compared with patients with EF >50%. Furthermore, these changes in mRNA levels were paralleled by immunohistologically observed increases in Col I in the patients with DCM as well as a significant correlation between the biochemically determined ratio of hydroxyproline concentration/total protein concentration and the mRNA abundance of Col I. In addition, these changes in collagen expression and deposition were matched by a trend toward increased expression of TGF-ß1 and TGF-ß2 in combination with a significantly increased CD3 T-lymphocyte count. Thus, we have demonstrated that it is possible to determine from small endomyocardial biopsies various structural and humoral factors that may play key roles in the development of functional abnormalities.
Collagen mRNA Abundance
The shift in the Col I/Col III ratio from 6.4:1 to 8.4:1 in the
patients with DCM may derive from different molecular mechanisms.
Either there could be a stimulation of collagen mRNA expression through
the activation of various cytokines or there could be a reduced
turnover of Col I and Col III mRNAs. Although we found no significant
increase in the mRNA abundance of either TGF-ß1
or TGF-ß2, 2 cytokines intimately
involved with collagen production, this does not rule out the
former pathway. It is possible that we missed the peak in
TGF-ß1 and TGF-ß2
expression and detected only its consequences. In addition, there are
other possible cytokines that may be equally or even more
important.9 10
Our study showed a trend toward increased myocardial TGF-ß1 and TGF-ß2 mRNA abundance in DCM. This, however, could possibly translate into a significant increase in collagen gene expression, because both the promotors of Col I and Col III contain TGF-ß–activatable elements.15 16 17 Because the Col I promotor also contains several SP-1 binding sites, which are not found in the Col III promotor, the differential gene expression levels of Col I and Col III might rely on their respective promotors.18 19 Stimulation of myocardial TGF-ß expression itself may result from a number of factors that are not known at present. Because Hinglais et al20 demonstrated the colocalization of myocardial fibrosis and predominantly T helper lymphocytes and macrophages in spontaneously hypertensive rats, the local myocardial expression of TGF-ß1 and TGF-ß2 could have been stimulated by the significantly increased CD3 T lymphocytes cell count as seen in our DCM patients with EF <50%.21 22
Collagen Protein Detection
The increase in collagen mRNA that we found was mirrored by an
increase in collagen detected at the protein level by histochemistry
(picrosirius red staining) and immunohistochemistry. We were able to
document a significant increase of fibrosis in patients with DCM
because of a trend toward an increase in Col I but not Col III in
patients with DCM, which is in line with earlier reports by
Marijianowski et al.4 This increase in collagen content
appeared to be uniform throughout the biopsy samples. The inability to
detect an increase of Col III by immunohistochemical techniques as well
might be influenced by the availability of Col III epitopes for
antibody binding. If the Col III fibers are covered by type I collagen,
it may be difficult to accurately measure the amount of Col III in
these biopsies. Despite this principal methodological problem of
immunohistological staining, the immunohistochemical
results are consistent with the mRNA findings and indicate a
shift in the Col I/Col III ratio on both the mRNA and protein levels.
In addition, the biochemical results of the significant correlation
between the ratio of hydroxyproline concentration to total protein
concentration and the mRNA abundance of Col I emphasize these findings.
Although the increase in collagen detected at the protein level may
reflect an increase in synthesis, it is also possible that differences
in degradation may play a role. Differences concerning protein levels
of Col I and Col III may depend not only on differential expression but
also on differential degradation. Two different matrix
metalloproteinases (MMPs) are described as being active in degrading
collagens. MMP 1 is synthesized by fibroblasts and shows equal affinity
for Col I and Col III degradation. MMP 8, however, which is synthesized
by neutrophils, has a higher affinity for Col III. Therefore, either
differential activities or differential expression levels of MMP may
also contribute to changes in myocardial Col I and Col III content, in
addition to the changes in gene expression shown in our own
study.23 24 25
Potential Significance of Collagen Changes
Col I and Col III are essential components of the
myocardium, maintaining its structural and functional
integrity. Because of their different physical properties, the altered
Col I/Col III ratio may therefore have a major impact on the
diastolic and systolic function of the heart.
Whereas Col III forms an elastic network storing kinetic energy as an
elastic recoil, Col I represents a stiff fibrillar protein
providing tensile strength. Therefore, increasing Col I protein levels
might impose increasing myocardial stiffness, compromising
diastolic and systolic function of the
myocardium.6
Limitations
A limitation of this study is that only
endomyocardial biopsies from patients with
different degrees of impaired left ventricular function
were analyzed, and not explanted hearts with end-stage heart
failure. Therefore, the analysis of regional distribution of
collagen content (septal versus posterior region, endocardial versus
epicardial region, right ventricle versus left ventricle), which would
have strengthened our study, was not possible. However, this type of
analysis of regional matrix remodeling is possible only in
explanted hearts with end-stage heart failure, which was not the target
group of patients to be evaluated in our study. The aim of the study
was to analyze, in particular, the collagen remodeling in
endomyocardial biopsies of patients with mild to
severe left ventricular dysfunction and not in terminally
failing explanted hearts. In addition, postmortem studies by Baandrup
et al26 demonstrated identical collagen contents in the
right ventricular septum and the left ventricle as assessed
in histological studies of autopsy samples of normal
hearts. Therefore, the use of right ventricular
endomyocardial biopsies from the right
ventricular septum should also be
representative for left ventricular matrix
remodeling. Further studies will be necessary to determine whether the
collagen changes reported here are specific for DCM or whether similar
changes occur in heart failure as a result of other cardiac pathology.
Another limitation of this study is that we did not obtain
endomyocardial biopsies from normal hearts for
ethical reasons. However, the mean total collagen content in the group
of patients with mild left ventricular dysfunction in this
study measured by picrosirius red staining in circularly polarized
light is similar to the total collagen content measured with the same
technique in normal hearts from the right ventricular
septum27 as well as from the free left
ventricular wall.28 Therefore, we also did not
analyze the collagen expression in normal hearts. The reported
differences in collagen expression between DCM and the patients with
mild left ventricular dysfunction are relevant and may have
major implications for systolic and in particular
diastolic dysfunction as seen in DCM.
Summary
Our results showed increased mRNA abundances for Col I and Col III
in endomyocardial tissues of patients with DCM. In
addition, a significant shift of the Col I/Col III ratio was found both
at the level of gene expression and at the protein level. Finally, the
increased mRNA abundances of TGF-ß1 and
TGF-ß2 in DCM suggest that these growth factors
could play a role in the myocardial matrix remodeling seen in DCM. An
interesting secondary finding was the possibility to establish a method
for the analysis of mRNA abundances of 5 different genes in one
single human endomyocardial biopsy with a wet
weight of 3 to 5 mg.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (DFG Pa 369/2-1, Pa 369/2-3). We thank Andrew Remppis, Lübeck, Germany, for his critical review of the manuscript, and we wish to gratefully acknowledge the technical assistance of Enrico Wilke.
Received September 15, 1998; revision received March 12, 1999; accepted March 23, 1999.
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