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Introduction
Members of the small heat shock protein (sHSP) family,
including HSP20, HSP25, HSP27, aB-crystallin, and myotonic
dystrophy kinase binding protein, are expressed in muscle
tissues and share a homologous sequence of approximately
80-100 amino acids at the C-terminus, known as the
a crystallin domain[1,2]. The past decade has witnessed the
discovery of new mammalian sHSP, of which HSP20 is the best
characterized. HSP20 was co-purified from skeletal muscle
with aB-crystallin and HSP27 by affinity chromatography
on a column of immobilized antibodies against
aB-crystallin[3]. Exposure of rat diaphragm tissue to heat stress
in vitro results in the redistribution of HSP20, as well as
aB-crystallin and HSP27, from the cytosol into insoluble fractions, and
enhanced dissociation of the aggregated form to the small
form, which is characteristic of stress
proteins[4]. Stable overexpression of HSP20 in Chinese hamster ovary cells
results in enhanced survival after heat shock, which is similar
to results obtained with
aB-crystallin[5]. Chu et
al[6] were the first to identify the
de novo phosphorylation of cardiac HSP20 in mouse cardiomyocytes after prolonged activation
of the b-adrenergic signaling pathway. The
adenovirus-mediated overexpression of HSP20 in adult rat cardiomyocytes
increases cell contractility, which indicates that HSP20 is
involved in the regulation of myocardial
contractility[6,7].
Heat shock proteins have been implicated in modulating
the cellular response to many stressors, and as molecular
chaperones in suppressing the aggregation or assisting in
the refolding of partially denatured proteins. They usually
protect against ischemic/reperfusion (I/R) injury
in vitro[8-11] and
in vivo[12-14]. However, whether gene transfer of the
HSP20 gene into the beating heart produces a myocardial
protective effect has not been shown. In the present study,
we transferred the HSP20 gene through a recombinant
adeno-virus encoding HSP20 into the myocardium, and showed
that HSP20 protected against I/R injury, probably by
reducing myocardial apoptosis and necrosis in rats.
Materials and methods
Animals and experimental
protocols Male adult Sprague-Dawley rats (230 g-280 g) received a standard diet and free
water. The treatment of the animals and experimental
protocols adhered to the guidelines of the Health Sciences Center
of Peking University (Beijing, China). The animals were
allowed to readjust to the new housing environment for 1 week
before the experiments.
Rats were assigned randomly to 4 groups. In the no-vector control group, the chest was opened and injected
with saline. The I/R control group was also injected with
saline. The third group received the recombinant
adenovirus encoding wild-type HSP20 (Ad.HSP20), and the fourth
group received the recombinant adenovirus encoding green
fluorescent protein (Ad.GFP).
Construction of recombinant
adenoviruses The recombinant Ad.HSP20 and Ad.GFP were prepared as described
previously[15]. Adenovirus was propagated in 293 cells and
purified by 2 rounds of CsCl density ultracentrifugation (4 °C,
13 000×g for 105 min and 16 h, respectively). Viral stocks
were then desalted through a PD-10 desalting column
(Amersham Biosciences, Buckinghamshire, UK) into a
Tris-buffered solution (10 mmol/L Tris, pH 8.0, 2 mmol/L
MgCl2 and 4% sucrose)[16], plaque-titered, aliquoted, and stored at
-80 °C with 4% sucrose until use.
In vivo intracoronary delivery of
adenoviruses The surgical procedures were carried out as described
previously[17]. Donor rats were anesthetized with sodium pentobarbital
(50 mg/kg, ip). Further injections were given as needed
throughout the surgical procedure. Animals were placed
supine on a thermoregulated table (37 °C) . The surgery was
carried out under sterile conditions. The animals were
intubated and ventilated on a positive-pressure ventilator. The
tidal volume was set at 1.5 mL-2.5 mL, and the respiratory
rate was adjusted to within the range of 80 cycles/min to
90 cycles/min to maintain normal arterial
paO2, paCO2, and pH.
The chest was entered through a left intercostal approach.
Before virus infusion, adenosine (0.15 mg), lidocaine
(0.03 mg), and heparin (50 U) were administered via the
jugular vein. With the use of a 26 gauge needle, 200 µL diluted
replication-deficient adenovirus
(2.2×1010 pfu) or 200 µL
sterile saline were injected from the apex of the left ventricle into
the left ventricular cavity while the aorta and pulmonary
arteries were clamped just above the aorta root. The clamp
was maintained for 15 s when the heart pumped against a
closed system. After injection, the exposed heart was
monitored for 5 min for resumption of normal sinus rhythm.
Hemodynamic indices were measured and electrocardiography
was carried out throughout the experimental period.
Myocardial infarction
protocol Four days after the injection of saline or virus, the animals were re-anesthetized
and ventilated artificially with room air. The thorax was
reopened and the heart were exposed to identify the left
anterior descending coronary artery (LAD). A 7-0 silk suture was
passed around the LAD with an atraumatic needle just 4 mm
inferior to the left auricle, and the artery was occluded by
snaring with a vinyl tube through which the ligature had
been passed. The coronary artery was occluded by pulling
the snare tight and securing it with a hemostat. Ischemia
was confirmed by myocardial blanching and
electrocardiography evidence of injury. After 20-min ischemia, the
ligature was released and the heart was reperfused for
2 h. Reperfusion was identified by an obvious ST segment
change.
Measurement of infarction At the end of the infarction
protocol, the ligature around the LAD was retightened and
0.1 mL of 10% Evans blue dye was injected as a bolus into
the left ventricle (LV) cavity with a 26-gauge needle
positioned in the apex of the heart. When the eyes turned blue,
the animals were euthanized immediately, the heart was
excised and rinsed in water to remove excess dye, the atria and
right ventricular free wall were removed, and the remaining
LV was frozen. The LV was then cut from apex to base into
4-6 transverse slices of 2 mm thick. Each slice was weighed
and then incubated in 4% triphenyltetrazolium chloride
solution (TTC) in isotonic pH 7.4 phosphate buffer at 37 °C for
30 min. The slices were subsequently fixed in 10% formalin
solution for 24 h. Viable tissue (red-stained by the TTC) was
distinguished easily from the infarcted regions (pale or
unstained by the TTC) and the risk area (unstained by Evans
blue). The total slice area, the infarcted area, and the risk
area of each slice were determined by computer-assisted
planimetry (Leica Qwin image analysis software; Leica,
Cambridge, UK). During planimetry, the operator was blinded
as to the type of animal. The ratios of risk area to total slice
area, infarct area to total slice area, and infarct area to risk
area were calculated and multiplied by the weight of the slice
to determine risk and infarct weight per slice. Infarct size
was expressed as a proportion of LV mass or risk area mass.
Hemodynamic studies Hemodynamic measurements
were taken at 0 min, 10 min and 20 min ischemia and 30 min,
60 min and 120 min reperfusion. A 1.5 F
micronanometer-tipped catheter was advanced into the LV through the right
carotid artery. The heart rate, blood pressure, left
ventricular end diastolic pressure (LVEDP), left ventricular end
systolic pressure (LVESP) and maximal rates of pressure increase
(+dp/dtmax) and decrease
(-dp/dtmax) were recorded on a
polygraph (NEC San-ei Instruments, Japan).
Measurement of serum cardiac troponin T (cTnT) and
creatine phosphokinase (CK) levels At the end of the
myocardial I/R experiment, a 1-mL blood sample was obtained
from the carotid cannula, stored at 4 °C for 30 min, and
centrifuged at 3000×g for 10 min. The serum was stored at
-40 °C prior to analysis. The concentration of serum cTnT
was determined by the short-turn-around-time (STAT)
assay (Roche Diagnostics, Basel, Switzerland), with use of the
Roche Elecsys 2010 immunoassay analyzer (Roche
Diagnostics). The serum was also analyzed
spectrophotometrically for CK activity (Roche Diagnostics).
Western blot analysis Three hearts from each of the
experimental groups were used separately for measurement of
HSP20 by Western blot analysis. After 4 d of injection of
saline or virus, the hearts were removed quickly, and the LV
was separated and frozen in liquid nitrogen. The frozen LV
tissue was homogenized in protein extraction buffer
containing 20 mmol/L Tris-HCl, pH 7.4, 1% Trion X-100,
150 mmol/L NaCl, 1 mmol/L ethylenediaminetetracetic acid, 2.5 mmol/L
sodium pyrophosphate, 1 mmol/L NaF, 1 mmol/L
Na3VO4 and 0.1 mmol/L phenylmethylsulfonyl fluoride. Aliquots were
resolved on sodium dodecyl sulphate-polyacrylamide gel
electrophoresis. Proteins were transferred to polyvinylidene
difluoride membranes (Schleicher & Schuell, Keene, NH,
USA) and incubated with primary polyclonal anti-HSP20
antibodies (1:1000) (presented by Prof Rui-ping XIAO, NIH,
USA), which recognized HSP20, at 4 °C overnight. Bound
antibodies were detected using a secondary antibody
conjugated to horseradish peroxidase (Santa Cruz Biotechnology,
Inc, CA,USA) and visualized by use of an enhanced
chemiluminescence kit
(SuperSignal® West Pico Trial Kit, Pierce
Biotechnology, Inc, IL, USA) and exposed to X-ray film for
the appropriate time.
Terminal dUTP nick-end labelling staining
Hearts were isolated from each group after I/R for analysis using the
terminal dUTP nick-end labeling (TUNEL) assay. Tissue
samples were fixed in a 4% paraformaldehyde solution,
paraffin embedded, and cut transversely into 6-µm sections.
The assay was operated according to the manufacturer¡¯s
instructions (DeadEnd™ Fluorometric TUNEL System;
Promega, WI, USA). Stained samples were analyzed using a
confocal microscope; at least 500 cells were counted in
randomly selected views.
Statistical analysis Data were expressed as mean±SD.
Differences were analyzed for significance by one-way
repeated-measures ANOVA and further analyzed with the use
of the Newman-Keuls test for multiple comparisons between
treatment groups. The results were considered significant
at P<0.05.
Results
Expression of HSP20 Intraventricular injection of
Ad.HSP20 in vivo resulted in increased HSP20 expression in the
LV as compared with vector and Ad.GFP treatments (Figure 1A). The hearts treated with Ad.GFP showed only
low-level of HSP20 expression, which indicates that
treatment with viral vectors has no significant effect on HSP20
expression in the rat myocardium. LV treated with Ad.HSP20
showed homogenous expression of GFP, whereas those
treated with vector showed no background fluorescence
(Figure 1B).
Myocardial infarction Ad.HSP20-treated hearts showed
a significant reduction in infarct size (39.2%±4.3% risk area)
compared with vector- and Ad.GFP-treated hearts (56.3%±2.9% and 54.9%±8.1%, respectively; P<0.01; Figure 2A).
Infarct size did not differ between vector- and Ad.GFP-treated
hearts (P>0.05). A similar result was observed when infarct
size was expressed as a proportion of LV (Figure 2B). Both
results suggest that the reduced infarct size observed in
Ad.HSP20-injected hearts is entirely due to the
overexpres-sion of HSP20. However, the risk areas (% of LV) were not
significantly different among the groups (ie 53.2%±6.5%, 57.9%±7.3%, and 56.4%±7.5% in the vector-, Ad.GFP-,
and Ad.HSP20-treated groups, respectively;
P>0.05; Figure 2C).
HSP20 gene delivery reduced serum cTnT and CK
levels Ad.HSP20-treated hearts showed a significant
reduction in cTnT release (2.2 µg/L±1.7 µg/L) compared with
vector- and Ad.GFP-treated hearts (12.9 µg/L±3.2 µg/L, and
11.8 µg/L±3.1 µg/L, respectively;
P<0.01; Figure 3A). Similar results were observed for CK release (Figure 3B).
HSP20 gene delivery attenuated apoptosis in the acute
ischemia/reperfusion rat model Figure 4A shows
representative apoptotic cardiomyocytes identified by TUNEL
staining in the I/R-injured region. The ratio of TUNEL-positive
cardiomyocytes to total number of cardiomyocytes in the
Ad.HSP20 group was significantly reduced as compared with
the vector and Ad.GFP groups (15.4%±3.2%
vs 25.7%±4.5% and 27.6%±2.2%,
P<0.01; Figure 4B).
HSP20 gene delivery improved cardiac function
in vivo LVEDP, LVESP,
+dp/dtmax and
-dp/dtmax values are shown in Figure 5. All parameters were comparable among
the 3 groups before and during ischemia and during
reperfusion. All parameters, except LVEDP, declined in value
after ischemia. LVEDP in HSP20-treated rats was
significantly decreased after 60-min reperfusion compared with that
in vector- and Ad.GFP-treated rats (P<0.05, Figure 5A).
LVESP, +dp/dtmax, and
-dp/dtmax values in HSP20-treated rats
were significantly increased after 60-min reperfusion
compared with those in vector- and Ad.GFP-treated rats
(P<0.05, Figure 5B, 5C, 5D).
Discussion
Gene therapy has emerged as a genuine alternative
therapy in coronary artery disease, including ischemic heart
disease. One of the commonly used intramyocardial gene
transfer methods is direct intramyocardial injection. There
have been a few promising trials involving the use of direct
intramyocardial injection in this
area[18,19]. However, the technical problems with this method are that only a small volume
of the myocardium is accessible for transfection and the
distribution of transgenes in the myocardium is not
homo-geneous. To overcome these problems, we used an
in vivo intracoronary gene delivery method that modified the
approach of Hajjar et al[17] to transduce the HSP20 gene into
the ventricular muscle with the use of recombinant
adenoviral vectors. The adenoviral vectors are delivered into the
myocardium via the coronary circulation. Using this
delivery method, we sought to elucidate a direct cause and effect
relationship between HSP20 and cardioprotective effects in
the intact rat heart.
The present study demonstrates, for the first time, that
gene transfer of Ad.HSP20 into the LV muscle causes robust
expression of HSP20 as compared with vector or Ad.GFP
transfer. Ad.GFP-treated LV showed no significant increase
in HSP20 expression compared with vector-treated LV. These
results suggest that the increased expression in the
Ad.HSP20-treated hearts is not due to virus-related stress.
Infarct size was reduced significantly in the I/R hearts
injected with Ad.HSP20. We also examined serum cTnT and
CK levels independently. cTnT originating exclusively from
the myocardium clearly differs from skeletal muscle troponin
T. As a result of its high tissue specificity, cTnT is a
cardio-specific, highly sensitive marker for myocardial
damage[20]. Our results showed that the cTnT level in Ad.HSP20-treated
hearts was reduced significantly as compared with that in
vector- and Ad.GFP-treated hearts. Similar results were
shown with CK. TUNEL staining showed that apoptosis of
cardiomyocytes was reduced in Ad.HSP20-treated hearts.
The decrease of LVEDP and increase of LVESP,
+dp/dtmax and
-dp/dtmax in HSP20-treated hearts may be explained by
HSP20 being an actin-associated protein. It is biochemically
associated with aB-crystallin and localized to distinct
transverse bands in a pattern similar to aB-crystallin and
sarcomeric actin[21,22]. Phosphorylated HSP20 increases the
contractility rate of cardiac myocytes, which indicates that HSP20
is involved in the regulation of myocardial
contractility[6,7].
Heat shock proteins are a family of endogenous
protective proteins. Various HSP have protective effects against
stress injury. HSP70 prevents cell death by inhibiting
apoptosis via associating with apoptosis protease
activating factor-1 (Apaf-1) and blocking the assembly of a
functional apoptosome[23]. Combined and individual mitochon
drial HSP60 and HSP10 expression in cardiomyocytes
protects mitochondrial function and decreases apoptotic cell
death induced by simulated I/R accompanied by decreased
mitochondrial cytochrome c release and caspase-3
activity[11]. HSP60 interacts with Bax and Bak to regulate
apoptosis[24]. Overexpression of aB-crystallin in transgenic mice hearts
provides resistance to I/R injury by negatively regulating
myocyte and non-myocyte apoptosis[25]. HSP27 binds to
cytochrome c released from the mitochondria into the
cytosol and prevents cytochrome c-mediated interaction of Apaf-1
with procaspase-9[26]. These results highlight the notion
that the protective effects of HSP are closely related to
mitochondrial function. Thus, HSP are anti-apoptotic proteins in
cardiomyocytes.
Myocardial ischemia is followed frequently by reperfusion. Reperfusion and the resultant re-oxygenation
lead to the generation of oxygen radicals that can cause
reperfusion injury. Our results
in vivo are consistent with those of Fan
et al[15], who showed that HSP20 and its
phosphorylation at Ser16 might provide protective effects against
b-agonist-induced apoptosis in vitro.
Death of cardiomyocytes due to I/R injury is caused by 2
distinct mechanisms, necrosis, and apoptosis, which
contribute independently to myocardial
infarction[25,27]. The infarct area represents cell death, including necrotic cell death
and apoptotic cell death. cTnT and CK are indicators of
myocardial necrosis, whereas TUNEL staining can reveal
apoptosis. Thus, our results suggest that the protective
effect of HSP20 is attributed to a reduction of necrosis and
apoptosis in cardiomyocytes. In addition, our recent data
shows that lactate dehydrogenase release and caspase-3
activity in H9c2 cells infected with Ad.HSP20 are also
decreased. Therefore, the cardioprotective effect of HSP20
in vivo might be mediated mainly by inhibiting both
cardio-myocyte necrosis and apoptosis.
In conclusion, our results show that overexpression of
HSP20 protects against I/R injury
in vivo, not only by inhibi-ting cardiomyocyte necrosis and apoptosis but also by
increasing myocardial contractility. Our data suggest that
HSP20 is a potential therapeutic protein for ischemic
diseases and additional experiments should be carried out.
Acknowledgements
We are grateful to Prof Rui-ping XIAO (Laboratory of
Cardiovascular Science, Gerontology Research Center,
National Institutes of Health, Bethesda, MD, USA) for her
advice during this research.
References
1 Boelens WC, Croes Y, de Ruwe M, de Reu L, de Jong WW.
Negative charges in the C-terminal domain stabilize the
aB-crystallin complex. J Biol Chem 1998; 273: 28085-90.
2 Suzuki A, Sugiyama Y, Hayashi Y, Nyu IN, Yoshida M, Nonaka I,
et al. A novel member of the small heat shock protein family,
binds and activates the myotonic dystrophy protein kinase. J
Cell Biol 1998; 140: 1113-24.
3 Kato K, Shinohara H, Goto S, Inaguma Y, Morishita R, Asan T.
Copurification of small heat shock protein with alpha B
crystallin from human skeletal muscle. J Biol Chem 1992; 267:
7718-25.
4 Kato K, Goto S, Inaguma Y, Hasegawa K, Morishita R, Asano T.
Purification and characterization of a 20-kDa protein that is
highly homologous to aB crystallin. J Biol Chem 1994; 269:
15302-9.
5 van de Klundert FAJM, van den IJssel PRLA, Stege GJ, de Jong
WW. Rat Hsp20 confers thermoresistance in a clonal survival
assay, but fails to protect coexpressed luciferase in Chinese
hamster ovary cells. Biochem Biophys Res Commun 1999; 254:
164-8.
6 Chu G, Egnaczyk GF, Zhao W, Jo SH, Fan GC, Maggio JE,
et al. Phosphoproteome analysis of cardiomyocytes subjected to
b-adrenergic stimulation: identification and characterization of a
cardiac heat shock protein p20. Circ Res 2004; 94: 184-93.
7 Pipkin W, Johnson JA, Creazzo TL, Burch J, Komalavilas P,
Brophy C. Localization, macromolecular associations, and
function of the small heat shock-related protein HSP20 in rat heart.
Circulation 2003; 107: 469-76.
8 Mestril R, Chi SH, Sayen MR, O'Reilly K, Dillmann WH.
Expression of inducible stress protein 70 in rat heart myogenic cells
confers protection against simulated ischemia-induced injury. J
Clin Invest 1994; 93: 759-67.
9 Lau S, Patnaik N, Sayen MR, Mestril R. Simultaneous
over-expression of two stress proteins in rat cardiomyocytes and
myogenic cells confers protection against ischemia-induced injury.
Circulation 1997; 96: 2287-94.
10 Martin JL, Mestril R, Hilal-Dandan R, Brunton LL, Dillmann
WH. Small heat shock proteins and protection against ischemic
injury in cardiac myocytes. Circulation 1997; 96: 4343-8.
11 Lin KM, Lin B, Lian IY, Mestril R, Scheffler IE, Dillmann WH.
Combined and individual mitochondrial HSP60 and HSP10
expression in cardiac myocytes protects mitochondrial function
and prevents apoptotic cell deaths induced by simulated
ischemia-reoxygenation. Circulation 2001; 103: 1787-92.
12 Okubo S, Wildner O, Shah MR, Chelliah JC, Hess ML, Kukreja
RC. Gene transfer of heat-shock protein 70 reduces infarct size
in vivo after ischemia/reperfusion in the rabbit heart.
Circulation 2001; 103: 877-81.
13 Hutter JJ, Mestril R, Tam EK, Sievers RE, Dillmann WH, Wolfe
CL. Overexpression of heat shock protein 72 in transgenic mice
decreases infarct size in vivo. Circulation 1996; 94: 1408-11.
14 Zhong N, Zhang Y, Fang QZ, Zhou ZN. Intermittent hypoxia
exposure-induced heat-shock protein 70 expression increases
resistance of rat heart to ischemic injury. Acta Pharmacol Sin
2000; 21: 467-72.
15 Fan GC, Chu G, Mitton B, Song Q, Yuan Q, Kranias EG. Small
heat-shock protein Hsp20 phosphorylation inhibits
b-agonist-induced cardiac apoptosis. Circ Res 2004; 94: 1474-82.
16 Nyberg-Hoffman C, Aguilar-Cordova E. Instability of
adenoviral vectors during transport and its implication for clinical studies.
Nat Med 1999; 5: 955-7.
17 Hajjar RJ, Schmidt U, Matsui T, Guerrero JL, Lee KH, Gwathmey
JK, et al. Modulation of ventricular function through gene
transfer in vivo. Proc Natl Acad Sci USA 1998; 95: 5251-6.
18 Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD,
Maysky M, et al. Gene therapy for myocardial angiogenesis.
Initial clinical results with direct myocardial injection of
phVEGF165 as sole therapy for myocardial ischemia.
Circulation 1998; 98: 2800-4.
19 Rosengart TK, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman
GW, et al. Angiogenesis gene therapy: phase I assessment of
direct intramyocardial administration of an adenovirus vector
expression VEGF121 cDNA to individuals with clinically
significant severe coronary artery disease. Circulation 1999; 100: 468-74.
20 Zacharowski K, Otto M, Hafner G, Chatterjee PK, Thiemermann
C. Endotoxin induces a second window of protection in the rat
heart as determined by using p-nitro-blue tetrazolium staining,
cardiac troponin T release, and histology, Arterioscler Thromb
Vasc Biol 1999; 19: 2276-80.
21 Brophy CM, Lamb S, Graham A. The small heat shock-related
protein-20 is an actin-associated protein. J Vasc Surg 1999; 29:
326-33.
22 Rembold CM, Zhang E. Localization of heat shock protein 20 in
swine carotid artery. BMC Physiol 2001; 1: 10.
23 Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana
T, et al. Heat-shock protein 70 inhibits apoptosis by preventing
recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat
Cell Biol 2000; 2: 469-75.
24 Kirchhoff SR, Gupta S, Knowlton AA. Cytosolic heat shock
protein 60, apoptosis, and myocardial injury. Circulation 2002;
105: 2899-904.
25 Ray PS, Martin JL, Swanson EA, Otani H, Dillman WH, Das DK.
Transgene overexpression of aB crystallin confers simultaneous
protection against cardiomyocyte apoptosis and necrosis during
myocardial ischemia and reperfusion. FASEB J 2001; 15:
393-402.
26 Bruey JM, Ducasse C, Bonniaud P, Ravagnan L, Susin SA,
Diaz-Latoud C, et al. Hsp27 negatively regulates cell death by
interacting with cytochrome C. Nat Cell Biol 2000; 2: 645-52.
27 Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH,
Krajewski S, et al. Apoptotic and necrotic myocyte cell deaths
are independent contributing variables of infarct size in rats. Lab
Invest 1996; 74: 86-107.
28 Zhu YH, Wang X. Overexpression of heat-shock protein 20 in
rat heart myogenic cells confers protection against simulated
ischemia/reperfusion injury. Acta Pharmacol Sin 2005; 26: 1076-80.
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