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Introduction
Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in the world. In recent years, the study of
the molecular mechanisms of CVD has accelerated the discovery of many new potential therapeutic targets. Fire
et al[1] injected double-stranded RNA (dsRNA) into the nematode worm
Caenorhabditis elegans, resulting in silencing the gene
whose sequence was complementary to that of the dsRNA. This post-transcriptional gene silencing (PTGS) phenomenon
was named RNA interference (RNAi). Since then, it has become clear that RNAi is a defensive process of cells used to
degrade invasive genetic materials and inhibit endogenous genes that should be controlled. This technology is an extremely
useful tool for identifying gene functions and evaluating potential therapeutic targets. So, an increasing number of
biotechnological and pharmaceutical companies are attempting to develop RNAi-based drugs for the prevention and treatment of
human disease such as viral infections, tumors and
CVD[2_4].
Many CVD are chronic in nature and worsen progressively over a long period of time. Despite the significant advances
that have been made in the therapy of CVD, current available drugs are associated with many undesirable factors such as
toxicity, complexity and resistance. As a natural self-defense mechanism of eukaryotic cells, RNAi can offer major
advantages over pharmacological therapy by targeting pathogenic genes specific for CVD with high potency and low toxicity.
RNAi has been used in models of hyperlipemia, ischemia-reperfusion, and choroidal neova-scularization and for
identification of those genes involved in
CVD[5,6]. This review will discuss some of the more subtle mechanistic aspects of
RNA-induced PTGS, and present
recent work that focuses on the potential application of this breakthrough technology to CVD such as congenital heart
disease, hypertension, atherosclerosis, cardiac hypertrophy, myocarditis, and heart failure.
Molecular mechanisms
Biochemical and genetic studies have revealed the molecular mechanisms by which homologous dsRNA cause
degradation of target messenger RNA. RNAi includes 2 main steps: an initiator step for generation of small-interfering RNAs
(siRNAs) from long dsRNA or mature microRNAs (miRNA) from their precursors, and an effector's step for cleavage or
repression of target RNA[7].
In the initiator step, the dsRNA-specific endonuclease Dicer binds with high affinity to dsRNA of more than 38 bp in
length and chops long dsRNA (introduced directly or via a transgene or virus) into fragments of ~22
nt[8]. The primary structure of Dicer includes an ATP-dependent RNA helicase domain, a Piwi/Argonaute/Zwille (PAZ) domain, two RNase
III-like domains and a COOH-terminal dsRNA-binding domain. The recently identified Dicer 1 and Dicer 2 are responsible for the
production of mature miRNAs from their precursors and for the cleavage of long dsRNA into siRNAs,
respectively[9]. siRNA is composed of 21_23 nt dsRNA duplexes with a 5'-monophosphate, a 3'-hydroxyl group and 2-nt 3' overhangs, and this
configuration plays an important role in the efficacy of silencing.
In the effector's step, the siRNA duplexes are incorporated into the RNA-induced silencing complex (RISC), a
protein-RNA effector nuclease complex that recognizes and destroys passenger-strand of siRNA
duplexes[10]. After releasing that cleaved strand, the guide-strand in active RISC becomes available to interact with target mRNA. The phosphorylation of
siRNA 5'-terminal is required to entry into RISC. The PAZ and PIWI domains of RISC can recognize the 3'-terminus and the
5'-terminus of the guide strand, respectively, and then targets the homologous transcript by base-pairing interactions with
the guide strand. Finally, an RNase H region at the PIWI domain cleaves the mRNA between the tenth and eleventh
nucleotide from the 5' terminus of the
siRNA[11,12].
One of the most important characters of RNAi is the ability to amplify and sustain gene silencing induced by dsRNA in
many organisms, even when triggered by minute quantities of aberrant RNA. Recently genetic studies have found that
siRNA might act as mRNA-specific primers that are incorporated during the subsequent conversion of the target mRNA into
dsRNA. Nascent dsRNA is then cleaved by RNase III-related enzymes to degrade the mRNA while generating new siRNAs
in the process. RNA-directed RNA polymerase (RdRP) plays the crucial role in mediating the incorporation of a synthetic
siRNA into nascent dsRNA. This enzyme is discovered in most eukaryote except mammals and
insects[13]. It is not clear if the cardiac myocytes connected by gap-junctions contain RdRP and display RNAi spread between cells. Through this approach,
aberrant RNAs can be degraded efficiently through a cycle of "degradative-PCR" (Figure 1).
Advantages
In comparison with other conventional drugs, siRNA have many advantages: (1) The selection of target sites is much
easier and more flexible because target mRNA and siRNA are sequences-specific and complementary. For a given mRNA
molecule, the inhibitory effects of siRNA can be achieved by targeting different regions of target
mRNA[4]; (2) For gene silencing, only a substoichiometric amount of siRNA is enough to decrease homologous mRNA drastically within 24 h; (3)
siRNA can knockdown the expression of any cognate genes in cells of different species even though that gene is critical for
animal development; (4) siRNA do not seem to adversely affect the physiological functions of the cells. The given length and
high level of homology of siRNA to the target region of cognate transcription ensure the selective destruction of only the
transcript of interest. siRNA without suitable targets seems to remain inert within cells. This exclusive specificity without
adverse side effects is the most attractive feature of using RNAi for an antiviral
approach[6,7]; (5) siRNAs can silence genes
stably. With the application of plasmid vectors and viral vectors, siRNA can display their long-term biological
effects[14]. Taken together, siRNAs produced
in vivo or in vitro transfected into cultured cells or animals could result in the
sequence-specific silencing of mRNA. With the proof-of-concept studies, siRNA-based gene drugs will be used as an alternative
therapeutic strategy in the future.
Disadvantages and improvements
Although siRNA-mediated RNAi has become a functional genomic tool, recent studies
in vitro have revealed that some duplex siRNA sequences have off-target effects, trigger an interferon
response[15] and possess other drawbacks such as
higher cost, short duration, and hard delivery into specific tissues and resistances. Therefore, many new methods are being
developed to overcome these disadvan-tages.
Researchers have proposed general guidelines for designing siRNA
oligonucleotides[16]. Recently, Heale
et al proposed a novel approach for the determination of mRNA secondary structures and
showed, that in combination with duplex-end
energies, the predicted strong secondary structures could account for 80% of non-functional
siRNA target sites[17]. Bioinformatics have also been used to design siRNA, and many websites provide methods to pick siRNAs. Our laboratory
has applied for a patent to design siRNA, and the success rate of gene silencing is more than 90%.
In order to improve the efficiency and duration of small RNA in gene silencing research, several methods to produce
siRNAs have been proposed, such as chemically synthesized
siRNA[18], DNA vector-based siRNA, and siRNA
cassette[19]. The chemical modifications of siRNA has been shown to be necessary for increasing the efficiency of small RNAs such as the
stability of siRNAs within the body, bioavailability to different tissues, affinity for the blood proteins, and specific delivery
to the chosen site[20]. Chemically stabilized siRNAs with partial phosphorothioate backbone and
2'-O-methyl sugar modifications on the sense and the antisense strands showed significantly enhanced resistance towards degradation by
exonucleases and endonucleases in serum and in tissue homogenates. Taken together, the chemical modifications of siRNAs may
improve the stability and utility of siRNAs for therapeutic application
in vivo.
Delivery system
Recent rapid progress in the application of RNAi to mammalian cells offers new approaches to drug target identification
and validation; however, the use of RNAi in mammals has been hindered by the inability to deliver siRNAs effectively. To
make RNAi highly efficient in cardiomyo-cytes, vascular smooth muscle cells (VSMC), and vascular endothelial cells with
low transfection efficacy, virus-mediated RNAi has been
developed[18,19]. The effects of RNAi on GAPDH transcripts were
reduced by 90% in the primary cultured cells, indicating that virus-mediated gene silencing is a promising technique for gene
suppression in cardiovascular
studies[21].
As an emerging technique with potential use as a therapy for CVD, ultrasound-targeted microbubble destruction (UTMD)
has proven to be an efficacious method for delivering genes such as decoy oligodeoxynucleotides and anti-sense
oligonucleotide for TNF-a[22,23]. For the first time, Shohet
et al found that UTMD could deliver recombinant adenovirus containing
b-galactosidase to the heart, achieving transient transgene expression with striking tissue
specificity[24]. Recently, UTMD has been used to deliver gene to cure ischemic heart
diseases[25,26]. It appears that this new technique can be used for the
delivery of siRNA to take advantage of its favorable characteristics such as organ specificity, low level of toxicity, no
immunogenicity, repeatable applicability, and low costs.
Injection, a novel therapeutic method of siRNA delivery, includes local and systemic injection. Chae
et al found that local injection of siRNA targeting S1P, a
family of S1P G protein-coupled receptors required for the stabilization of nascent
blood vessels during embryonic development, could silence the cognate transcript in endothelial cells and
inhibited endothelial cell
migration[27]. As a kind of systemic injection, hydrodynamic injection involves the rapid injection of a large-volume bolus to
cause transient high venous pressures, which can facilitate the delivery of siRNAs. Hamar
et al used hydrodynamic transfection to administer siRNAs targeting Fas into mice after ischemic reperfusion injury and demonstrated a 4-fold
reduction of Fas mRNA and protein expression, and a substantial reduction of renal tubular
apoptosis[28].
More interestingly, Soutschek
et al developed a new delivery system by conjugation of cholesterol to siRNAs. They
found that Chol-apoB-siRNAs, but not unconjugated apoB-siRNAs, could effectively degrade apolipoprotein B mRNA by
more than 50% in the liver and by 70% in the jejunum via injection into a tail
vein[6]. The plasma levels of the apoB protein
were reduced also by more than 60%. Furthermore, chol-apoB-siRNA resulted in about a 40% reduction in the levels of LDL
and total plasma cholesterol. Soutschek's work is of significance for the systemic
in vivo application of RNAi technology, because it shows for the first time a new class of therapeutics that harnesses the RNAi mechanism and suggests the
therapeutic potential of RNAi for the treatment of CVD. Recently, it has been reported that apoB-specific siRNAs were
encapsulated in stable nucleic acid lipid particles and administered by intravenous injection to cynomolgus monkeys. The
results demonstrated that the apoB protein, serum cholesterol and low-density lipoprotein levels significantly decreased as
early as 24 h after treatment for 11
d[5]. Although these delivery systems are effective in animal models, they still need detailed
safty testing and efficacy evaluation before they are used for human clinical trials.
RNAi application in CVD
It has been established that the activity of a gene can be inhibited by the introduction of dsRNA with the sequence
specific to the gene. The specificity and potency of RNAi make it ideal for investigating human disease-susceptibility
genes[29]. In this way, some CVD-specific molecular targets have been identified (see Table 1 for a summary). So RNAi-based gene
drugs can be developed in the near future.
Apoptosis in CVD
Compelling evidence has accumulated indicating that apoptosis may play a critical role in the pathogenesis of a variety
of CVD including atherosclerosis, myocardial infarction, ischemia followed by reperfusion, and heart
failure. Naturally, there is intensive apoptosis research in the field of CVD therapy.
Evidence has confirmed that Fas-induced apoptosis can enlarge infarct size during reperfusion of ischemic tissue in
multiple tissues such as the heart[30], kidney and brain. Hamar
et al found that siRNAs targeting Fas could inhibit Fas
expression in the murine kidney
in vivo and protect mice from postischemic acute renal
failure[28]. Another good example is that
in vivo silencing the Fas gene protects mice from liver failure and fibrosis by intravenous injection of Fas siRNA.
Therefore, these findings suggest that the heart or brain might also be protected from ischemia reperfusion injury by
silencing Fas, and offer a novel therapeutic that is prophylactic against ischemia-perfusion disease. On the other hand,
experimental results show that excessive apopto-sis of vascular smooth muscle cells (VSMCs) plays a key role in the
progression of atherosclerotic lesions, resulting in many cardiovascular
events[32]. GADD153, a member of the
CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, has been linked to apoptosis in VSMCs. Blaschke
et al found that inhibition of GADD153 by siRNAs reduces C-reactive protein-induced GADD153 mRNA expression and apoptosis.
Moreover, several recent investigations have demonstrated that Omi/HtrA2, a serine protease, could be released into the
cytosol from mitochondria and promote caspase-dependent apoptosis after an apoptotic insult. Liu
et al found that the introduction of siRNA molecules against Omi into cardiomyocytes effectively eliminated Omi/HtrA2 protein expression and
reduced hypoxia reoxygenation-induced cardiomyocyte apoptosis with decreased caspase-3
activity[33]. Their findings might lead to a
new strategy for the treatment of cardiovascular
disorders[34].
Congenital heart disease
Identifying genetic components plays a key role in understanding development processes that can be useful for the
discovery of targets in the treatment of CVD. Some laboratories have utilized the technology of RNAi to identify genes
involved in heart development. With the use of an RNAi-based genome wide loss-of-function screen, Kim's group identified
a variety of genes encoding cell-signaling proteins and transcription factors for different steps during the development of the
Drosophila embryonic heart[35]. Recently, Qian et al revealed that neuromancer (nmr), a
homolog of Tbx20, is another determinant of morphogenesis of the
Drosophila heart by conducting RNAi experiments with
related genes. For example, reducing nmr function in the absence of pannier further aggravates the deficit in cardiac
mesoderm specification[36]. Furthermore, another research group found that RNAi-mediated depletion of Smarcd 3, a 60-kDa
subunit of the BAF complexes, in mouse embryos derived from embryonic stem cells caused defects in heart morphogenesis.
The defect displays an impaired expansion of the anterior/secondary heart field that can lead to abnormal cardiac muscle
differentiation[37]. More interes-tingly, Chartier
et al reported that embryos in which pericardin, an extracellular matrix
component, silenced via RNAi, could exhibit severe defects in the formation of the heart epithelium because pericardin can
mediate the crosstalk between the dorsal ectoderm and cardioblasts required to regulate their coordinated movement during
dorsal closure. In addition, they found that the heart epithelium displayed a disorganized appearance during its migration to
the dorsal midline in these embryos following the knockdown of pericardin
mRNA[38]. Obviously, the silencing of pericardin
is associated with pathologic holes in the walls of the heart. Taken together, these studies demonstrate that RNAi is an
efficient reverse genetic tool for producing and identifying the loss-of-function mutant phenotypes of genes involved in
CVD.
Hypertension
Hypertension is one of the most important risk factors for stroke, congestive heart failure, myocardial infarction, and
peripheral vascular disease. Several lines of experimental results have revealed that renin-angiotension systems (RAS) are
involved in the development and maintenance of hypertension. Angiotensin (Ang) II is a potent vasoconstrictor peptide
produced by the RAS, and binds to 2 distinct receptor subtypes, namely type 1
(AT1) and type 2 (AT2). Mice with a
homozygous deletion of the AT1A subtype
(AT1A_/_) exhibit reduced blood pressures without pressor responsible for Ang II
infusion[41]. It implicates AT1A
as the primary subtype accountable for Ang II actions in mice. Although several
AT1 receptor antagonists are used for the treatment of
hypertension, they pose significant limitations. So, it is important to develop new ways to improve hypertension control by
providing longer-lasting effects with a single dose and reducing side effects that lead to poor com-pliance. Recently, Vazquez
et al employed RNAi technology to explore these
possibilities[42]. They selected
AT1R as the target gene to design corresponding dsRNAs, and then transfected them into Chinese hamster ovary cells (CHO), which express rat
AT1R. They found that transfection of
AT1R-expressing CHO cells with
AT1R-dsRNA resulted in an 80% decrease in the level of
AT1R mRNA. Furthermore, dsRNA caused a dose-dependent decrease in the specific binding of Ang II to
AT1R-expressing CHO cells. To determine whether the decrease in
AT1R -specific binding was associated with a reduction in functional
AT1R, they examined the effects of dsRNA transfection on Ang II-stimulated calcium uptake. The Ang II-induced increase in calcium uptake was
completely abolished in AT1-dsRNA
transfected cells. On the other hand, Chen
et al found that knocking down the expression of heat shock factor-1 (HSF-1) with RNAi technology exacerbated Ang II-induced inflammatory injury by causing
significantly higher activation of NF-kB in
VSMCs[43]. Such evidence would support the notion that heat shock proteins play
a direct role in suppressing Ang II-induced inflammatory signaling pathways and subsequent inflammation. Furthermore,
recent research on the mechanism of hypertension showed that the RhoA-Rho kinase pathway was the important
pathogenesis of the abnormal contraction of the VSMCs in
CVD[44]. Bi et al discovered that the knockdown of RhoA by RNAi
decreased the level of RhoA mRNA and the contractility of the cultured
VSMCs[45]. Their result indicated that the expression
level of RhoA played a critical role in the regulation of contractility in the de-differentiated VSMC, and RhoA could be a new
therapy target of hypertension. Taken together, these findings suggest that RNAi might have potential as an alternative to
drug therapy for hypertension.
Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of the arterial intima, resulting from a concerted action of multiple
factors[46]. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis
of the disease. Further molecular analysis indicates that the nuclear factor-kappa B
(NF-kB) plays a prominent role in the formation of atherosclerosis because of its ability to adhere to elements in the promoters of key inflammatory and
atherosclerosis genes[47]. Dwarakanath
et al synthesized siRNA targeting NF-kB by the use of a rapid PCR-based approach that
generates sense and antisense siRNA separated by a hairpin loop downstream of the U6 promoter, and then transfected them
into the VSMC derived from 12/15-Loko mice versus genetic control wild type mice in relation to cellular growth and
migration[48]. They found that the mRNA and protein of
NF-kB and NF-kB-dependent transcriptional responses were reduced
markedly by the siRNA. On the other hand, Kobashi
et al found that adiponectin enhances Akt
phosphorylation[49]. The inhibitory effect of adiponectin on
TNF-a-induced interleukin (IL)-8 synthesis was blocked in part by pretreatment with the
PI3 kinase inhibitor LY294002 or by Akt siRNA transfection. It suggests that Akt activation might inhibit IL-8 synthesis, a
pro-inflammatory chemokine that plays a role in atherogenesis. These observations may suggest new options for the
treatment of atherosclerosis.
Cardiac hypertrophy
Cardiac hypertrophy is a compensatory response to a variety of physiological or pathological stimuli, and prolonged
hypertrophic responses may eventually lead to arrhy-thmia, heart failure and sudden
death[50]. So identifying the cardiac hypertrophy-related novel human genes will provide important insights into the mechanisms that regulate hypertrophic cell
growth and assist in development of new pathway for treatment of cardiac hypertrophy heart failure.
Liu et al identified a novel human gene, myofibrillogenesis regulator-1 (MR-1) from a human skeletal muscle cDNA
library that interacts with contractile proteins and exists in human myocardial
myofibrils[51]. They established a hypertrophy
model in which hypertrophic cell growth can be
induced by Ang II incubation in cultured neonatal rat cardio-myocytes. By transfecting neonatal cardiomyocytes with a
pSi-1 targeting the MR-1 sequence, Liu found that the
MR-1 mRNA and protein expression were greatly decreased. Furthermore,
compared with the Ang II-treated group, the MR-1 RNAi+Ang II group showed a decrease on the surface area of cells by 36%.
More interestingly, Pedram et
al[52]
reported that the translocation of the hypertrophic transcription factor, NF-AT, to the nucleus of the cardiomyocyte and the
enhancement of NF-AT transcriptional activity induced by Ang II could be prevented by
17b-estradiol (E2). Ang II also stimulated the activation of ERK and protein kinase C, contributing to cardiac hypertrophy. E2 inhibited these pathways,
related to the stimulation of atrial natriuretic peptide production and secretion. These observations were further supported
by the evidence that siRNA against the MCIP1 gene significantly reversed both the E2 restraint of protein synthesis and the
inhibition of Ang II-induced calcineurin activity. Accordingly, their findings may provide a better understanding of the
mechanism of cardiac remodeling and new insight into the development of novel therapeutic strategies in cardiac hypertrophy.
Myocarditis
Evidence has accumulated that viral myocarditis is an
important cause of heart failure and dilated
cardiomyopathy[53]. More effective approaches are needed to treat viral infection. If genes responsible for affective disorders are identified, gene
silencing could be an alternative therapeutic tool,
especially for cases of drug therapy resistance.
Coxsackievirus B3 (CVB3) has been identified as the most common causal agent of viral myocarditis, but existing drug
therapies are of limited value[54]. Many studies have shown that RNAi can control viral infection by targeting viral genes.
Recently, Schubert et al found that 2 independent siRNA targeting the 3D RNA-dependent RNA polymerase were able to
reduce virus titre by 80% and 90%,
respectively[55]. Their results demonstrate the enormous potential of the RNAi approach.
More interestingly, Schubert and colleagues constructed a siRNA double expression vector (SiDEx) to achieve simultaneous
expression of both siRNA from 1 plasmid. Compared with conventional expression vectors, SiDEx showed substantial gene
regulation of the mutated target RNA. So it is believed that SiDEx may be a helpful tool to achieve sustained silencing of
viruses, ultimately reducing the risk of emergence of viable mutants. Recently, Yan
et al reported that the siRNA targeting the
viral protease 2A displayed a 92% inhibition of CVB3 replication and a 90% protection of the siRNA-pretreated cells.
Moreover, they found that administration of the siRNA after viral infection could effectively inhibit CVB3 replication,
indicating its therapeutic
potential[56]. These findings imply that siRNA-based gene drugs may be an effective therapy for
viral myocarditis.
Heart failure
Investigation shows that heart failure is a common lethal condition associated with various CVD and remains the leading
cause of morbidity and mortality. One of the important features of heart failure is a decreased
Ca2+ uptake into the sarcoplasmic reticulum (SR) by the SR
Ca2+-ATPase 2 (SERCA2), which is negatively regulated by phospholamban (PL), a key regulator
of cardiac calcium homeostasis[57].
Recent findings demonstrated that the development of severe heart failure in the genetic MLP (-/-) animal model could be
abolished completely by the targeted ablation of PL. PL has been considered as the potential therapeutic target for the
improvement of SR Ca2+ uptake and cardiac function. Watanabe
et al synthesized 21 nt siRNA duplexes with symmetric 2-nt
3'-overhangs targeting PLB mRNA and introduced them into neonatal rat cardiac myocytes by use of the HVJ envelope
vector[58]. They found that PLB siRNA resulted in a significant decrease in the levels of both PLB mRNA and protein, while
the mRNA and protein of SERCA2 calsequestrin were not affected. The affinity of SERCA2 for
Ca2+ was also increased. In order to determine the effect of PLB RNAi on the cardiac myocytes in which
Ca2+ handling was impaired, Watanabe
et al exposed myocytes to
H2O2, a reactive oxygen species. The same result with decreased PLB mRNA and protein levels was
achieved. So Watanabe's strategy used for the PL ablation may be considered as a novel and attractive candidate for clinical
therapy in heart failure.
Conclusion
CVD is a severe public health problem with significant personal, social, and economical consequences. More effective
approaches are urgently needed to treat CVD. It is evident from the above discussion that the use of RNAi may hold great
promise for a permanent treatment of CVD by targeting special pathogenic genes. Given the relative ease with which one can
design one to several siRNA to target a gene of interest, there is no need for time-consuming pharmacological analyses and
drug specificity studies. One can simply target the specific genes in the cardiovascular system by using chemically
synthesized siRNA or viral vectors expressing hsRNA. The potential targets in CVD are numerous. However, careful assessment
is required for the potential of RNAi as a gene therapy approach for controlling CVD, because there are some questions to be
solved such as the delivery of vector and dosage required to elicit the desired effect. In addition, because each gene may be
a point of signaling networks, and cross talk with other signal pathways, inhibition of the signaling certainly affects the
intricate networks and may leads to unwanted side effects. So it has no exaggeration to say that careful planning is necessary
before any clinical trial, although a report has shown an encouraging result that a siRNA targeting vascular endothelial
growth factor receptor-1 has potential for clinical use in preventing aging-related macular degeneration.
Even although RNAi-based therapy is still in infancy stage, it holds tremendous promise for use in routine clinical
practice as an adjunct to existing procedures since it can help to overcome the limitations associated with current therapeutic
regimen[59,60]. The increasing knowledge of small RNA will help us to better understand the mechanism of gene expression
regulation in eukaryotes. Therefore, modulation of transcripts of interest may be an alternative molecular strategy for
experimental research and treatment of pathophysiological
states[61,62]. Scientists have developed many RNAi libraries to
study the function of genes in nematodes. With the advent of an RNAi library in mammals and the refinement of techniques
to silence gene, siRNA-based drugs will make great advances in the prevention and treatment of CVD.
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