Effect of Phosphorothioated Oligonucleotides on Neointima Formation in the Rat Carotid Artery
Dissecting the Mechanism of Action
Abstract Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-myc suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a “neonatal” phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.
- Received May 16, 1996.
- Accepted November 28, 1996.
Animal studies using antisense oligonucleotides to genes associated with cell proliferation demonstrate that adventitial or intraluminal application of these agents inhibits neointima formation after injury in both the rat carotid and pig coronary arteries.1 2 3 4 5 6 Despite this apparent success of animal studies, a number of important concerns have been raised regarding the use of oligonucleotides as a potential therapy to inhibit restenosis following angioplasty in humans. First, there is increasing evidence that many of the antiproliferative effects in vitro and in vivo are not specific to the target sequence. Although the antiproliferative effect of the 4G motif in phosphorothioated oligonucleotides is the most well documented,7 8 antisense oligonucleotides have been associated with a multitude of nonspecific actions (reviewed in Bennett and Schwartz9 ). While the fact that oligonucleotides inhibit cell proliferation nonspecifically does not exclude their clinical use to block restenosis, this observation has raised wider concerns over the mechanism of action of oligonucleotides on the cellular kinetics after arterial injury.
The premise behind the use of oligonucleotide agents is that a single dose delivered at the time of injury can profoundly affect neointima formation and thus suppress late restenosis. There are a number of flaws behind this argument. First, this mechanism of action supposes that inhibition of the initiation of the response to injury, ie, medial smooth muscle cells entering the cell cycle, can affect later stages of the process. Conceptually, there is no reason why this should be so. The intimal lesion forms by cell migration and intimal cell proliferation. Blocking medial proliferation per se can thus affect intimal formation only if it also leads to suppression of cell migration. Second, if the effect of the agent is maintained beyond 4 days, the time at which the intima is beginning to form, then agents that inhibit medial cell proliferation may also inhibit intimal cell proliferation. However, there is no evidence of this from the published studies of inhibition of either cell migration or intimal cell proliferation using antisense agents, and in most studies, the antisense agent is either not present or no longer active at 4 days. Furthermore, there is evidence from studies of nonantisense agents that profound suppression of medial cell proliferation after injury, eg, by antibodies to bFGF, does not suppress overall neointima formation at late time points.10 In contrast, antibodies to platelet-derived growth factor that have minimal effects on medial replication but marked effects on migration do inhibit neointima formation.11
We previously showed that a 4G-containing phosphorothioated antisense oligonucleotide directed against the proto-oncogene c-myc suppresses neointima formation after injury in the rat carotid artery.2 In this study we attempted to determine how this oligonucleotide impairs the cellular responses of the artery to injury.
Synthesis of Antisense Oligonucleotides
Phosphorothioate-derived oligodeoxynucleotides were synthesized by Operon Technologies (Alameda, Calif). After deprotection, oligodeoxynucleotides were dissolved in water, extracted with phenol/chloroform/isoamyl alcohol (49.5:49.5:1), precipitated with ethanol, and redissolved in water. Concentrations were determined spectrophotometrically by assuming 1 A260 unit=33 μg DNA. All oligodeoxynucleotides used were 15 mer. The c-myc sense oligodeoxynucleotide sequence comprised the first five codons of human c-myc mRNA (5′ ATGCCCCTCAACGTT 3′), and antisense c-myc oligodeoxynucleotides comprised the complementary sequence AACGTTGAGGGGCAT. Although the human sequence has a 1-bp mismatch compared with the rat sequence, we previously showed that this oligonucleotide suppresses rat c-myc expression in vitro and in vivo.2 The choice of this sequence therefore allows direct comparison with our earlier study. Antisense oligonucleotides were also biotinylated at the 5′ end for studies on the distribution of oligonucleotides in the vessel wall.
Injury to the Rat Carotid Artery
Male Sprague-Dawley rats (3 to 4 months old) (Bantin and Kingman Laboratories, Edmonds, Wash) were used in all experiments. Rats were anesthetized by intraperitoneal injection of xylazine (Anased, 4.6 mg/kg body weight) and ketamine (Ketaset, 70 mg/kg body weight). A midline incision in the neck was made to expose both carotid arteries. Common carotid injury was induced on both sides by the passage of a Fogarty balloon embolectomy catheter (FG2) that was inserted via the external carotid artery and distended with 20 μL of saline as described by Clowes et al.12 The catheter was rotated while it was pulled back to denude the vessel of endothelium and to injure the media. This procedure was repeated two more times. After arterial injury, antisense c-myc (n=6) or sense c-myc (n=6) oligodeoxynucleotide was applied to the adventitial surface of the vessel wall in a pluronic gel solution, or vessels were treated with the gel alone. Sense and antisense c-myc oligodeoxynucleotides were dissolved in a 0.25% pluronic gel solution at 4°C at 1 mg/mL. Two hundred milliliters of the gel solution was applied to the distal third of the common carotid artery immediately after vessel injury. For measurement of cells entering S phase during the last 24 hours before death, BrdU (25 mg/kg body weight) was injected subcutaneously 17, 9, and 1 hour before killing. Rats were killed by repeated administration of anesthetic cocktail and exsanguination via the jugular vein. Arteries were pressure-fixed in vivo with 0.1 mmol/L phosphate-buffered 4% paraformaldehyde at 110 mm Hg perfusion pressure for 10 minutes. Vessels were excised and placed in the same fixative for 1 hour, then placed in Ringer’s solution. Cross sections were cut and immunostained for BrdU as previously described.13 For assessment of c-myc, osteopontin, and tropoelastin mRNA expression, carotid arteries were injured, oligonucleotides were applied (antisense, sense, or pluronic gel alone, n=6 for each group), and vessels were removed at 4 days. Vessels were divided in half and snap-frozen in liquid nitrogen for RNA extraction. To assess the effect of oligonucleotide application on neointima formation, animals were killed at 14 days, and vessels were fixed in situ by perfusion under systolic pressure using a manometer to measure perfusion pressure. Two-micron sections were stained with orcein, and the cross-sectional area of neointima was measured for the proximal (untreated) and distal (treated) segments of the artery. Measurements were made only on vessel profiles that were almost completely spherical, ensuring near perpendicular alignment to the cutting blade. A cross section of the artery was examined by light videomicroscopy using a computerized morphometry system. Medial area was defined as the area enclosed by the external and internal elastic laminae, whereas intimal area was defined as the area between the internal elastic lamina and the perimeter of the lumen.
Assessment of Endothelial Cell Proliferation
For studies investigating the effects of oligonucleotides on endothelial cell proliferation, the carotid artery was injured by introducing the catheter to approximately half-way down the vessel, then gently inflating and rotating the balloon. This produced an obvious interface between areas of vessel with endothelium and areas without it. After 2 days, animals received three injections of BrdU as described above and 0.5 mL of 5% Evan’s blue into the tail vein just before killing. Vessels were removed, and en face preparations were made for endothelial cell BrdU staining as previously described.14 The total number of endothelial cells and the number of labeled endothelial cells were counted using an eyepiece reticule in a 10× eyepiece with a 40× objective. Quantification of replicating endothelial cells was restricted to an area that was within 1 mm of the interface, equivalent to four reticule fields deep in the endothelial monolayer.
Distribution of Oligonucleotides in the Vessel Wall
Biotinylated oligonucleotides were used to track the distribution of sequences 2 days after injury. Vessels were gently injured as described above, and biotinylated oligonucleotides were applied at 1 mg/mL in a pluronic gel. Vessels were fixed as described above, and biotinylated sequences were detected using an avidin-peroxidase detection system.
Northern Hybridization for c-myc, Osteopontin, and Tropoelastin
Proximal (untreated) or distal (treated) segments of carotid artery were pooled for each treatment group for extraction of RNA. Frozen arterial tissue was ground to a fine powder under liquid nitrogen, and total cellular RNA was prepared by acid thiocyanate extraction.15 The pulverized tissue was homogenized in 4 mmol/L guanidine isothiocyanate and 0.5% sarkosyl in 25 mmol/L sodium citrate with a Polytron homogenizer. This was followed by two phenol/chloroform extractions and ethanol precipitation. Equal amounts of total cellular RNA (15 μg), as determined by absorption of extracts at 260 nm, were loaded onto a 1.2% agarose gel. Formaldehyde-phosphate gel electrophoresis and RNA transfer to nylon membranes were performed as previously described.16 After transfer, RNA was bound and cross-linked using ultraviolet radiation. c-myc mRNA was detected using a ribonucleotide probe from a linearized human c-myc fragment (D414-433),17 osteopontin was detected using a random primed rat cDNA probe (2B7),18 and tropoelastin was detected using a random primed rat probe.19 Filters were prehybridized in 6× SSC, 50% Denhardt’s solution, 5 μg/mL denatured salmon sperm DNA, 0.2% SDS, and 50% formamide. Prehybridization was performed at 65°C (c-myc) or 42°C (osteopontin and tropoelastin) for 1 hour, and hybridization was performed for 16 hours at the above temperatures. The filters were then washed with 6× SSC, 0.1% SDS, and 2× SSC, 0.1% SDS, at 58°C and exposed using a Cronex intensifying screen and Kodak-X-omatic AR film at −70°C overnight. For repeat hybridizations, the filters were stripped by boiling in 0.1% SDS for 1 hour and exposing them overnight to verify loss of signal. Densitometric analysis of the Northern blots was used to assess specific mRNA signals against a signal from the 28S subunit.
Comparisons between treatment groups used ANOVA of means for multiple comparisons following a Bonferroni correction. Paired analysis between two groups, eg, between antisense- and sense-treated groups, was performed using Student’s t test when ANOVA indicated significance for the multiple comparison.
Oligonucleotide or pluronic gel alone was applied to the adventitia of the distal segment of the rat common carotid artery. We assayed replication of smooth muscle and endothelial cells, intimal and medial areas, and cell numbers and expression of the c-myc, osteopontin, and tropoelastin genes. An effect mediated by the antisense oligonucleotide was concluded only if there was a significant difference in the variable measured in the antisense-treated group compared with both gel alone and sense treatment and only if it occurred in the treated (distal) segment of the vessel and not in the untreated segment.
Effect of Oligonucleotides on Cell Replication
As described in “Methods,” all data are based on the administration of oligonucleotides at the time of injury. Thus, assays performed at later time points reflect persistent effects. Cell replication in the arterial media was assessed 2, 4, and 14 days after injury and in the arterial intima 14 days after injury by BrdU labeling of cells for the 24 hours before killing. The percentage of cells positive for BrdU in the total cell population was then estimated. At 2 days, there was a mean reduction of 25.1% (±3.1% SEM in medial cell replication in the antisense-treated group compared with both the sense and pluronic gel treatment groups (P<.05) (n=6 for each group) (Table 1⇓). By 4 days, however, no difference could be found. At 14 days there was no difference in medial or intimal replication rates between any of the groups.
Effect of Oligonucleotides on Cell Number in the Vessel Wall and Neointima Formation
Despite the reduction in medial cell replication in vessels treated with antisense oligonucleotides, there was no significant difference in the number of medial cells between the groups at day 2 or 4 (Table 2⇓). At day 14, however, antisense-treated vessels showed a significant increase in the number of medial cells compared with both control groups, and the same vessels showed a significant decrease in the number of intimal cells (P<.05 in both instances). Of considerable interest is that the total number of cells (medial plus intimal cells) of the vessel wall was not different in the antisense-treated vessels at 14 days.
At day 14, the early treatment with antisense oligonucleotides also reduced neointimal area by 24% to 26% and the intima/media ratio by 28% to 31% compared with either the sense- or gel-treated vessels (P<.05) (Figs 1⇓ and 2⇓). The suppression of intimal area was also associated with an increase in lumen area in the antisense-treated group of approximately 23% (Fig 1⇓, C). The effect of antisense oligonucleotides on intimal area, intima/media ratio, and lumen area was significant only in the distal portion of the vessel, the region to which the oligonucleotide had been applied, and not in the proximal region, which had been injured but had not received oligonucleotide treatment (Figs 1⇓ and 2⇓). Similarly, antisense treatment had no significant effect on the number of intimal or medial cells at 14 days in the proximal segment of the vessel (data not shown).
Effect of Oligonucleotides on Endothelial Cell Proliferation
The presence of an intact endothelium is associated with a lower rate of proliferation in the intima than in areas in which the intima remains exposed.12 Therefore, we considered the possibility that suppression of endothelial proliferation by antisense oligonucleotides could reduce the effect of this agent on suppression of neointima formation. Endothelial cell proliferation was assessed at 2 days after injury by creating an interface between areas of the vessel containing an intact endothelium and one in which the endothelium had been removed. This was achieved by gentle rotation of the balloon catheter in the middle portion of the vessel without overdistension of the artery. Just before killing, the animals received an intravenous injection of Evan’s blue stain to delineate this interface, in addition to 24 hours of BrdU labeling. BrdU-positive cells were then counted in an area of intact endothelium distal to this interface.14 Antisense treatment did not affect the percentage of endothelial cells replicating in this assay compared with either the sense oligonucleotide or gel alone treatments (mean±SEM: gel, 51.6±5.3%; sense, 62.6±4.5%; and antisense, 55.3±4/1%; n=3; data not shown). In addition, no effect of sense oligonucleotides on endothelial cell replication could be demonstrated. We also examined arterial sections from each end of the distal (treated) arterial segment and a region in the center of this segment for the presence of endothelial cells at 14 days. However, because the balloon injury was performed over the whole common carotid artery and the treated section comprised only a segment of this artery, endothelial cells were not seen in any arterial profile in any treatment group at this time point (not shown).
Localization of Oligonucleotides in the Vessel Wall
To assess whether the lack of any effect of antisense oligonucleotides on endothelial cell proliferation was due to failure of adventitial application of the oligonucleotide to penetrate to the luminal surface, oligonucleotides were biotinylated, applied to the adventitial surface of an injured vessel, then detected histochemically at 2 days. The oligonucleotide was readily detectable at 2 days and was evident throughout the vessel wall up to the luminal surface (Fig 3⇓).
Effect of Oligonucleotides on c-myc mRNA Expression
We previously demonstrated that application of antisense c-myc oligonucleotides to the adventitial surface of the artery after injury suppresses induction of c-myc mRNA at 2 hours.2 Other studies have demonstrated that pluronic gel delivery of the same antisense sequence suppresses target mRNA expression at 24 hours.20 Although neither of these findings proves that the oligonucleotide is inhibiting the target mRNA by an antisense mechanism, the expression of target mRNA does provide some indication of how long the oligonucleotide remains active in the vessel wall. Because we could not detect a biological effect of antisense oligonucleotides at 4 days and onward in this study, in terms of measurements of cell proliferation, we examined the effect of the oligonucleotides on c-myc expression at 4 days. Because only part of the vessel was treated with oligonucleotides, vessel segments were divided into proximal (untreated) segments and distal (treated) segments, and the distal segments were analyzed for mRNA expression by Northern blotting. By 4 days, we could find no significant difference in expression of c-myc between sense-, antisense-, or gel-treated vessels (Fig 4⇓). Thus, it is likely that the effectiveness of the oligonucleotide is limited to the period before 4 days, ie, before migration of smooth muscle cells.
Effect of Antisense Oligonucleotides on Expression of Osteopontin and Tropoelastin
Tropoelastin and osteopontin show minimal expression in the uninjured vessel, but expression appears between 1 and 4 days after balloon injury as medial smooth muscle cells migrate and form the neointima.19 21 Tropoelastin and osteopontin mRNAs are also preferentially expressed by cultured fetal or neointimal smooth muscle cells compared with adult cells, suggesting that phenotypic modulation may be required for neointima formation.18 We therefore used expression of osteopontin and tropoelastin as markers of a neonatal phenotype to determine whether oligonucleotides suppress a change in phenotype of medial smooth muscle cells. Fig 4⇑ demonstrates a Northern blot of mRNA at 4 days after injury for osteopontin and tropoelastin, demonstrating that there was no significant difference in either mRNA expression in vessels treated with antisense or sense oligonucleotides compared with gel alone in the distal segments of the artery.
Replication of medial smooth muscle cells after balloon catheter injury to the rat carotid artery can be blocked by antibodies or inhibitors directed against specific agonists or receptors (reviewed in Schwartz et al22 ). Effective inhibitors of medial smooth muscle cell replication include α1-adrenergic receptor blockade,23 AT1 receptor inhibitors,24 and antibodies to bFGF.10 However, continuous application of these agents may be necessary to reduce neointima formation. More recently, intima formation has been shown to be inhibited by a series of antisense oligonucleotides directed at genes associated with cell replication delivered as a single dose.3 20 These agents are of potential value because of their theoretical ability to target specific gene sequences and also because their effects may be localized to the site of delivery, reducing systemic side effects.
The data found in this study suggest that the long-term effects of oligonucleotides applied at the time of injury are mediated by a previously unexplored action. Application of a phosphorothioated oligonucleotide directed against c-myc immediately after injury resulted in suppression of medial replication at 2 days but not at 4 or 14 days. This time course is to be expected because the half-lives of c-myc mRNA and protein are short, of the order of 20 to 30 minutes.25 26 Thus, continued suppression of expression of this gene requires the constant presence of the antisense oligonucleotide. Our earlier study2 showed that the oligonucleotide inhibited target mRNA expression early, at 2 hours. However, a recent study indicated that pluronic delivery of the same oligonucleotide suppresses target mRNA and protein levels only at time points before 4 days.20 Thus, any effect of the antisense oligonucleotide on the number of intimal or medial cells at times after 4 days must be due to an action on medial cell replication or migration before 4 days.
The antisense-treated animals did show a reduction in the size of the neointima at 14 days (assessed by a decrease in intimal area and intima/media ratio and an increase in lumen area) and a reduction in the number of intimal cells. This effect of the antisense oligonucleotide occurred despite no apparent effect on intimal or medial cell proliferation after 4 days. Surprisingly, there was also an increase in the number of medial cells at 14 days in the antisense group. The total number of cells in the vessel (intima plus media) at 14 days was not different after antisense treatment compared with either gel alone or sense treatment, but cells were distributed more in the media and less in the intima. Although it is possible that an increase in the number of medial cells at 14 days after antisense treatment may result from a rebound increase in cell replication in the media following initial suppression at 2 days, our data do not show such an increase in medial replication at either 4 or 14 days. Thus, we suggest that the increase in the number of medial cells is due to an inhibition in cell migration after the antisense oligonucleotide. Such an effect on cell migration is consistent with in vitro data indicating that the antisense c-myc oligonucleotide suppression of smooth muscle cell migration is a far more potent action of the oligonucleotide than suppression of proliferation.27
The possibility that antisense agents inhibit cell migration directly may not be the full explanation of our data, however. Cell migration becomes apparent in the rat model from 4 days onward, a time at which we have shown that the oligonucleotide is no longer effective at reducing cell proliferation or suppressing target mRNA. Although it is theoretically possible that nonantisense effects of the oligonucleotide continue to suppress migration after 4 days, it is also possible that the oligonucleotide is acting through suppression of medial replication in a subpopulation of smooth muscle cells that then go on to migrate. Such an action is consistent with the data, showing that antisense oligonucleotides suppress medial replication at 2 days but not at 4 to 14 days, and would explain how a modest effect on medial cell replication can be translated into a significant reduction in neointima formation.
The concept of a subpopulation of medial smooth muscle cells that migrate in response to injury comes from studies indicating that cells that migrate and proliferate in the intima may possess an mRNA phenotype paralleling that of a “neonatal” phenotype, characterized by expression of genes such as osteopontin and tropoelastin.18 19 Under this hypothesis, suppression of medial replication in the subpopulation can inhibit migration without a direct effect on molecules that mediate migration itself. Equally, suppression of medial cell replication without suppression of the replication of this subpopulation would not be effective in inhibiting neointima formation because the same number of cells will migrate and then proliferate in the intima. The presence of a small, phenotypically modulated subpopulation in the media may explain why there is no difference in the expression of the “neonatal” phenotype markers at 4 days on Northern blots of vessels in the antisense-treated group; at this time the bulk of the cells in the vessel do not possess this phenotype. Rather than inhibiting medial proliferation alone, an effect of antisense oligonucleotides limiting the population of migrating cells would also reinforce results of other studies indicating that profound suppression of medial replication may not inhibit neointimal formation10 and that changes in neointima formation can be achieved by agents that inhibit migration alone.28
Other explanations for our data must also be considered. For example, antisense c-myc oligonucleotides have been shown to suppress synthesis of extracellular matrix components,6 and suppression of matrix synthesis can inhibit neointima formation without any effect on cell proliferation.29 However, matrix is synthesized by smooth muscle cells. Thus, although there may be changes in the composition of the matrix following antisense oligonucleotide treatment,6 any effects of the antisense oligonucleotide on total extracellular matrix content in the intima may be secondary to changes in cell number, such as those seen in the antisense-treated animals.
Although it is possible that oligonucleotide treatment had effects on endothelial cell migration in this model, it is important to point out that endothelial cell proliferation did not appear to be suppressed by any oligonucleotide treatment at 2 days after injury, a time at which medial proliferation was suppressed in the antisense group. Oligonucleotides were detectable close to the luminal surface of the vessel at 2 days after injury, so it is likely that endothelial cells were exposed to the oligonucleotide. Although the detection of biotin in this instance does not prove that the biotin is still attached to a full-length oligonucleotide, suppression of medial proliferation at this time implies that at least some of the oligonucleotide detectable by immunocytochemistry is functional. Furthermore, in other studies, oligonucleotides delivered by pluronic gel have been shown to be intact up to 72 hours after delivery.30 The huge excess of oligonucleotide to mRNA used in this and other studies also makes it unlikely that the resistance of endothelial cells to oligonucleotide action is due to a concentration gradient across the wall of the vessel. Thus, it is possible that endothelial cells are intrinsically more resistant to the effects of oligonucleotides than smooth muscle cells. Although such a property has not been well documented, it would be potentially beneficial in any therapy using oligonucleotides aimed at treating restenosis due to angioplasty.
Finally, some words of caution are necessary in interpreting the results of this study, which was not designed to investigate specific, antisense-mediated effects of the oligonucleotide. We used an antisense sequence containing a 4G motif in an attempt to explain the apparent potent effect of these oligonucleotides in inhibiting neointima formation.1 2 8 Many, if not all, of the effects of the oligonucleotide may thus be mediated by non-sequence-specific effects. In this respect, the choice of target gene or the demonstration of any effect of the oligonucleotide on target gene expression may be irrelevant to attempts at understanding the effects of oligonucleotides on cellular kinetics in the vessel wall. For example, demonstration of suppression of target mRNA or protein at any time after administration does not indicate that this is the mechanism of action of the oligonucleotide. In contrast, we attempted here to explain the cellular effects of a single administration of the oligonucleotide in vivo after arterial injury. Our studies cannot necessarily be extrapolated to the mechanism of action of other oligonucleotides to cell proliferation genes that act only specifically or are administered continuously. However, a recent consensus suggests that it is virtually impossible to prove specificity of action of oligonucleotides and that a wholly specific action may not exist at all.31 Furthermore, nonspecific mechanisms of action do not necessarily diminish the potential usefulness of oligonucleotides to inhibit neointima formation clinically.8
In conclusion, we demonstrated that a phosphorothioated antisense oligonucleotide to c-myc suppresses formation of a neointima after injury in the rat. Our findings suggest that a major action of the oligonucleotide may be suppression of migration of smooth muscle cells rather than suppression of medial replication alone.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
M.R.B. is supported by a British Heart Foundation Clinical Scientist Fellowship, and D.D., by a fellowship from the Medical Research Council of Canada. This research was also supported by National Institutes of Health grants HL-18641 (to S.M.S.) and HL-03174 (to M.A.R.).
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