Cyclooxygenase-2 Is Required for Tumor Necrosis Factor-α– and Angiotensin II–Mediated Proliferation of Vascular Smooth Muscle Cells
Abstract—Tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) induced a transient increase in vascular smooth muscle cell (VSMC) cyclooxygenase-2 (COX-2) mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 mRNA accumulation were similar in VSMCs challenged with either TNF-α or Ang II; mRNA accumulation peaked at 2 hours and decreased to control levels by ≈6 hours. Accumulation of COX-2 mRNA was associated with a time-dependent increase of COX-2 protein expression that displayed similar kinetics in response to either TNF-α or Ang II. Both the increase in COX-2 mRNA accumulation and protein expression in response to either TNF-α or Ang II were inhibited by the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD098059. In addition, the AT1-selective receptor antagonist losartan attenuated the Ang II–mediated increase in COX-2 mRNA accumulation; the AT2-selective antagonist PD123319 had no effect. Prostacyclin I2 synthesis was tightly coupled to expression of COX-2, whereas prostaglandin E2 and thromboxane A2 (TXA2) synthesis may be associated with differential usage of COX-1 and COX-2. The COX-2–selective inhibitors NS-398 and nimesulide and the TXA2 receptor antagonist BMS 180,291 inhibited TNF-α– and Ang II–mediated increases in DNA content and cell number by ≈95%. These findings suggest that a prostanoid derived from COX-2, possibly TXA2, may contribute to VSMC hyperplasia in vessel injury or pathophysiological conditions associated with elevated levels of either TNF-α or Ang II.
Angiotensin II (Ang II)–dependent forms of hypertension are associated with the growth of vascular smooth muscle cells (VSMCs) and infiltration of mononuclear cells into blood vessels and the kidney.1 Activated neutrophils and monocytes produce several cytokines, including tumor necrosis factor-α (TNF-α), which participates in inflammatory, immune, and pathophysiological events.2 Previous studies indicated that Ang II increased TNF-α production by monocytes.3 These data suggest that conditions in which Ang II contributes to ongoing cardiovascular disease also may result in enhanced TNF-α production via activation of infiltrating mononuclear cells by the peptide. Alternatively, TNF-α, which stimulates migration of VSMCs and increases expression of inducible NO synthase and adhesion molecules, may be produced by VSMCs.4 5
Recent studies from our laboratory have shown that Ang II can stimulate TNF-α production in renal epithelial cells isolated from the medullary thick ascending limb (mTAL).6 This interaction contributed to a cyclooxygenase-2 (COX-2)–dependent mechanism by which the effects of Ang II on ion transport in mTAL cells were modulated by TNF-α production.7 However, TNF-α–Ang II interactions are not limited to the kidney, as TNF-α participates in a counterregulatory mechanism that opposes the pressor effects of Ang II.8 Thus, the demonstration that some effects of Ang II are associated with increases in TNF-α production and, hence, COX-2–mediated prostanoid synthesis, prompted us to determine whether either Ang II or TNF-α regulated COX-2 expression in VSMCs.
There are 2 isoforms of prostaglandin (PG) H synthase, PGH synthase 1 and PGH synthase 2, which are commonly referred to as COX-1 and COX-2, respectively. The amino acid sequences for COX-1 and COX-2 are similar (≈75% homology), and the residues that are important for the catalytic activities of these enzymes are highly conserved. Thus, these enzymes catalyze the same reactions; ie, arachidonic acid is converted to PGG2 via the cyclooxygenase reaction, followed by a peroxidase reaction in which the 15-hydroperoxyl group of PGG2 is reduced to the 15-hydroxyl group of PGH2. The latter is metabolized by specific isomerases to prostanoids in a cell-specific manner. Regulation of the COX isoforms is quite different, as COX-1 is constitutively expressed in many cell types, whereas COX-2 expression is increased after exposure to cytokines and mitogens. These 2 isoforms also may subserve different functions, as the role of COX-2, for instance, in the dysregulated proliferation of several tumor cell types has recently been examined.9 10 The present study demonstrates that both TNF-α and Ang II increase COX-2 mRNA accumulation and protein expression in VSMCs. Prostacyclin I2 (PGI2) synthesis was tightly coupled to COX-2 expression, whereas formation of PGE2 and thromboxane (TX) A2 occurred by either COX-1 or COX-2, depending on the stimulus. Moreover, the proliferative response of VSMCs to both TNF-α and Ang II was COX-2 dependent, suggesting that this isoform may contribute to the pathophysiological responses of VSMCs to these molecules.
Materials and Methods
Male Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA) weighing 100 to 115 g were maintained on standard rat chow (Ralston-Purina Co) and given tap water ad libitum. Care and use of animals were according to guidelines of the National Institutes of Health.
Isolation and Culture of VSMCs
Thoracic aortae were removed, minced in digestion medium (DMEM-F12 containing 0.58 mg/mL collagenase IA, 0.33 mg/mL soybean trypsin inhibitor, and 1.67 mg/mL BSA), and incubated for 3 hours in digestion medium containing 0.17 mg/mL elastase III. Cells (>95% purity) were cultured in DMEM-F12 containing 10% FBS, streptomycin-penicillin (100 U/mL), and fungizone (1 μg/mL). After cells became confluent, they were passaged using trypsin-EDTA. Passages 3 to 8 were used for the experiments performed in this study.
Isolation of Total RNA/Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was isolated by lysing cells in Trizol reagent (Life Technologies) and by precipitation with isopropyl alcohol, as previously described.7 A 5-μg aliquot of total RNA was used for cDNA synthesis with the Superscript preamplification system (Life Technologies). An aliquot of cDNA was then amplified (30 cycles) using Taq DNA polymerase in the presence of sense and antisense primers for murine COX-1, COX-2, or GAPDH, as previously described.7
Western Blot Analysis of COX Proteins
Cells were lysed with 10 mmol/L Tris-HCl (pH 7.5) and 1% SDS. Protein concentrations of the supernatants were measured using a detergent-compatible Bio-Rad protein assay kit, and 50 μg of cell lysate was mixed with 5× SDS-PAGE sample buffer (500 mmol/L DTT, 0.2% bromphenol blue, and 50% glycerol) and boiled for 3 minutes. Proteins were separated on a 10% SDS-PAGE gel, transferred to nitrocellulose or polyvinylidene difluoride membranes, and immunoblotted with a 1:1000 dilution of a rabbit anti-mouse COX-2 polyclonal antibody (Cayman). Membranes were washed with Tris-buffered saline with Tween 20 and incubated with horseradish peroxidase (HRP)–conjugated antisera, and COX-2 protein was detected by enhanced chemiluminescence. Alternatively, incubation with alkaline phosphatase–conjugated antisera and subsequent detection with enhanced chemifluorescence and phosphor imaging on a Molecular Dynamics Storm 860 phosphor imager was used for the analysis of COX-2 protein expression.
Diluted medium (50 μL) and 50 μL of HRP-conjugated PGE2 or 6-keto-PGF1α were added to wells of a 96-well plate previously coated with either anti-PGE2 or anti–6-keto-PGF1α antibody, respectively. Substrate for HRP was added and the reaction stopped by addition of 1N HCl. Quantification was achieved by measuring absorbance at 450 nm. Analysis of TXB2 was performed as previously described.11
Cellular DNA Content
DNA content was assessed using the CyQUANT assay kit (Molecular Probes). Briefly, cells were challenged and then lysed with CyQUANT lysis solution. Lysates were then transferred to black 96-well microtiter plates and read on a CSA fluorescence plate reader at 485 nm excitation and 535 nm detection.
Analysis of Cell Number
Cells were seeded at 2×104 cells/well in 24-well plates, quiesced for 24 hours, and then challenged. Cells were harvested, and a 10-μL aliquot was mixed with trypan blue and counted twice on a hemocytometer. Alternatively, cell number was determined using the CellTiter proliferation assay (Promega).
The responses of control and treated VSMCs were compared by a 1-way ANOVA and Neuman-Kuels post-test; a probability value of ≤0.05 was considered statistically significant.
Ang II and TNF-α Increase Accumulation of COX-2 mRNA in VSMCs
Confluent, quiescent VSMCs were incubated in the absence or presence of Ang II (10–7 mol/L) for varying times from 30 minutes to 6 hours. Cells were lysed with Trizol, and total RNA was extracted for analysis by RT-PCR, which was performed using primers specific for COX-2, COX-1, and GAPDH. Ang II increased COX-2 mRNA accumulation when compared with untreated cells, whereas COX-1 and GAPDH mRNA accumulation did not change after challenge with Ang II (Figure 1A⇓). COX-2 mRNA accumulation was detectable 30 minutes after stimulation with Ang II, was substantially increased at 2 hours, and was diminished by 6 hours after challenge with the peptide. TNF-α (1 nmol/L) also increased COX-2 mRNA accumulation and exhibited kinetics similar to those observed for cells challenged with Ang II; ie, mRNA accumulation was detectable at 30 minutes, was maximal at 2 hours, and then decreased from peak levels by 6 hours (Figure 1A⇓). No effects on either COX-1 or GAPDH mRNA accumulation were observed when cells were challenged with TNF-α.
Ang II Stimulates COX-2 mRNA Accumulation via AT1 Receptors
The AT1 and AT2 receptor antagonists, losartan and PD123319, respectively, were used to determine the role of Ang II receptor subtypes in COX-2 mRNA accumulation. Preincubation for 15 minutes with losartan (1 μmol/L) attenuated the Ang II–mediated increase in COX-2 mRNA accumulation without affecting the levels of either COX-1 or GAPDH mRNA (Figure 1B⇑). In contrast, PD123319 (1 μmol/L) had no effect on the Ang II–mediated increases in COX-2 mRNA accumulation. These data suggest that Ang II increases COX-2 mRNA accumulation in VSMCs via AT1 receptors.
Ang II and TNF-α Increase COX-2 Protein Expression
The expression of COX-2 protein was investigated by incubating confluent, quiescent VSMCs in the absence or presence of either Ang II or TNF-α; serum was included as a positive control. Western blot analysis showed that exposure of the cells to either Ang II or TNF-α for 4 or 8 hours substantially increased COX-2 protein expression (Figure 2⇓). COX-2 protein expression induced by either Ang II or TNF-α was decreased subsequent to 8 hours of exposure but was still elevated relative to control cells at each time point tested. Similar results were obtained when cells were challenged with serum, which previously was shown to increase COX-2 expression in VSMCs.12
Relative Influence of COX-2 on Ang II– and TNF-α–Mediated Prostanoid Production
The effects of Ang II and TNF-α on the prostanoid profile of VSMCs were determined by assessing levels of PGI2, TXA2, and PGE2, 3 major arachidonic acid metabolites produced by VSMCs. Levels of the hydrolysis product of PGI2, 6-keto-PGF1α, were measured in confluent, quiescent cells incubated in the absence or presence of either Ang II or TNF-α. Levels of 6-keto-PGF1α were not different from control levels after a 4-hour exposure to either Ang II or TNF-α (data not shown). However, 6-keto-PGF1α levels increased ≈8-fold and 3-fold when VSMCs were challenged with either Ang II or TNF-α, respectively, for 8 hours (Figures 3A⇓ and 4A⇓). Pretreatment with the COX-2–selective inhibitor, NS-398 (0.1 μmol/L), significantly attenuated basal levels of 6-keto-PGF1α as well as those induced by either Ang II or TNF-α (Figures 3A⇓ and 4A⇓). In contrast, despite significant increases in COX-2 protein expression in response to both Ang II and TNF-α, only Ang II significantly increased PGE2 levels in a COX-2–dependent manner (Figures 3B⇓ and 4B⇓). Moreover, NS-398 did not significantly alter basal levels of PGE2, suggesting a role for COX-1 in basal synthesis of this prostanoid. Ang II– and TNF-α–mediated increases in TXB2 levels were reduced to control levels by NS-398 and another COX-2–selective inhibitor, nimesulide, suggesting a role for COX-2 in TXA2 synthesis in response to Ang II and TNF-α (Figures 3C⇓ and 4C⇓). Collectively, these data suggest that there may be a preferential coupling of COX-2 to PGI2 synthase in VSMCs, as both control and stimulated levels of 6-keto-PGF1α were markedly reduced after addition of NS-398. In addition, the inhibitory effect of NS-398 on Ang II–mediated PGE2 levels, but not on basal levels, suggests that this prostanoid may be formed via the combined actions of COX-1 and COX-2. Finally, although Ang II– and TNF-α–mediated TXA2 syntheses were COX-2 dependent, basal synthesis of this prostanoid may be coupled to COX-1, as neither NS-398 nor nimesulide affected basal TXA2 synthesis.
Ang II and TNF-α Increase COX-2 mRNA Accumulation and Protein Expression Through Activation of Mitogen-Activated Protein (MAP) Kinase
Activation of MAP kinase has been implicated as a signaling pathway for COX-2 gene transcription and protein expression and the proliferative response of VSMCs to Ang II.13 14 Therefore, the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD098059 (10–4 mol/L) was used to assess the contribution of signaling via MAP kinase to COX-2 mRNA accumulation and COX-2 protein expression. PD098059 attenuated Ang II– and TNF-α–mediated increases in COX-2 mRNA accumulation and COX-2 protein expression, suggesting a role for MAP kinase activation in these events (Figure 5⇓).
Ang II and TNF-α Increase Proliferation of VSMCs via COX-2/Role of TXA2
Previous studies have shown that Ang II causes hypertrophy and hyperplasia of VSMCs. The contribution of COX-2–derived prostanoids on VSMC proliferation was evaluated by measuring cellular DNA content and changes in cell number. An increase in DNA content was observed after exposure of the cells to either Ang II or TNF-α for 48 hours (Figure 6⇓). Coincubation with the COX-2 inhibitor NS-398 (0.1 μmol/L) or nimesulide (1 μmol/L) abolished the Ang II– and TNF-α–mediated increase in DNA content but did not affect DNA content when added to the cells in the absence of either stimulus (Figure 6⇓). Cell number was determined to distinguish between an increase in DNA content caused by polyploid hypertrophy of the cells rather than cell division (hyperplasia). Indeed, in every instance, the increase in DNA content was directly proportional to the increase in cell number in response to challenge of the cells with either Ang II or TNF-α (Figures 6⇓ and 7⇓). The increase in cell number in response to either Ang II or TNF-α was inhibited by nimesulide (Figures 7A⇓ and 7B⇓) and NS-398 (Figures 7C⇓ and 7D⇓). Neither COX-2 inhibitor affected cell number when added to the cells in the absence of either Ang II or TNF-α. These data suggest that the increase in DNA content reflects an increase in the cellular proliferation after exposure to either Ang II or TNF-α and that COX-2–derived prostanoids may be involved in the proliferative response to these stimuli.
On the basis of previous work describing the effects of prostanoids on VSMC proliferation, TXA2 was considered a likely mediator of the proliferative response to Ang II and TNF-α. Thus, VSMCs were challenged with either Ang II or TNF-α in the absence or presence of the TXA2 receptor antagonist BMS 180,291. In each instance, BMS 180,291 reduced the increases in DNA and/or cell number induced by both Ang II and TNF-α to levels that were not significantly different from control values (Figures 8A⇓ through 8C). These data suggest that a COX-2–dependent prostanoid, possibly TXA2, contributes to the proliferative response to Ang II and TNF-α.
We demonstrated that Ang II and TNF-α increased COX-2 mRNA accumulation and protein expression in VSMCs. The effects of Ang II were dependent on activation of AT1 receptors and stimulation of the MAP kinase pathway. Moreover, the increases in mRNA accumulation and protein expression were transient, with maximal effects observed after exposure of cells to Ang II for 2 and 4 hours, respectively. TNF-α–mediated effects on VSMCs were similar to those observed for Ang II with respect to the kinetics of COX-2 mRNA accumulation, COX-2 protein expression, and activation of the MAP kinase pathway. Both Ang II and TNF-α preferentially elevated PGI2 synthesis in a COX-2–dependent manner, whereas production of PGE2 and TXA2 occurred via both COX-1 and COX-2 pathways, depending on the stimulus and activation status of the cells. Ang II and TNF-α increased VSMC DNA content and cell number in a COX-2–dependent manner. These responses may have been mediated by COX-2–derived TXA2, despite the predominance of PGI2 synthesis by these cells in response to either Ang II or TNF-α.
Ang II induces the expression of several autocrine factors that affect VSMC growth, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β). Moreover, net VSMC hyperplasia and/or hypertrophy is the result of dynamic interactions between several growth-stimulating and growth-inhibiting autocrine factors.15 For instance, TGF-β has an antiproliferative action that modulates the mitogenic effects of bFGF.15 The actions of TXA2 and PGI2 on VSMC growth also are consistent with a paradigm that invokes the balance of pro- and antimitogenic molecules as part of a regulatory mechanism.16 A possible role of COX-2 in this process is suggested in the present study, which demonstrated that both Ang II and TNF-α induced COX-2 mRNA and protein expression. Moreover, the COX-2 inhibitors NS-398 and nimesulide, at doses that do not affect COX-1 activity,17 18 inhibited the proliferative response of VSMCs to these stimuli. The exact sequence of events in which COX-2 is involved is not clear. However, COX-2 expression may contribute to either a proximal step, given that prostanoids such as TXA2 can increase the production of autocrine growth factors by VSMCs, or a distal step, as some autocrine growth factors increase COX-2 expression.13 15
The relative contribution of COX-2 to Ang II– and TNF-α–mediated prostanoid synthesis in VSMCs appears to differ for PGI2, TXA2, and PGE2. PGI2 synthesis appeared to be tightly coupled to COX-2 expression, as both basal production and either Ang II– or TNF-α–mediated production of this prostanoid were inhibited by NS-398. In contrast, although Ang II–stimulated TXA2 and PGE2 levels were inhibited by NS-398, selective COX-2 inhibition did not significantly reduce basal levels of these prostanoids. These data suggest that activation of VSMCs by either Ang II or TNF-α may influence the utilization of COX isoforms with respect to formation of TXA2 and PGE2. Several recent studies have suggested the possibility of a close coupling between COX-2 and PGI2 synthase in both healthy humans and in those with cardiovascular disease associated with elevated expression of COX-2.19 20 For instance, in advanced atherosclerosis, PGI2 synthesis associated with activation of the COX-2 gene was significantly elevated in VSMCs of the plaque, in both the intima and the media.21 Moreover, in enterocytes, the COX-2–selective antagonist SC58125 was shown to inhibit basal and lipopolysaccharide-stimulated 6-keto-PGF1α levels but not PGE2, suggesting that PGI2 production occurred mainly by a COX-2–dependent mechanism, whereas PGE2 formation occurred via a COX-1-mediated mechanism.22
PGI2 has several vasoprotective effects, including vasodilation, anti-platelet aggregation, and inhibition of smooth muscle cell proliferation. Thus, the increase in PGI2 production in response to either Ang II or TNF-α, in the context of an increase in proliferation, may be indicative of a compensatory mechanism that limits the mitogenic activity of these molecules. Alternatively, PGI2 produced in response to either Ang II or TNF-α may have minor antiproliferative effects under these experimental conditions. For instance, previous studies indicated that the antiproliferative effects of PGI2 were most pronounced when cells were exposed to exogenous PGI2 after an initiating mitogenic event had occurred. For instance, in vivo transfer of the PGI2 synthase gene into balloon-injured rat carotid arteries substantially reduced 5-bromo-2′-deoxyuridine staining and neointimal formation.23 The PGI2 analog iloprost also was an effective inhibitor of proliferation induced by PDGF-BB when added 20 to 24 hours after stimulation of the cells. However, when added to cells together with growth factors, the inhibitory effect of iloprost was markedly reduced, suggesting the development of tolerance to this prostanoid.24 The latter data are consistent with studies showing that although PGI2 is antimitogenic for VSMCs, tolerance can develop toward PGI2 receptors in a number of experimental settings.21 24 Possible desensitization at the receptor level also has been observed when iloprost therapy was administered to humans for 1 to 4 weeks.21 In the present study, endogenous formation of COX-2–derived PGI2 occurred after exposure of the cells to either Ang II or TNF-α, a sequence that may have limited the antiproliferative effects of this prostanoid. However, the increase in COX-2–derived PGI2 synthesis in VSMCs may be important in conditions such as Ang II–dependent hypertension, or other conditions associated with the infiltration of mononuclear cells, given that damage of endothelial cells may preclude the production of PGI2 at the site of injury. Thus, release of TNF-α by infiltrating mononuclear cells, possibly via stimulation by Ang II, into vascular tissue could contribute to the net effects on VSMC growth, as this cytokine has been shown to participate in VSMC migration and proliferation.4 5 25 As PGI2 inhibits the chemotaxis of monocytes and inhibits TNF-α production,26 these protective properties may be part of a compensatory mechanism to limit the proliferative effects of cytokines such as TNF-α or vasoactive peptides such as Ang II.
Previous work demonstrated that in early-passage, quiescent VSMCs (conditions similar to those used in the present study), TXA2 acts as a hypertrophic factor rather than as a complete mitogen.27 28 These data are consistent with the observation in the present study that the TXA2 receptor antagonist, BMS 180,291, prevented the proliferative response to Ang II and TNF-α but did not affect proliferation of unstimulated cells. Indeed, TXA2 could contribute to the proliferative response of Ang II or TNF-α that may be mediated by growth factors such as bFGF and PDGF. For instance, TXA2 increases DNA synthesis 3- to 4-fold in bovine coronary artery smooth muscle cells and rat aortic smooth muscle cells by increasing the G1-to-S transition29 and potentiates the mitogenic effect of thrombin in VSMCs.27 30 TXA2 also may promote growth via an interaction with bFGF and via expression of several genes involved in growth such as c-fos and early growth response gene-1.31 As both Ang II– and TNF-α–mediated TXA2 synthesis are COX-2 dependent, TXA2 may contribute to an environment that favors proliferation; however, in the absence of a stimulus, such as Ang II or TNF-α, this prostanoid alone is not sufficient to induce proliferation.
PGE2 has little effect on VSMC proliferation.32 However, in some cell types PGE2 has been identified as the prostanoid responsible for a growth-promoting effect via overexpression of COX-2. For instance, in HCA-7 cells, PGE2 rescued cells from apoptotic cell death after selective inhibition of COX-2 with SC-58125.33 The proposed mechanism for this effect was via a PGE2-mediated increase in the expression of Bcl-2, which inhibits apoptosis. The present findings, however, suggest that PGE2 may not be the final mediator of the COX-2–dependent proliferative response, as only Ang II, but not TNF-α, significantly increased levels of this prostanoid in a COX-2–dependent manner. Notwithstanding, the possibility of a prostanoid-dependent effect on some aspect of apoptosis cannot be ruled out.
Mitogenic growth factors such as PDGF, epidermal growth factor, and bFGF increase COX-2 mRNA accumulation in VSMCs,34 and COX-2 protein expression increased after stimulation of VSMCs either with serum or after balloon de-endothelialization in vivo, conditions associated with VSMC growth.12 These data are consistent with those in the present study showing that COX-2 expression was required for VSMC proliferation in response to either Ang II or TNF-α. Previous work from our laboratory demonstrated that anti–TNF-α antisera exacerbated mean arterial pressure in an Ang II–dependent model of hypertension, suggesting that TNF-α participates in a counterregulatory mechanism that opposes the pressor effects of Ang II.8 Several sites of action, including the heart and vasculature, may be involved in this mechanism. Thus, local production of TNF-α at crucial sites that contribute to blood pressure homeostasis and Ang II– and TNF-α–mediated PGI2 production in VSMCs may be components of a mechanism that limits pathophysiological responses to elevated blood pressure.
Note Added in Proof
A recently published study (Ohnaka K, Numaguchi K, Yamakawa T, Inagami T. Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells. Hypertension. 2000;35[1 pt 1]:68–75) contains findings similar to those in the present study.
This work was supported by NIH Grant RO1HL56423 (to N.R.F.) and by American Heart Association Grant 9740001N (to N.R.F.). N.R.F. is an Established Investigator of the AHA.
Presented in part at the 1998 Experimental Biology meeting in San Francisco, Calif, April 18–22, 1998, and published in abstract form (FASEB J. 1998;12:A403).
- Received January 19, 2000.
- Accepted February 25, 2000.
- © 2000 American Heart Association, Inc.
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