The selective cyclooxygenase-2 inhibitor celecoxib modulates the formation of vasoconstrictor eicosanoids and activates PPARγ. Influence of albumin☆

Article Outline

• Abstract
• 1. Introduction
• 2. Material and methods
  • 2.1. Cell culture
  • 2.2. Cell incubations
  • 2.3. RT-PCR
  • 2.4. Analysis of eicosanoids and renin levels
  • 2.5. PPARγ trans-activation assay
  • 2.6. α-Smooth muscle actin protein expression
• 3. Results
• 4. Discussion
• Acknowledgements
• References
• Copyright

Background/Aims

Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions.

Methods

Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and α-smooth muscle actin (α-SMA) expression were determined. The effects of celecoxib on PPARγ were assessed in RMC co-transfected with PPARγ and luciferase reporter constructs.

Results

Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E₂, thromboxane (TX)B₂, 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF_2α. Celecoxib, in addition to reducing PGE₂, significantly decreased 8-epi-PGF_2α formation. In the presence of albumin, celecoxib also reduced TXB₂ and 12-HETE. Albumin per se inhibited PGE₂ as well as renin release. In trans-activation assays, celecoxib acted as a PPARγ agonist whereas albumin inhibited PPARγ as well as 15d-PGJ₂-induced PPARγ activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ₂ on α-SMA expression.

Conclusions

These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.

Keywords: Cyclooxygenase-2, PPARγ, Albumin, Prostaglandins, Renin, 8-Isoprostanes

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1. Introduction 

Selective cyclooxygenase (COX)-2 inhibitors effectively inhibit inflammation while sparing physiologic prostaglandin (PG) production [1], [2]. This new class of anti-inflammatory drugs are of interest in diseases such as decompensated cirrhosis, in which, renal function is critically dependent on PGs and the administration of non-steroidal anti-inflammatory drugs (NSAIDs) is frequently associated with acute renal failure [3]. In fact, recent studies have demonstrated that, unlike conventional NSAIDs, selective COX-2 inhibitors do not impair renal function in rats with experimental cirrhosis and ascites [4], [5].

Although, the mechanism of action of selective COX-2 inhibitors is based on its ability to inhibit the formation of COX-2-derived PGs, additional actions cannot be excluded. Recent reports have shown that these compounds regulate the binding of transcriptional factors such as NF-κB and AP-1 [6], inhibit the phosphorylation of protein kinase B/Akt [7], [8], block the activity and nuclear translocation of ERK2 [9] and induce apoptosis via a novel mitochondrial pathway [10]. Moreover, we have recently demonstrated that selective COX-2 inhibitors are able to modulate inflammatory genes such as cytokine-induced neutrophil chemoattractant-1, a member of the interleukin-8 family [11]. These findings are consistent with an emerging appreciation that the full spectrum of properties of these compounds has only been partially appreciated.

In the current study, we investigated the molecular mechanisms of the selective COX-2 inhibitor, celecoxib, in rat mesangial cells. Mesangial cells constitute a useful cellular model to explore the renal actions of celecoxib because these myofibroblasts are essential for maintaining the structural, glomerular and immunological functions of the kidneys and are the main producers of vasoactive and inflammatory mediators including eicosanoids [12]. In these cells, we firstly assessed the effects of celecoxib on the biosynthesis of eicosanoids, specially on those carrying vasoactive properties such as PGE₂, thromboxane (TX) B2, 8-epi-PGF_2α (8-isoprostane), 12-hydroxyeicosatetraenoic acid (12-HETE) and cysteinyl-leukotrienes (Cys-LTs) [13], [14]. Secondly, we explored whether, similar to other anti-inflammatory compounds, celecoxib is able to bind and activate PPARγ [15], [16], [17]. Finally, in order to further extend our investigation on celecoxib to renal complications other than the hepatorenal syndrome, we assessed the effects of celecoxib on α-smooth muscle actin (α-SMA) expression, a well established marker of mesenchymal cell de-differentiation and activation [12], [18].

In addition to studying the molecular mechanisms of celecoxib, we also explored the effects of albumin. Albumin is a major protein component with remarkable biological effects [19]. For instance, albumin is a generic stabilizing protein, a major contributor to osmotic pressure, is involved in the maintenance of blood pH and protects cells from injury [20], [21], [22]. Moreover, albumin has been shown to bind many fatty acids and NSAIDs and to be a major determinant of eicosanoid formation in platelets and leukocytes [23], [24], [25], [26], [27]. On the basis of these observations and considering that cirrhotic patients may require anti-inflammatory therapy while receiving albumin (which is clinically used as a plasma expander for preventing post-paracentesis circulatory dysfunction and for reducing the incidence of renal impairment [28], [29]), we questioned whether the molecular actions of celecoxib are influenced by this protein.

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2. Material and methods 

2.1. Cell culture 

Rat RMC 85/4 cells were grown in RPMI 1640 supplemented with 10% FCS, l-glutamine (2mM), penicillin (50U/ml) and streptomycin (50μg/ml) at 37°C in a 5% CO₂ atmosphere. This established rat mesangial cell line was provided by Dr. M. Martin (University of Hannover) who obtained it from a cell preparation from glomerular outgrowths of male Sprague–Dawley rats after 6 months of culture in the presence of 20% FCS [30], [31]. The mesangial phenotype of RMC 85/4 cultures was routinely characterized by optical visualization and immunocytochemistry showing positive staining for desmin and vimentin and negative staining for cytokeratin and factor VIII (Fig. 1(A)).

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• Fig. 1. 

  Immunocytochemical characterization and profile of eicosanoids formed by rat RMC 85/4 cells. (A) Expression of characteristic cellular markers of mesangial cells. Cells were cultured in RPMI 1640 supplemented with 10% FCS and characterized by immunocytochemistry using specific antibodies against desmin, vimentin, cytokeratin and factor VIII. (B) Detection of COX-1, COX-2 and 12/15-LO mRNA expression by RT-PCR. RNA was reverse-transcribed and COX-1, COX-2 and 12/15-LO amplified by PCR. Representative results obtained from two separate samples (samples 1 and 2) are shown in this ethidium bromide-stained gel; m, size marker (100-base pair DNA ladder). (C) Generation of arachidonic acid metabolites. Eicosanoid levels in supernatants from RMC 85/4 cells growing in serum-free RPMI 1640 medium for 24h were determined by EIA. Results are expressed as mean±SEM of three different experiments with duplicate determinations. 8-iso, 8-epi-PGF_2α.

2.2. Cell incubations 

RMC 85/4 cells were serum-deprived for 24h, exposed to vehicle (0.1% ethanol) or the selective COX-2 inhibitor, celecoxib, (3μM) for 20min and subsequently incubated in the presence or absence of albumin (Cohn V fraction, fatty acid-depleted, 10mg/ml) for 16h at 37°C.

2.3. RT-PCR 

RNA was isolated by using the trizol reagent. PCR was performed using oligonucleotides specific for rat COX-1, COX-2, 5-LO, 12/15-LO, PPARγ and GAPDH (GenBank Accession Numbers NM_017043, NM_017232, NM_012822, NM_031010, NM_013124 and NM_017008, respectively). COX-1, COX-2, PPARγ and GAPDH were amplified at 96°C (30s), 60°C (1min) and 72°C (1min) for 35 cycles. 5-LO and 12/15-LO were amplified for 35 cycles at 94°C (30s), 58°C (1min) and 72°C (30s) and 94°C (30s), 56°C (45s) and 72°C (1min), respectively. PCR products were analyzed by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining.

2.4. Analysis of eicosanoids and renin levels 

PGE₂, TXB₂, 12-HETE, Cys-LTs, LTB₄, lipoxin A₄ (LXA₄) and renin levels were determined in unextracted supernatants of RMC 85/4 cells by specific enzymeimmunoassays (EIAs) (Amersham, Buckinghamshire, UK; DRG Instruments, Marburg, Germany; Cayman, Ann Arbor, MI; Neogen, Lexington, KY and DiaSorin, Stillwater, MN, respectively). 8-epi-PGF_2α levels were determined by EIA after extraction of supernatants in 8-isoprostane affinity columns (Cayman, Ann Arbor, MI).

2.5. PPARγ trans-activation assay 

A fusion protein containing the yeast GAL4 DNA-binding domain linked to the ligand-binding domain of PPARγ (PPARγ-GAL4 plasmid) and a luciferase reporter construct containing four copies of a GAL4 upstream activating sequence (UAS_G) and a thymidine kinase (tk) promoter (MH100-tk-luc plasmid) were kindly provided by Dr R. Evans (Salk Institute). This chimeric PPARγ activates transcription through a heterologous response element and allows PPARγ activity to be assayed independently of endogenous receptors [32]. Transient transfections in mesangial cells were conducted by using the Effectene^® transfection reagent (Qiagen, Hilden, Germany) in a ratio 1:10 to DNA. Briefly, a total of 5.0×10⁴ RMC 85/4 were seeded in each well of 12-well plates and when cells reached 70% confluence were incubated in RPMI 1640 containing 0.3μg of the luciferase reporter construct MH100-tk-luc, 0.1μg of PPARγ-GAL4 and 0.025μg of β-galactosidase expression vector pCMV (an internal control plasmid containing a cytomegalovirus promoter). After 42h of incubation, cells were washed twice with DPBS and treated with vehicle (0.5% ethanol), 15d-PGJ₂ (1μM), celecoxib (3 and 10μM) and albumin (1 and 10mg/ml) for another 18h in serum-free RPMI 1640 medium. Cells were then harvested in luciferase lysis buffer and light units from firefly luciferase and β-galactosidase activities measured in a Lumat LB 9507 luminometer (Berthold, Bad Wildbad, Germany). Relative luciferase activity was obtained by normalizing the luciferase activity against the internal control β-galactosidase activity. Results were presented as ‘fold induction’ relative to the vehicle control values.

2.6. α-Smooth muscle actin protein expression 

RMC 85/4 cells growing in serum-free medium were incubated with vehicle (0.5% ethanol), 15d-PGJ₂ (1 and 10μM), celecoxib (3 and 10μM) and albumin (10mg/ml) for 18h at 37°C. At the end of the incubation, total protein was extracted and equal amounts of protein (10μg) were resuspended in SDS-containing Laemmli sample buffer, heated at 100°C for 5min, electrophoresed on 10% SDS–polyacrylamide gels and transferred overnight to PVDF membranes. Gels were stained with Coomassie blue to visualize loading differences and membranes were submitted to Ponceau S staining to monitor the efficiency of the transfer. The blots were subsequently blocked for 1h in tris-buffered saline (20mM tris/HCl pH 7.4 and 0.5M NaCl) containing 5% non-fat dry milk and 0.5% Tween 20, followed by incubation for 2h with a mouse monoclonal antibody specific for α-SMA (1:5000 dilution) (Sigma, St Louis, MO). After extensive washes, the blots were incubated for 1h at room temperature with a sheep anti-mouse secondary antibody conjugated to horseradish peroxidase (1:10000 dilution) and bands visualized by an enhanced chemiluminescence detection system.

Statistical analysis of the results was performed using the unpaired Student's t-test and differences considered significant at P<0.05.

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3. Results 

Under resting conditions, RMC 85/4 cells constitutively expressed COX-1, COX-2 and 12/15-LO (Fig. 1(B)) and mainly generated PGE₂ together with lower quantities of TXB₂, 12-HETE, 8-epi-PGF_2α, Cys-LTs and LTB₄ (Fig. 1(C)). LXA₄ was not detected in these samples.

We next assessed the effects of the selective COX-2 inhibitor, celecoxib, on the formation of vasoactive eicosanoids, namely PGE₂, TXB₂, 8-epi-PGF_2α, 12-HETE and Cys-LTs by RMC 85/4 cells. As shown in Fig. 2, celecoxib significantly decreased PGE₂ and 8-epi-PGF_2α concentrations without modifying TXB₂, 12-HETE and Cys-LT biosynthesis. Celecoxib did not modify LTB₄ biosynthesis (from 40±9 to 46±1pg/incubation).

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• Fig. 2. 

  Effects of the selective COX-2 inhibitor, celecoxib, on eicosanoid formation in rat RMC 85/4 cells. Cells were exposed to vehicle (empty bars) or celecoxib (3μM) (dashed bars) for 16h at 37°C and eicosanoid levels measured in cell supernatants by specific EIAs. Results represent the mean±SEM of 3–6 different experiments with duplicate determinations. *P<0.01 and **P<0.005 versus vehicle.

We also examined how the effects of celecoxib on arachidonic acid metabolism in RMC 85/4 cells were influenced by the presence of albumin in the culture medium. Albumin had a major impact on the COX and LO pathways since this protein significantly inhibited PGE₂ and markedly increased 12-HETE and Cys-LTs (Table 1). Albumin did not affect TXB₂ and 8-epi-PGF_2α. In the presence of albumin, celecoxib, in addition to inhibiting PGE₂, it also induced inhibition of TXB₂ formation (Table 1). Interestingly, celecoxib significantly inhibited the increase in 12-HETE production induced by albumin (Table 1). LTB₄ levels were not modified by albumin either alone or in combination with celecoxib (from 40±9 to 25±11 and to 37±14pg/incubation).

Table 1. Influence of albumin on the actions of the selective COX-2 inhibitor, celecoxib, on eicosanoid formation in rat RMC 85/4 cells

RMC 85/4 cells were exposed to vehicle (0.1% ethanol), albumin (10mg/ml) and combinations of celecoxib (3μM) with albumin (10mg/ml) for 16h at 37°C and levels of PGE₂, TXB₂, 8-epi-PGF_2α, 12-HETE and Cys-LTs were measured in cell supernatants by specific EIAs. The results represent the mean±SEM of 3–6 different experiments with duplicate determinations. Values are given as ng/incubation. *P<0.05, **P<0.025, ***P<0.005 versus vehicle.

We next evaluated whether celecoxib modulates the renin–angiotensin system by testing the effects of this compound on renin release in RMC 85/4 cells. As shown in Fig. 3, celecoxib did not modify renin release. Conversely, a significant reduction in renin release was observed with albumin alone or in combination with celecoxib (Fig. 3).

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• Fig. 3. 

  Effects of the selective COX-2 inhibitor, celecoxib, on renin release by rat RMC 85/4 cells. Cells were exposed to vehicle or celecoxib (3μM) for 16h at 37°C in the absence or presence of albumin (10mg/ml) and renin concentrations in cell supernatants determined by EIA. Results represent the mean±SEM of three different experiments with duplicate measurements. *P<0.025 and **P<0.01 versus vehicle.

To assess the effects of celecoxib on PPARγ, we transiently co-transfected RMC 85/4 cells with PPARγ-GAL4 and luciferase reporter constructs. Since, PPARγ is constitutively expressed in RMC 85/4 cells (data not shown), this strategy allowed us the measurement of PPARγ activity without interference from the endogenous receptor. As shown in Fig. 4(A), celecoxib significantly increased, in a concentration-dependent fashion, PPARγ activity. Celecoxib did not alter the agonistic effect of the PPARγ natural ligand, 15d-PGJ₂ (Fig. 4(B)). Interestingly, a significant inhibition of PPARγ activity was exerted by albumin (Fig. 4(C)), which in addition, abolished, in a concentration-dependent manner, the response of PPARγ to 15d-PGJ₂ (Fig. 4(D)).

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• Fig. 4. 

  Effects of the selective COX-2 inhibitor, celecoxib, on PPARγ in RMC 85/4 cells. Cells were co-transfected with PPARγ-GAL4, luciferase and pCMV-βGal plasmids and treated for 18h with increasing concentrations of celecoxib (3 and 10μM) (A), albumin (1 and 10mg/ml) (C) and combinations of 15d-PGJ₂ (1μM) with celecoxib (3 and 10μM) (B) or albumin (1 and 10mg/ml) (D). Luciferase values were normalized to the level of β-galactosidase activity and results plotted as fold activation relative to untreated cells (vehicle, V) arbitrarily set to a value of 1. Results are the mean±SEM of five different experiments. *P<0.025, **P<0.01 and ***P<0.005 versus vehicle. ^aP<0.01 and ^bP<0.025 versus 15d-PGJ₂.

Fig. 5 shows the effects of celecoxib on α-SMA expression in RMC 85/4 cells and their modulation by albumin and 15d-PGJ₂. Celecoxib at concentrations of 10μM had no effect on α-SMA protein levels (Fig. 5). Similar findings were obtained with lower concentrations of celecoxib (i.e. 3μM) (data not shown). In contrast, the expression of α-SMA was significantly decreased by albumin, either alone or in combination with celecoxib and 15d-PGJ₂ (Fig. 5). A significant inhibition of α-SMA was observed with high (10μM) but not low (1μM) concentrations of 15d-PGJ₂ (Fig. 5). Interestingly, celecoxib potentiated the effect of this cyclopentenone PG, since a significant inhibition in α-SMA was observed following the addition of this selective COX-2 inhibitor together with lower concentrations of 15d-PGJ₂ (Fig. 5).

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• Fig. 5. 

  Effects of the selective COX-2 inhibitor, celecoxib, on α-SMA protein expression in rat RMC 85/4 cells. Cells were exposed to vehicle, 15d-PGJ₂ (1 and 10μM), celecoxib (10μM), albumin (10mg/ml) and combinations of 15d-PGJ₂ (1μM) with either celecoxib (10μM) or albumin (10mg/ml) for 18h at 37°C. Expression of α-SMA was analyzed by Western blot and band intensities determined by scanning densitometry. This figure shows a representative blot and the mean±SEM of four different experiments. *P<0.05, **P<0.020 and ***P<0.01 versus vehicle.

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4. Discussion 

Celecoxib is a selective COX-2 inhibitor with proven anti-inflammatory and analgesic efficacy and low gastrointestinal toxicity compared with conventional NSAIDs [1], [2]. The pharmacological properties of celecoxib are based on its ability to inhibit COX-2, but the complete mechanism of action is poorly understood. The results of the current study indicate that, in addition to its primary inhibitory action on COX-2 and PG formation, celecoxib also modulates the biosynthesis of vasoconstrictor eicosanoids and is able to bind and activate PPARγ.

Our results demonstrate that RMC 85/4 cells mainly convert arachidonic acid into COX-derived products and 12-HETE. Although, we were not able to detect 5-LO expression in RMC 85/4 cells (data not shown), minor quantities of Cys-LTs and LTB₄ were found in their supernatants. These findings are consistent with previous studies implying the presence of 5-LO activity in rat glomeruli [33]. It must be taken into consideration that our results reflect eicosanoid formation by non-dividing cells since cells were grown in serum-deprived medium for 24h. On the other hand, RMC 85/4 cells also generated detectable amounts of the free radical-catalyzed product of arachidonic acid, 8-epi-PGF_2α (8-isoprostane). Isoprostanes are PG isomers that, unlike the enzymatic products of COX, are initially formed in situ in the phospholipid domain by free radical-mediated peroxidation [34]. Indeed, isoprostanes are reliable and specific markers of non-enzymatic lipid peroxidation [34]. Since, COX-2 by itself has been postulated as a source not only of 8-isoprostanes but also of oxygen radicals [35], [36], the inhibitory actions of celecoxib on these eicosanoids could reflect either a direct inhibition of COX-2-dependent 8-isoprostane biosynthesis or a reduction in oxidative stress levels or both mechanisms.

A major finding of the current study was that albumin modulated eicosanoid biosynthesis, celecoxib actions and renin release in RMC 85/4 cells. In fact, in these cells, albumin significantly inhibited PGE₂ levels and markedly increased 12-HETE and Cys-LT formation. These changes are consistent with earlier observations demonstrating that albumin diverts free arachidonate to lipoxygenation [24]. In our study, albumin also influenced celecoxib actions. Albumin is the most abundant protein in plasma and is extremely important from a biopharmacological point of view because it is the major transporter of non-esterified fatty acids as well as of different drugs and metabolites. There is a growing body of evidence that albumin binds NSAIDs and influences their pharmacokinetic properties [37]. Moreover, celecoxib is extensively protein bound, primarily to albumin [38]. Therefore, although our incubations were carried out in serum-deprived medium and cells were exposed to celecoxib for 20min before the addition of albumin, we cannot rule out the possibility that the observed effects are secondary to binding of celecoxib to albumin. These findings together with the observation that albumin reduced renin release in RMC 85/4 cells, support the notion that albumin displays remarkable biological properties.

A finding of interest is that celecoxib activated PPARγ in RMC 85/4 cells. Moreover, celecoxib did not alter the stimulatory actions of 15d-PGJ₂, a cyclopentenone PG resulting from the dehydration of PGD₂ and a natural ligand and potent activator of PPARγ [32]. Interestingly, the metabolism of PGD₂ to form 15d-PGJ₂ is catalyzed in vitro by albumin [39]. In our cell-based reporter assay, albumin significantly inhibited the induction of PPARγ by 15d-PGJ₂, an effect previously reported by Person et al. [40]. Our results differ from those previously reported by Yamazaki et al. [8] and Kusunoki et al. [41] who failed to demonstrate transcriptional activation of PPARγ in the luciferase reporter gene assay in response to celecoxib and NS-398. Whether differences in cell type and/or methodology may explain these controversial findings remain, at present, unknown. Taken together, the finding that celecoxib binds and activates PPARγ is of importance for the understanding of the molecular mechanisms of celecoxib. Thus, similar to conventional NSAIDs, which also bind PPARγ [15], [16], [17], the pharmacological properties of celecoxib are mediated, at least in part, by activation of this nuclear receptor.

Our data are of clinical interest and may be relevant for disease states with altered effective blood volume. In this regard, our investigation is particularly pertinent to the pathogenesis of renal complications in patients with decompensated liver cirrhosis in which the renal effects of selective COX-2 inhibitors are still a matter of discussion. For instance, it is well established that renal function in cirrhosis depends upon a critical equilibrium between the activity of endogenous vasoconstrictor systems and the renal production of vasodilator PGs. Given that celecoxib, in addition to inhibiting PGs also reduces the formation of 8-isoprostanes, TXB₂ and 12-HETE, which are potent renal vasoconstrictors [14], [42], [43], [44], this selective COX-2 inhibitor is expected, in theory, to produce less renal side effects than conventional NSAIDs. In fact, a recent double-blind, randomized, placebo-controlled study in patients with cirrhosis and ascites has shown that celecoxib does not impair renal function to a similar extent than the NSAID, naproxen [45]. On the other hand, the modulation of α-SMA expression by celecoxib and albumin extends our results to clinical scenarios other than cirrhosis and ascites, in which impairment of renal function is largely determined by overexpression of this phenotypic marker of mesangial cell activation [12], [18]. Overall, the in vitro results reported herein provide evidence for novel mechanisms of action underlying the biological effects of celecoxib and albumin in renal cells and provide support for additional evaluation of these compounds in vivo.

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Acknowledgements 

We thank Dr M. Martin (Medical School of Hannover, Germany) for the RMC 85/4 cells and Dr R. Evans (Salk Institute, La Jolla, USA) for providing the PPARγ constructs. Supported in part by grants from the Ministerio de Ciencia y Tecnología (SAF03/0586) and Instituto de Salud Carlos III (C03/02).

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