# Cholesterol Accumulation Is Increased in Macrophages of Phospholipid Transfer Protein-Deficient Mice

Nicolas Ogier, Alexis Klein, Valérie Deckert, Anne Athias, Ginette Bessède, Naig Le Guern, Laurent Lagrost, Catherine M Desrumaux

► To cite this version:

Nicolas Ogier, Alexis Klein, Valérie Deckert, Anne Athias, Ginette Bessède, et al.. Cholesterol Accumulation Is Increased in Macrophages of Phospholipid Transfer Protein-Deficient Mice. Arteriosclerosis, Thrombosis, and Vascular Biology, 2007, 27, pp.2407 - 2412. 10.1161/atvbaha.107.151753. hal-04200513

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# Cholesterol Accumulation Is Increased in Macrophages of Phospholipid Transfer Protein-Deficient Mice
## Normalization by Dietary Alpha-Tocopherol Supplementation

Nicolas Ogier, Alexis Klein, Valérie Deckert, Anne Athias, Ginette Bessède, Naig Le Guern, Laurent Lagrost, Catherine Desrumaux

**Objective**—Phospholipid transfer protein (PLTP) is a multifunctional, extracellular lipid transport protein that plays a major role in lipoprotein metabolism and atherosclerosis. Recent in vivo studies suggested that unlike systemic PLTP, macrophage-derived PLTP would be antiatherogenic. The present study aimed at characterizing the atheroprotective properties of macrophage-derived PLTP.

**Methods and Results**—Peritoneal macrophages were isolated from PLTP-deficient and wild-type mice and their biochemical characteristics were compared. It is shown that macrophages isolated from PLTP-deficient mice have increased basal cholesterol content and accumulate more cholesterol in the presence of LDL compared with wild-type cells. Cholesterol parameters in macrophages of PLTP-deficient mice were normalized by dietary α-tocopherol supplementation.

**Conclusions**—The antiatherogenic properties of macrophage-derived PLTP are related at least in part to its ability to reduce cholesterol accumulation in macrophages through changes in the α-tocopherol content and oxidative status of the cells. (Arterioscler Thromb Vasc Biol. 2007;27:2407-2412.)

**Key Words:** phospholipid transfer protein ■ vitamin E ■ tocopherol ■ macrophage

Phospholipid transfer protein (PLTP) is a multifunctional, extracellular lipid transport protein that plays a major role in lipoprotein metabolism and atherosclerosis.¹⁻⁴ In addition to phospholipids, PLTP transfers several amphipatic compounds including unesterified cholesterol, lipopolysaccharides, diacylglycerides, and α-tocopherol, the main isomer of vitamin E.⁵⁻⁷ PLTP is produced by a variety of tissues and secreted by macrophages, the hallmark cells of atherosclerotic lesions.⁸⁻¹¹ Several recent studies indicated that PLTP not only affects plasma lipid and lipoprotein profiles, but also modulates cell physiology.¹²⁻¹⁶ To date, the question of the pro- or antiatherogenicity of PLTP has not been fully elucidated. On the one hand, recent in vivo studies indicated that systemic PLTP deficiency is atheroprotective in different strains of hypercholesterolemic mice, and sustained overexpression of plasma PLTP in PLTP transgenic or bone marrow-transplanted mice leads to an increased risk of atherosclerosis.¹⁷⁻²¹ In further support of the proatherogenic potential of plasma PLTP in vivo, a positive correlation between PLTP activity and the risk of coronary artery disease was observed in humans.²² On the other hand, recent observations from our group²³ as well as from Liu et al²⁴ indicated that the onset of atherosclerosis is significantly delayed in low density lipoprotein-receptor-deficient and apoE-

deficient mice that were reconstituted with PLTP-expressing compared with PLTP-deficient (PLTP⁻/⁻) bone marrow cells. In these experiments, PLTP expression was mostly restricted to macrophages with only minimal modification of plasma PLTP level, suggesting that unlike systemic PLTP, macrophage-derived PLTP would be atheroprotective in vivo. In other words, the consequences of PLTP on atherosclerosis in experimental models would result from a combination of antiatherogenic effect of macrophage PLTP and proatherogenic effect of systemic plasma PLTP. To assess selectively the impact of macrophage-derived PLTP on cell properties or cholesterol accumulation, peritoneal macrophages were isolated from PLTP-deficient and wild-type (WT) mice, and their biochemical characteristics were compared in the present study. It is shown that macrophages isolated from PLTP-deficient mice have increased basal cholesterol content and accumulate more cholesterol in the presence of LDL compared with WT cells. Dietary α-tocopherol supplementation to PLTP-deficient mice allowed us to demonstrate that the proatherogenic potential of macrophages isolated from PLTP-deficient mice is related at least in part to reduced α-tocopherol content and increased oxidative stress in these cells. Together with observations made in PLTP-deficient and PLTP-transgenic mice, the present findings suggest that the
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Cholesterol accumulation in WT and PLTP-deficient macrophages. Peritoneal macrophages were incubated for 2 hours, 4 hours, 6 hours, 24 hours, or 48 hours with [³H]CE-AcLDL (A) or freshly isolated, native [³H]CE-LDL (B; 80 µg protein/mL). Cholesterol accumulation was expressed as the percentage of radioactivity recovered in cells/µg cell protein. Values are mean±SEM of n=6 WT and n=6 PLTP-deficient mice (A) or n=4 WT and n=4 PLTP-deficient mice (B). \*P<0.005 vs WT (Mann-Whitney *U* test).</figcaption></figure>

pro- or antiatherogenicity of PLTP depends on its site of expression.

## Materials and Methods

### Mice

All mice were on an homogeneous C57Bl/6 background. PLTP-deficient mice were a kind gift from Drs X.-C. Jiang and A.R. Tall (Columbia University, New York). The mice were matched for sex and age and fed either a standard chow diet or an α-tocopherol-supplemented (800 mg/kg) chow diet (2 months) before harvest. Blood samples were collected from fasted animals by retro-orbital puncture using heparin-coated capillary tubes. All procedures were performed in accordance with institutional guidelines.

### Statistical Analysis

Results are expressed as mean±SEM. The statistical significance of differences between data means was determined with the nonparametric Mann-Whitney *U* test.

Supplemental Materials and Methods (collection of elicited peritoneal macrophage, culture conditions and phenotypic characterization) are available online at http://atvb.ahajournals.org.

## Results

### Cholesterol Accumulation in Macrophages Isolated From PLTP-Deficient and WT Mice

Cholesterol accumulation was measured in macrophages isolated from PLTP-deficient and WT mice after 2-hour, 4-hour, 6-hour, 24-hour, or 48-hour incubation in the presence of radiolabeled AcLDL. As shown in Figure 1A, both short- and long-term exposure to AcLDL resulted in significantly higher cholesterol accumulation in macrophages from PLTP-deficient mice compared with those from WT mice. When freshly isolated, native LDL were used instead of acetylated LDL to promote cholesterol accumulation, again a significantly higher accumulation of cholesterol was observed in macrophages from PLTP-deficient mice as compared with WT cells (Figure 1B).

### Scavenger Receptor Expression in Macrophages Isolated From PLTP-Deficient and WT Mice

SR-A and CD36 mRNA quantification indicated no difference in the basal expression of the 2 receptors in PLTP-deficient and WT cells (CD36 ΔCP: 3.75±2.05 and 3.05±1.96 for PLTP-deficient and WT cells (n=5), respectively, n.s.; SR-A ΔCP: 4.45±2.77 and 4.35±2.55 for PLTP-deficient and WT cells (n=5), respectively, n.s.). When cells were incubated in the presence of AcLDL, again no difference in the expression level of the 2 receptors between

PLTP-deficient and WT cells was observed (results not shown).

### Cholesterol Efflux in Macrophages Isolated From PLTP-Deficient and WT Mice

Several in vitro studies led to demonstrate that PLTP can promote cholesterol efflux from macrophages both through the generation of preβ-HDL cholesterol acceptors and through the stabilization of ABCA1 and enhancement of cellular cholesterol efflux. To assess whether under our experimental conditions cholesterol efflux is impaired by lack of PLTP, cholesterol-loaded peritoneal macrophages were incubated in the presence of lipid-free apoAI or acetylated LDL as cholesterol acceptors. As shown in supplemental Figure I, we observed no difference in cholesterol efflux from WT and PLTP-deficient elicited peritoneal macrophages toward lipid-free apoAI or acetylated LDL.

### Effect of Dietary α-Tocopherol Supplementation on α-Tocopherol Content in Plasma, Erythrocytes, and Macrophages of PLTP-Deficient Mice

When PLTP-deficient mice were fed an α-tocopherol-enriched diet for 2 months, a significant increase in the α-tocopherol content of plasma, red blood cells, as well as of peritoneal macrophages was observed (plasma (mean±SEM, n=5): 6.51±2.28 mg/L in supplemented versus 0.87±0.28 mg/L in nonsupplemented PLTP-deficient mice, *P*<0.0001; red blood cells (mean±SEM, n=7): 1165.23±102.52 ng/mL in supplemented versus 582.36±113.88 ng/mL in nonsupplemented mice, *P*<0.05; macrophages (mean±SEM, n=5): 28.17±4.90 ng/10⁶ cells in supplemented versus 9.19±1.26 ng/10⁶ in non-supplemented mice, *P*<0.02).

### Effect of Dietary α-Tocopherol Supplementation on Oxidative Stress in Macrophages

Previous results from our group indicated that oxidative stress, as measured by DCFH-DA oxidation rate, is significantly increased in macrophages isolated from PLTP-deficient mice compared with WT cells²³ (Figure 2A).

In the present study, we moreover observed a significant, 2-fold increase in the oxysterol content of macrophages isolated from PLTP-deficient mice compared with WT cells (Figure 2B).

After dietary α-tocopherol supplementation, oxidative stress (as measured by DCFH-DA oxidation rate) and cellular oxysterols were significantly reduced in macrophages iso-
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Effect of dietary α-tocopherol supplementation on oxidative stress in peritoneal macrophages from PLTP-deficient mice. A, Intracellular hydroperoxides were quantified by fluorimetry using DCFH-DA. Values are expressed as fluorescence intensity/µg cell protein. B, Levels of intracellular oxysterols were determined by GC-MS. Values are expressed as ng/µg cholesterol. All values are mean±SEM of n=13 WT, n=11 PLTP-deficient, and n=7 α-tocopherol-supplemented/PLTP-deficient mice. a, *P*<0.05 vs WT; b, *P*<0.05 vs PLTP-/- (Mann–Whitney *U* test).</figcaption></figure>

lated from PLTP-deficient mice, reaching values found in WT macrophages (Figure 2A and 2B).

## Effect of Dietary α-Tocopherol Supplementation on LDL Oxidation by Macrophages

The ability of macrophages from PLTP-deficient mice to oxidize LDL is significantly higher than that of WT cells.²³ As shown in Figure 3, dietary α-tocopherol supplementation led to a significant decrease in LDL oxidation by macrophages from PLTP-deficient mice, with values returning to those observed in WT cells.

## Effect of Dietary α-Tocopherol Supplementation on Cholesterol Accumulation in Macrophages

As shown in Figure 4, dietary α-tocopherol supplementation led to a significant decrease in cholesterol accumulation in macrophages from PLTP-deficient mice. After 24-hour incubation with labeled AcLDL, cholesterol accumulation in the supplemented group was less than half that in the nonsupplemented group (% influx/µg cell protein [mean±SEM, n=7]: 0.212±0.021 versus 0.462±0.070%/µg cell protein in supplemented and nonsupplemented groups, respectively; *P*<0.005) and was not significantly different from that observed with WT macrophages (0.163±0.028%/µg cell protein).

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Effect of dietary α-tocopherol supplementation on LDL oxidation by macrophages from PLTP-deficient mice. LDL oxidation was determined by measurement of TBARS. Results are expressed as nanomoles of malondialdehyde in LDL per microgram of cell protein and are mean±SEM of n=13 WT, n=11 PLTP-deficient, and n=7 α-tocopherol-supplemented/PLTP-deficient mice. a, *P*<0.05 vs WT; b, *P*<0.005 vs PLTP-/- (Mann–Whitney *U* test).</figcaption></figure>

## Effect of PLTP Deficiency and Dietary α-Tocopherol Supplementation on Cholesterol Content in Macrophages

Additional experiments were conducted to determine the impact of PLTP deficiency and α-tocopherol supplementation on cholesterol content in macrophages. As shown in Figure 5A, cholesterol levels in PLTP-deficient macrophages were about twice that of WT macrophages under basal conditions (total cholesterol [mean±SEM, n=6]: 21.0±1.3 and 11.8±0.7 ng/µg protein in PLTP-deficient and WT cells, respectively; *P*<0.02). Esterified cholesterol, but not free cholesterol content, was significantly higher in PLTP-deficient compared with WT cells (Figure 5A). Cholesterol levels were markedly increased in both PLTP-deficient and WT cells on incubation with AcLDL. In accordance with observations made with the radioisotopic assay (Figure 1), we observed a much greater cholesterol (mainly esterified cholesterol) enrichment in PLTP-deficient compared with WT cells on incubation with AcLDL (cholesterol mass increment versus basal values [mean±SEM, n=6]: 40.1±12.8 ng/µg versus 17.3±2.9 ng/µg protein, respectively; *P*<0.02; Figure 5B and 5C). Dietary α-tocopherol supplementation led to a significant decrease in basal cholesterol content in PLTP-deficient macrophages, with values returning to those measured in WT cells (Figure 5A). In accordance with observations made with the radioisotopic assay (Figure 4), cholesterol enrichment was significantly reduced in the α-tocopherol-supplemented group compared with the nonsupple-

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Effect of dietary α-tocopherol supplementation on cholesterol accumulation in macrophages from PLTP-deficient mice. Peritoneal macrophages were incubated for 24 hours with 80 µg protein/mL of [³H]CE-AcLDL. Cholesterol influx was expressed as the percentage of radioactivity recovered in cells/µg cell protein. Values are the mean±SEM of n=7 mice in each group. a, *P*<0.005 vs WT; b, *P*<0.05 vs PLTP-/- (Mann–Whitney *U* test).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Cholesterol content of macrophages isolated from wild-type, PLTP-deficient, and α-tocopherol-supplemented/PLTP-deficient mice. A, Basal cholesterol content of freshly isolated peritoneal macrophages B, Cholesterol enrichment (increment in cholesterol content) of macrophages after a 24-hour incubation with 80 µg/mL AcLDL. Results are expressed as ng cholesterol/µg protein and are the mean±SEM of n=6 mice in each group. C, Oil Red O staining of mouse macrophages after incubation with AcLDL, showing increased lipid content in macrophages from PLTP-deficient mice compared with WT cells, and normalization after dietary α-tocopherol supplementation. TC indicates total cholesterol; FC, free cholesterol; EC, esterified cholesterol. a, *P*<0.02 vs WT; b, *P*<0.005 vs PLTP⁻/⁻; c, *P*<0.05 vs PLTP⁻/⁻; d, *P*<0.02 vs PLTP⁻/⁻ (Mann–Whitney *U* test).</figcaption></figure>

mented group after 24-hour incubation with AcLDL, and was not significantly different from values measured in WT cells (cholesterol mass increment versus basal values [mean±SEM, n=6]: 18.9±4.1 ng/µg protein in supplemented versus 40.1±12.8 ng/µg in non-supplemented PLTP-deficient mice, *P*<0.05; Figure 5B and 5C).

## Discussion

Although PLTP was initially identified as a phospholipid transfer protein that can modify high density lipoprotein (HDL) structure and composition, its biological function recently appeared to be much more complex. Over the past few years, several studies have demonstrated that PLTP is expressed in a variety of tissues, and that beyond its role in the intravascular compartment, PLTP can affect the function of various organs, including liver,¹³ brain,¹⁴ and testis.¹⁵ Recent in vivo studies indicated that systemic PLTP deficiency is atheroprotective in different strains of hypercholesterolemic mice, and sustained overexpression of plasma PLTP in PLTP transgenic or bone marrow transplanted mice leads to an increased risk of atherosclerosis.¹⁷⁻²¹ PLTP is highly expressed in macrophages, ie, essential cellular actors in atherogenesis, and it is present in the intima of atherosclerotic arteries.⁹⁻¹¹ Recent observations from our group as well as from Liu et al indicated that the onset of atherosclerosis is significantly delayed in irradiated LDL-receptor deficient and apoE-deficient mice that were reconstituted with PLTP-expressing compared with PLTP-deficient bone marrow-derived cells.²³,²⁴ In these studies, alteration in PLTP expression level was mostly restricted to macrophages, with only minimal changes in systemic PLTP level. In contrast, Vikstedt et al reported a marked increase in plasma PLTP level together with increased atherosclerosis in irradiated LDL-receptor deficient mice transplanted with PLTP-expressing as opposed to PLTP-deficient bone marrow.²¹ Taken together, these observations suggested that the pro- or antiatherogenic-

ity of PLTP may actually depend on its site of expression. To test this hypothesis, the impact of macrophage-derived PLTP on cell properties and cholesterol accumulation was addressed in the present study in a plasma PLTP-free context. It is shown that endogenous PLTP deficiency leads to increased cholesterol content of macrophages, and cholesterol enrichment in the presence of modified LDL is markedly increased in cells from PLTP-deficient compared with WT mice. To assess further the impact of PLTP deficiency on cholesterol accumulation in macrophages, cholesterol uptake by peritoneal macrophages from wild-type and PLTP-deficient mice was subsequently measured in the presence of freshly isolated, native LDL. Again, and as observed earlier with acetylated LDL, a markedly higher cholesterol accumulation was observed in peritoneal macrophages from PLTP-deficient mice as compared with wild-type cells after long-term incubation with LDL particles. Because only native LDL were used in these experiments, their nonregulated uptake through scavenger receptors must have required their oxidation by macrophages in the incubation mixtures. We previously demonstrated that this key event indeed takes place in the experimental medium with a stronger oxidative potential of PLTP-deficient macrophages as compared with wild-type macrophages.²³ Thus, PLTP arises as a previously unrecognized factor that can modulate cholesterol accumulation in macrophages, a crucial step in the initiation of atherosclerosis, and increased LDL oxidation may contribute to increased cholesterol accumulation in PLTP-deficient macrophages.

The role of PLTP in α-tocopherol metabolism in vivo has been the subject of intense research over the past few years, and high α-tocopherol requirements were proven to be dependent in part on PLTP in several tissues,⁷,¹³⁻¹⁶,¹⁹,²⁵ including macrophages.²³ α-tocopherol is the principal and most potent lipid soluble antioxidant in plasma and LDL. Beyond its antioxidant properties, α-tocopherol also displays
nonantioxidant properties that may affect the biological
functions of various cell types including monocyte-macro-
phages.²⁶,²⁷ In particular, several studies have addressed the
effect of α-tocopherol on foam cell formation. Although
some studies showed no effect of α-tocopherol on the
accumulation of cholesteryl ester in macrophages,²⁸⁻³⁰ most
of these studies demonstrated beneficial effects with regard to
foam cell formation. For instance, Suzukawa et al reported
that enrichment of J774 cells with α-tocopherol inhibited
cholesteryl ester formation,³¹ and Shige et al reported that
pretreatment with α-tocopherol significantly reduced the
uptake of modified LDL and suppressed acyl cholesterol acyl
transferase (ACAT) activity, resulting in reduced cholesterol
esterification.³² Devaraj et al reported that α-tocopherol
enrichment of macrophages significantly decreased choles-
terol ester accumulation induced by oxidized LDL and
acetylated LDL.³³ Iuliano et al showed that injection of
autologous radiolabeled native LDL into 7 patients with
carotid stenosis resulted in cholesterol accumulation in the
foam cells of the atherosclerotic plaques.³⁴ Also, in 3 patients
treated for 4 weeks with vitamin E (900 mg/d), they observed
an almost complete suppression of radiolabeled LDL uptake
by macrophages.³⁴ In the present study, we sought to deter-
mine whether altered α-tocopherol content in macrophages
isolated from PLTP-deficient mice might affect their ability
to accumulate cholesterol. We demonstrated that dietary
α-tocopherol supplementation leads to a significant reduction
of cholesterol accumulation in macrophages of PLTP-
deficient mice, with values returning to those measured in
wild-type cells. Both the antioxidant and nonantioxidant
properties of α-tocopherol might be relevant in relating the
α-tocopherol deficit in macrophages of PLTP-deficient mice
to accelerated foam cell formation. Although several in vitro
studies suggested that α-tocopherol may downregulate SR-A
and CD36 activities through both transcriptional and post-
transcriptional mechanisms,³³,³⁵,³⁶ no evidence for the effect
of α-tocopherol on scavenger receptor activity has been
reported in vivo. Our results showed no difference in scav-
enger receptor expression in macrophages from PLTP-
deficient and WT mice. Thus, increased cholesterol uptake by
macrophages of PLTP-deficient mice compared with WT
cells would not rely on altered expression of classical
scavenger receptors.

Another hypothesis is that α-tocopherol may affect choles-
terol accumulation through its antioxidative properties. In-
deed, Suzukawa et al and Devaraj et al showed that high
doses of α-tocopherol significantly reduced oxidation of LDL
by J774 macrophages.31,33 In addition, previous work from
our group showed that α-tocopherol levels are reduced in
PLTP-deficient macrophages, and they are associated with
increased oxidative status, an established major contributing
factor to atherosclerosis.23 Finally, the ability of macrophages
from PLTP-deficient mice to oxidize LDL is significantly
increased compared with that of wild-type cells.²³ In the
present study, we brought the first evidence for a direct link
between the reduction in α-tocopherol content and the in-
crease in oxidative properties of macrophages. We found that
α-tocopherol supplementation leads to a significant reduction
in macrophage oxidative status, as assessed by DCFH-DA

oxidation rate and cellular oxysterols. After α-tocopherol
supplementation, both parameters returned to values close to
those measured in WT cells. In addition, the ability of
macrophages to oxidize LDL was markedly reduced after
α-tocopherol supplementation. Taken together, these observa-
tions suggest that in vivo, α-tocopherol can modulate macro-
phage oxidative status, thus decreasing their ability to generate
oxidized LDL and reducing cholesterol accumulation.

It is worthy of note that α-tocopherol-independent mech-
anisms may also contribute to the atheroprotective effect of
macrophage-derived PLTP. In particular, PLTP may be one
component of the ATP-binding cassette transporter A1
(ABCA1)-mediated cholesterol efflux pathway in macro-
phages.³⁷ Lee-Rueckert et al demonstrated that macrophage-
derived PLTP can act as a rate-limiting factor of ABCA1-
mediated cellular cholesterol efflux when ABCA1 is not
upregulated by cAMP treatment.³⁸ In a recent study, Liu et al
demonstrated that the atheroprotective potential of macro-
phage PLTP is related at least in part to its ability to increase
apoE secretion by these cells.²⁴ It is worthy of note that the
above mentioned studies were conducted with resident peri-
toneal macrophages. In the present study, we observed no
alteration in cholesterol efflux from elicited peritoneal mac-
rophages of PLTP-deficient mice, either to ApoAI or to
acetylated LDL. A potential explanation to these contradic-
tory findings is that the contribution of endogenous PLTP to
cholesterol efflux is blunted by responses induced by the
inflammatory thioglycollate exposure of macrophages. In
support of the latter view, previous observations from our
group²³ as well as from Cao et al³⁹ reported no alteration in
cholesterol efflux from elicited peritoneal macrophages of
PLTP-deficient mice as compared with WT cells.

In conclusion, we have shown in the present study that
PLTP can modulate cholesterol accumulation in macrophages,
ie, a crucial step in atherosclerosis. In a PLTP deficiency
context, macrophages contain lower amounts of α-tocopherol
and their oxidative state is increased, which results in in-
creased oxidation of LDL and its internalization via the
scavenger receptor pathway. Increased cholesterol accumula-
tion in macrophages of PLTP-deficient mice seems to occur
independently of any modulation of scavenger receptors
expression. These findings indicate that the pro- or anti-
atherogenicity of PLTP depends on its site of expression and
that interventions aimed at inhibiting PLTP should be re-
stricted to the plasma protein.

Acknowledgments

We thank Philip Bastable for critical reading of this manuscript.

Sources of Funding

This study was supported by INSERM, the Conseil Régional de
Bourgogne, the Université de Bourgogne, the Fondation de France,
and the Agence Nationale de la Recherche. Nicolas Ogier is the
recipient of a fellowship from the Ministère de l'Éducation Nation-
ale, de la Recherche et des Technologies.

Disclosures

None.
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