draft - university of toronto t-space · pdf filepharmacological interventions to increase hdl...

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The role of paraoxonase 1 in regulating HDL functionality

during aging

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2017-0117.R1

Manuscript Type: Review

Date Submitted by the Author: 12-May-2017

Complete List of Authors: Khalil, Abdelouahed; University of Sherbrooke Kamtchueng Simo, Olivier; Universite de Sherbrooke Faculte des Sciences Ikhlef, Souade; Universite de Sherbrooke Faculte de medecine et des sciences de la sante Berrougui, Hicham; Reserch Centre on Aging

Is the invited manuscript for

consideration in a Special Issue?:

IACS Sherbrooke 2016 special issue Part 2

Keyword: HDL functionality, PON1, Cholesterol efflux, aging

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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The role of paraoxonase 1 in regulating HDL functionality during aging

Abdelouahed Khalil1,2*, Olivier Kamtchueng Simo1, Souade Ikhlef1 and Hicham Berrougui3

1Research Centre on Aging, Sherbrooke, QC, Canada

2Department of Medicine, Geriatrics Service, Faculty of Medicine and Biological Sciences, University of

Sherbrooke, Sherbrooke, QC, Canada

3Department of Biology, Polydisciplinary Faculty, University Sultan Moulay Slimane, BP 592, 23000 Beni

Mellal, Morocco

* To whom correspondence should be addressed: Abdelouahed Khalil, CdRV-Health Campus, 3001 12th

North Avenue, Sherbrooke, QC, Canada, J1H 4N4. Telephone: 1-819-821-8000, ext. 70148, Email:

[email protected]

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Abstract

Pharmacological interventions to increase HDL concentrations have led to disappointing results and

have contributed to the emergence of the concept of HDL functionality. The anti-atherogenic activity of HDL

can be explained by their functionality or quality. The capacity of HDL to maintain cellular cholesterol

homeostasis and to transport cholesterol from peripheral cells to the liver for elimination is one of their

principal anti-atherogenic activities. However, HDL possess several other attributes that contribute to their

protective effect against cardiovascular diseases. HDL functionality is regulated by various proteins and

lipids making up HDL particles. However several studies investigated the role of paraoxonase 1 (PON1) and

suggest a significant role of this protein in the regulation of the functionality of HDL. Moreover, research on

PON1 attracted much interest following several studies indicating that it is involved in cardiovascular

protection. However, the mechanisms by which PON1 exerts these effects remain to be elucidated.

Keywords: HDL functionality, PON1, cholesterol efflux, aging

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During the last 20 years, several studies have been interested in the link between paraoxonases (PON),

and more particularly PON1, and the development of cardiovascular diseases (CVD). Results of animal

studies have clearly demonstrated a beneficial role of PON1 by protecting against atherosclerotic plaque

formation. In humans, epidemiological studies have provided convincing evidence for the involvement of

PON1 in the protection against CVD. The decrease of PON1 paraoxonase activity is increasingly

associated with a higher risk of incidence of cardiovascular complications. Moreover, the demonstration that

diet interventions may enhance PON1 activity has generated a huge interest for the development of

pharmacological agent to improve PON1 activity and particularly in the presence of cardiovascular risk

factor. The beneficial effect of PON1 is explained in large part by its ability to regulate the atheroprotective

properties of HDL. However, the mechanism of this regulation is not clearly established. Moreover, PON1 is

only one among the numerous proteins forming HDL. In this review we will discuss the HDL concentration

and HDL function in the protection against CVD. We will focus on the role of PON1 in the regulation of the

functionality of HDL and the mechanisms by which PON1 may regulate the atheroprotective activities of

HDL.

Biogenesis and HDL metabolism: concentration versus functionality

Pre-βHDL are considered the early forms of HDL particles. They result exclusively from the hepatic (~

70%) and intestinal (~ 30%) secretion of apolipoprotein A-I (apoA-I) (Brunham et al. 2006; Timmins et al.

2005). ApoA-I interacts with the ABCAI transporter, which results in its lipidation and the generation of pre-

βHDL, which in turn are gradually transformed into mature HDL by the action of a number of plasmatic

enzymes (Chroni et al. 2003; Haghpassand et al. 2001). During their maturation, HDL are enriched in

cholesterol that is esterified by lecithin cholesterol acyl transferase (LCAT) and transferred back to the liver

for elimination (Tsompanidi et al. 2010). This normal HDL maturation process, which is called reverse

cholesterol transport (RCT), is the only way that excess cholesterol in peripheral tissues can be transferred

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to the liver to be metabolized. RCT is one of the main atheroprotective activities of HDL (Esterbauer et al.

1997; Glomset 1968; Johnson et al. 1991).

Epidemiological studies have shown that there is an inverse relationship between HDL concentrations

and the risk of cardiovascular diseases (CVD) (Gordon et al. 1977). However, other studies raise doubts

about the validity of this observation: (1) carriers of the apoA-I Milano mutation have low HDL

concentrations and, paradoxically, enjoy a greater life expectancy and a total lack of atherosclerotic lesions

(Alexander et al. 2009; Frikke-Schmidt et al. 2008; Roma et al. 1993); (2) the Investigation of Lipid Level

Management to Understand its Impact in Atherosclerotic Events (ILLUMINATE) study reported a high

mortality risk with Torcetrapib™ despite a 4-fold elevation in normal HDL concentrations (Barter et al. 2007);

(3) an analysis of data from the Framingham study revealed that 44% of CVD occur in subjects with normal

HDL concentrations (Ansell et al. 2003); and (4) several in vitro studies have shown that there is a

significant reduction in the anti-inflammatory and antioxidant activities of HDL during aging, even in the

presence of normal HDL concentrations (Jaouad et al. 2006a; Khalil et al. 1998; Loued et al. 2013). The link

between HDL concentrations and HDL functionality is thus in doubt (Sviridov et al. 2002), and HDL

functionality is increasingly being seen as being more important than HDL concentrations with respect to

cardiovascular protection (Khera et al. 2011).

HDL have a number of potential anti-atherogenic activities, although the relative importance of these

activities remains a subject of debate. HDL possess antioxidant, anti-inflammatory, antithrombotic, and

immunomodulatory activities and also regulate endothelial function (Figure 1). However the participation of

HDL in RCT is one of the most important and least controversial anti-atherogenic activities of HDL. The

RCT activity of HDL is determined in vitro by the measurement of cholesterol efflux capacity from

macrophages and is inversely associated with both carotid intima-media thickness and the likelihood of

angiographic coronary artery disease (Khera et al. 2011).

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HDL cholesterol efflux capacity

Cholesterol efflux from macrophages and other tissues is the first and rate-limiting step of RCT (Wang X.

et al. 2007). RCT is modulated by both the predisposition of cells to release cholesterol and by the ability of

HDL to transport cholesterol to the liver for elimination (Yancey et al. 2003). Cholesterol efflux is mediated

via three pathways. The first cellular efflux pathway is via the ATP-binding cassette transporters (ABCA1

and ABCG1). ABCA1 and ABCG1 are members of a large family of ATP-dependent transporters that share

common structural motifs for the active transport of a variety of substrates. Lipid-poor apolipoproteins,

particularly apoA-1, are the preferred cholesterol acceptors for ABCA1 (Wang N. et al. 2000). ABCG1 is

another transporter that promotes mass cholesterol efflux from cells to mature HDL particles (HDL2 and

HDL3) but not to lipid-poor apoA-I (Yvan-Charvet et al. 2010). ABCA1 and ABCG1 promote unidirectional

cholesterol efflux to lipid-poor apoA-I and apoE, and HDL particles, respectively. The second cholesterol

efflux pathway involves the scavenger receptor class B type I (SR-BI). The movement of free cholesterol via

SR-BI is bidirectional and depends on the direction of the cholesterol gradient (Ji et al. 2011). The third

cholesterol efflux pathway is aqueous diffusion. This process involves the desorption of free cholesterol

molecules from donor lipid-water interfaces and the diffusion of these molecules through the intervening

aqueous phase until they collide with and are absorbed by an acceptor (Berrougui et al. 2012). In addition to

the interaction of apoA-1 and mature HDL particles with various membrane proteins, which mediates

cholesterol exchange, other HDL-associated proteins also regulate the cholesterol efflux capacity of HDL,

including lecithin-cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and

paraoxonase (PON1). These proteins also regulate the other anti-atherogenic activities of HDL. A reduction

in their enzymatic activities or protein concentrations may thus significantly impact the functionality of HDL

and their capacity to prevent the initiation of the atherosclerosis process.

Lecithin-cholesterol acyltransferase (LCAT)

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LCAT is synthetized principally in the liver and circulates in the plasma associated with HDL (α-LCAT)

and LDL (β-LCAT). LCAT esterifies free cholesterol with phosphatidylcholine, contributing to the formation

of cholesteryl ester and lysophosphatidylcholine (Jonas 2000), the maturation of HDL, and the conversion of

HDL from discoidal to spherical particles. LCAT thus plays an important role in maintaining serum

cholesterol homeostasis by regulating cholesterol transport to the liver. Nevertheless, the role of LCAT in

the pathogenesis of atherosclerosis remains a subject of debate. Animal studies have shown, on the one

hand, that the over-expression of LCAT in transgenic mice significantly increases HDL concentrations and,

surprisingly, the number and size of atherosclerotic lesions in these mice compared to wild-type mice

(Mehlum et al. 1997). The increased atherosclerosis in mice over-expressing LCAT has been explained by

the formation of dysfunctional HDL particles, which are less effective in mediating cholesterol transport to

the liver for elimination (Berard et al. 1997). On the other hand, LCAT knockout mice are more protected

against atherosclerosis than wild-type mice, even when fed a pro-atherogenic diet (Lambert et al. 2001). In

human studies, The Copenhagen City Heart and The Copenhagen General Population studies, which

included more than 50,000 subjects, showed that one of the four common variants of LCAT is responsible

for low HDL concentrations (Haase et al. 2012). However, the significant reduction in HDL concentrations

due to the LCAT variant has no effect on the incidence of CVD (Haase et al. 2012).

An α-LCAT mutation is at the origin of a rare metabolic disorder (fish-eye disease) associated with low

HDL concentrations and a visual complication (Asztalos et al. 2007; Calabresi et al. 2005). Paradoxically,

despite their very low HDL concentrations, individuals with this metabolic disorder have the same risk for

CVD as normal subjects (Calabresi et al. 2009; Calabresi et al. 2012). Interestingly, even though the HDL

concentrations of these individuals is very low, the ability of their HDL to mediate cholesterol efflux is not

affected (Berard et al. 2001; Calabresi et al. 2009; Elkhalil et al. 1997; Tanigawa et al. 2009). In addition,

LCAT-deficient plasma is as efficient as control plasma in mediating cholesterol efflux (Berard et al. 2001).

These results may explain the absence of premature atherosclerosis in LCAT-deficient patients and indicate

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that HDL functionality is more important than quantity in terms of cardiovascular protection.

Cholesteryl ester transfer protein (CETP)

CETP is a hydrophobic glycoprotein produced in the liver and secreted into the plasma where it is mainly

associated with HDL (Tall 1993). The importance of CETP in HDL metabolism was first evoked based on a

mutation in the Japanese population whose phenotype is associated with very high HDL concentrations and

a lower incidence of coronary heart diseases (CHD) (Akita et al. 1994; Boekholdt et al. 2003; Inazu et al.

1990; Koizumi et al. 1985). This aroused much interest in developing a CETP inhibitor that would increase

HDL concentrations. Torcetrapib was one of the first CETP inhibitors tested in the ILLUMINATE trial to

determine whether it would decrease cardiovascular events in high-risk populations (Barter et al. 2007).

Torcetrapib, by inhibiting CETP, which normally transfers esterified cholesterol from HDL to other

lipoproteins (LDL and VLDL), increased HDL concentrations by approximately 72% (Barter et al. 2007).

However, despite significantly increasing HDL concentrations, Torcetrapib did not reduce cardiovascular

risk, and the ILLUMINATE trial was terminated because of very serious side effects (Rader 2007).

Nevertheless, the results of the trial contributed to the emergence of the concept of the HDL functionality (or

quality). There is increasing evidence that HDL concentrations do not reflect HDL functionality, which may

be much more important than HDL concentrations in terms of cardiovascular protection (Hiura et al. 2009;

Sviridov et al. 2008). Other studies showed that the 4-fold increase in HDL concentrations observed in

Torcetrapib-treated patients does not improve HDL functionality in terms of the ability of HDL particles to

meditate cholesterol efflux (Yvan-Charvet et al. 2007).

Paraoxonase 1 (PON1)

PON1 is a member of a multigene family that also includes PON2 and PON3. PON1 was initially studied

for its capacity to neutralize organophosphate xenobiotics such as paraoxon, sarin, soman, VX, chlorpyrifos

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oxon, diazoxon, and other related toxic compounds. Since all these compounds are non-physiological

substrates, the physiological role of PON1 was not studied at the time. PON1 is a calcium-dependent serum

esterase that is exclusively synthesized in the liver and is found in the bloodstream associated with HDL.

Research on PON1 attracted much interest following several studies indicating that it is involved in

cardiovascular protection (Shih et al. 1998; Tward et al. 2002). PON1-deficient mice are more susceptible to

atherosclerosis, while the over-expression of PON1 in mice increases their resistance to CVD (Shih et al.

1998; Tward et al. 2002). PON1 also prevents the accumulation of oxidized lipids within LDL (Mackness M.

I. et al. 1991). Several studies have contributed to understanding how PON1 prevents the atherosclerosis

process and the associated clinical complications. The anti-atherosclerotic activities of HDL, especially their

antioxidant and anti-inflammatory activities and cholesterol efflux capacity, have been attributed, in large

part, to PON1. However, the mechanisms by which PON1 exerts these effects remain to be elucidated.

Paraoxonase 1 and the antioxidant activity of HDL

Antioxidant activity constitutes one of the anti-atherogenic properties of HDL. Several studies have

shown that HDL prevent the oxidation of LDL both in vitro and in vivo (Bonnefont-Rousselot, Khalil, Gardes-

Albert, et al. 1997). This antioxidant effect was attributed for a long time to vitamin E, particularly α-

tocopherol, which is found in HDL. However, a comparison of the α-tocopherol content of HDL and other

lipoproteins showed that HDL contain the lowest α-tocopherol content (less than one molecule of α-

tocopherol per HDL particle compared to VLDL and LDL, which contain 12 and 45 molecules of α-

tocopherol per particle, respectively) (Bonnefont-Rousselot, Khalil, Delattre, et al. 1997; Romanchik et al.

1995). This clearly indicates that α-tocopherol content alone cannot explain the antioxidant activity of HDL.

A study by Shih et al. demonstrated that the antioxidant effect of HDL is due principally to PON1 (Shih et al.

1998). These authors showed that HDL from PON1 knockout mice lose their capacity to protect LDL against

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oxidation, while enriching these HDL with PON1 restores their antioxidant activity (Shih et al. 1998).

Moreover, mice lacking serum PON1 are more susceptible to organophosphate toxicity and atherosclerosis

than wild-type mice (Shih et al. 1998). On the other hand, Tward et al. showed that HDL from mice over-

expressing human PON1 are better able to protect LDL against oxidation and that these mice are more

resistant to atherosclerosis (Tward et al. 2002). In addition, HDL from avian species, which lack

paraoxonase activity, are unable to prevent LDL oxidation in the presence of copper ions (Mackness B. et

al. 1998). We purified PON1 from the plasma of healthy subjects and determined the capacity of human

PON1 to prevent the oxidation of LDL in vitro (Jaouad et al. 2006a). Interestingly, PON1 purified from

human plasma and used at physiological concentrations (40 to 80 g/mL) is more effective in protecting LDL

against oxidation than 50 µM α-tocopherol, which is approximately twice its physiological concentration

(Jaouad et al. 2006a). Nevertheless, the antioxidant activity of PON1 has been questioned in other studies,

which attributed the activity to the contamination of PON1 with platelet-activating factor acetylhydrolase

(PAF-AH) or with tergitol during its purification from plasma (Connelly et al. 2005; Teiber et al. 2004).

However, further studies, including ours, showed that recombinant human PON1 protein also possesses

strong antioxidant activity, which negates the hypothesis that the antioxidant activity is due to contamination

by other plasma proteins (Brushia et al. 2001; Loued et al. 2012). We also showed that the oxidation of

PON1 with oxygen free radicals produced by the gamma radiolysis of water or the incubation of PON1 with

n-ethylmaleimide (NEM) significantly affects its antioxidant activity (Jaouad et al. 2006a). PON1 has a free

sulfhydryl group at position Cys284 that may be involved in its antioxidant activity. The Cys284 residue in

PON1 is one of the amino acids that is modified by oxygen free radicals and by the action of NEM (Jaouad

et al. 2006a). Cys-284 is also the active site for the antioxidant activity of PON1 (Aviram et al. 1998). The

oxidation of Cys-284 can also occur in vivo under oxidative stress conditions, which would affect the

antioxidant activity of PON1 and the anti-atherogenic properties of HDL. In agreement with this, we showed

that the antioxidant activity of PON1 is significantly reduced with aging and that this is due to a decrease in

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the number of free sulfhydryl groups on the PON1 of elderly subjects compared to young subjects (Jaouad

et al. 2006b). The paraoxonase activity of PON1 is also lower in elderly subjects. However, this decrease

cannot be explained by a reduction in its plasma concentration or a decrease in HDL concentrations (Seres

et al. 2004). The oxidative stress conditions that characterize the aging process may explain the decrease

in PON1 paraoxonase activity in the elderly (Seres et al. 2004). The susceptibility of HDL to lipid

peroxidation also increases with age (Khalil et al. 1998). The fact that PON1 is exclusively associated with

HDL and that it exhibits antioxidant activity suggests that the increase with aging of the susceptibility of HDL

to lipid peroxidation is largely due to an alteration of PON1 activity (Seres et al. 1996). The decrease in

PON1 activity has an impact on HDL functionality and may explain, at least in part, the development of the

atherosclerosis process in the presence of cardiovascular risk factors such as aging, diabetes, obesity, and

renal failure (Bounafaa et al. 2014; Cherki et al. 2007; Gbandjaba et al. 2012).

PON1 and inflammation

PON1-deficient mice display increased vascular inflammation and thrombogenesis, suggesting that

PON1 has anti-inflammatory activity (Ng et al. 2008). PON1 also has phospholipase-like activity that allows

it to hydrolyze phosphatidylcholine core aldehydes and to produce lysophosphatidylcholine (LysoPC) and

free oxidized fatty acids (Ahmed et al. 2002; Loued et al. 2012). It has been suggested that the hydrolysis of

these oxidized phospholipids may explain the anti-inflammatory activity of PON1 (Ahmed et al. 2003).

Intriguingly, the hydrolysis of oxidized phospholipids in oxLDL contributes to the formation LysoPC and

oxidized free fatty acids, both of which are pro-inflammatory (Ahmed et al. 2002; Rozenberg et al. 2003;

Schilling et al. 2009). This raises the question of how PON1 exerts its anti-inflammatory activity. In a recent

study, we measured the capacity of PON1 to hydrolyze oxidized phospholipids in oxLDL and oxHDL (Loued

et al. 2012). The addition of PON1 to oxLDL contributed to the hydrolysis of oxidized phospholipids and

significantly stimulated the production of LysoPC (Loued et al. 2012). On the other hand, the addition of

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PON1 to oxHDL resulted in a concentration-dependent reduction in the production of LysoPC (Loued et al.

2012). These results were confirmed by measurements of endothelial cell-associated intercellular adhesion

molecule 1 (ICAM1) expression. The incubation of PON1 with oxLDL resulted in a significant increase in

ICAM1 expression on endothelial cells, suggesting that PON1 possesses pro-inflammatory activity (Loued

et al. 2012), while incubating PON1 with HDL resulted in a significant decrease in ICAM1 expression on

endothelial cells, suggesting that PON1 also possesses anti-inflammatory activity (Loued et al. 2012). This

discrepancy can be explained by the fact that the anti-inflammatory effect of PON1 depends on its

association with HDL (Loued et al. 2012). PON1 interacts with other lipoprotein components to hydrolyze

and inactivate oxidized phospholipids. These interactions appear to be an intrinsic property of HDL (Loued

et al. 2012). α-LCAT may be one of the HDL-associated enzymes that interact with PON1 to reduce the

pro-inflammatory effect of oxidized lipids. oxHDL alone has a pro-inflammatory effect as measured by

ICAM1 expression on endothelial cells, while the enrichment of oxHDL with PON1 (20 µg/mL) reduces

ICAM1 expression (Figure 2). Interestingly, the anti-inflammatory activity of PON1 increases when LCAT (5

µg/mL) is added to the mixture (Figure 2). We hypothesize that LCAT, due to its lysolecithin acyltransferase

II (LATII) activity, may interact with PON1 by esterifying LysoPC with fragmented acyl groups from oxidized

phospholipids, thus reducing the pro-inflammatory activity of HDL (Goyal et al. 1997; Subbaiah et al. 1996).

PON1 also reduces monocyte chemotaxis and adhesion to endothelial cells (Ahmed et al. 2003), which may

be due to the capacity of PON1 to inhibit the biological activities of oxidized phospholipids and their

hydrolytic products (Ahmed et al. 2003). In addition to its capacity to hydrolyze oxidized lipids, PON1 also

suppresses the pro-inflammatory response of macrophages. This activity may be mediated via its

interaction with the SR-BI receptor on macrophages (Aharoni et al. 2013). We showed that the decrease in

the enzymatic activity of PON1 in elderly subjects significantly affects its anti-inflammatory activity (Loued et

al. 2013). HDL obtained from elderly subjects have a lower capacity to reduce ICAM1 expression on

endothelial cells than HDL obtained from young subjects (Loued et al. 2013). The enrichment of HDL with

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PON1 increases the anti-inflammatory activity of HDL as determined by the measurement of ICAM1

expression (Figure 2). Interestingly, extra virgin olive oil consumption enhances the enzymatic activity of

PON1 and the anti-inflammatory activities of both PON1 and HDL (Loued et al. 2013).

PON1 and the capacity of HDL to mediate cholesterol efflux

Although the ability of HDL to mediate cellular cholesterol efflux and transport cholesterol to the liver for

elimination is considered the main anti-atherogenic activity of HDL, very few studies have investigated the

role that PON1 may play in this process. The results of one in vitro study suggested that PON1 stimulates

cholesterol efflux from macrophages (Rosenblat et al. 2006) and that this efflux depends, in part, on the

capacity of PON1 to increase LysoPC formation, which may stimulate HDL binding to macrophages

(Rosenblat et al. 2006). PON1 purified from human plasma enhances cholesterol efflux from J774

macrophages and THP-1 macrophage-like cells (Berrougui et al. 2012). This effect increases with the level

of expression of the ABCA1 transporter on macrophages, suggesting that PON1 may stimulate cholesterol

efflux via an interaction with this transporter (Berrougui et al. 2012). ApoA-I is the only protein associated

with HDL that is known to interact with the ABCA1 transporter and to initiate cholesterol efflux. De Beer et

al. showed that, under inflammatory conditions, serum amyloid A (SAA) has an apoA-I-like effect by

mediating cholesterol efflux via the ABCA1 and ABCG1 transporters (de Beer et al., 2011). Remaley et al.

showed that ABCA1-mediated cellular binding to apolipoproteins and lipid efflux is not specific to apoA-I but

can also occur with other apolipoproteins that contain multiple amphipathic helical domains (Remaley et al.

2001). In addition, the apoA-I mimetic peptide has no sequence homology with the apoA-I protein but

mimics the class A amphipathic helixes and interacts with ABCA1 to promote cholesterol efflux via this

pathway (Xie et al. 2011). PON1, like apoA-I and SAA, also contains an amphipathic α-helix with

approximately 22 amino acids in its secondary structure that enables HDL particles to tightly bind to and

stabilize PON1 and stimulate its activity (Gu et al. 2016; Harel et al. 2004; Perez-Jimenez et al. 2005). The

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presence of this structure strengthens the hypothesis that an interaction between PON1 and ABCA1

mediates cholesterol efflux. We showed that PON1 purified from human plasma uses an apoA-I-like

mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and

non-raft domains of the plasma membrane, respectively (Berrougui et al. 2012). PON1 also stimulates the

expression of ABCA1 on macrophages (Berrougui et al. 2012), which may be due to the capacity of PON1

to hydrolyze oxidized phospholipids in oxLDL and to the formation of LysoPC (Ikhlef et al. 2016). A pre-

treatment of oxLDL with PON1 followed by an incubation with cholesterol-loaded macrophages stimulates

cholesterol efflux to HDL and apoA-I, inducing an over-expression of the ABCA1 transporter (Ikhlef et al.

2016). Ikhlef et al., who investigated the effects of PPAR gamma, LXR alpha, and ABCA1 inhibitors,

showed that PON1 stimulates cholesterol efflux by over-expressing ABCA1 via the PPARγ-LXRα-ABCA1

pathway (Ikhlef et al. 2016). This process can occur in vivo. In fact, PON1 and oxLDL levels are significantly

higher in atherosclerotic lesions (Mackness B. et al. 1997). The coexistence of PON1 and oxLDL decreases

the atherogenicity of plaque by inhibiting the bioactivity of oxidized lipids (Cohen et al. 2014), while the up-

regulation of ABCA1 expression on macrophages stimulates the efflux of cholesterol from these

macrophages (Ikhlef et al. 2016). An investigation by Ikhlef et al. using transgenic mice that over-express

human PON1 confirmed the involvement of this HDL-associated protein in the stimulation of RCT (Ikhlef et

al. 2017). RCT is significantly higher in transgenic mice over-expressing PON1 than in wild-type mice (Ikhlef

et al. 2017). In addition, HDL from PON1-Tg mice have a higher capacity to mediate cholesterol efflux than

HDL from wild-type mice (Ikhlef et al. 2017). Moreover, macrophages from PON1-Tg mice also express

significantly more ABCA1 transporter than macrophages from wild-type mice (Ikhlef et al. 2017). Thus,

PON1 may stimulate cholesterol efflux via two mechanisms; the first is that PON1 interacts directly with

ABCA1 transporter to allow the efflux of cholesterol to HDL particles (Berrougui et al. 2012) and the second

mechanism suggests that PON1 induces ABCA1 overexpression thereby facilitating the efflux of cholesterol

via this transporter (Berrougui et al. 2012). These in vitro and in vivo studies provided evidence of the

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involvement of PON1 in the regulation of many of the anti-atherogenic activities of HDL and showed that

PON1 also modulates HDL functionality.

PON1 and other HDL activities

PON1 has been also suggested to regulate the apoptotic activity of HDL. This process has been

explained by the capacity of PON1, at basal condition, to enhance SR-BI receptor expression on

macrophages via a mechanism implicating LysoPC formation. The increased atherosclerosis development

observed in PON1-deficient mice was explained as a result of reduced SR-BI mediated HDL protection

against apoptosis (Farid et al. 2012; Fuhrman et al. 2010). PON1 was also suggested to regulate the anti-

apoptotic activity of HDL and this effect was explained by the capacity of PON1 to reduce HDL oxidation

(Camps et al. 2010). Ferree et al 2000 demonstrated that enhanced PON1 protein expression was

associated with increased serum biomarker of antiapoptotic activity (Ferre et al. 2006). Some studies have

also associated PON1 to the anti-thrombotic and anti-adhesion activities of HDL and advanced that this

effect may be explained by the capacity of PON1 to protect against oxidation and protein modification.

However, there is not enough convincing data to confirm the involvement of PON1 in the regulation of these

activities of HDL (anti-apoptotic, anti-thrombotic, anti-adhesion activities).

Aging and HDL functionality

It is well established that LDL concentrations and the pro-atherogenic effects of LDL increase with aging

(Abbott et al. 1983; Heiss et al. 1980; Khalil et al. 1996). Conversely, there is no clear agreement regarding

the effect of aging on HDL concentrations (Frishman et al. 1992; Nikkila et al. 1990; Nikkila et al. 1991;

Schaefer et al. 1989; Schaefer, Lamon-Fava, Johnson, et al. 1994; Wallace et al. 1992; Wilson et al. 1994).

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While prospective studies have shown that plasma HDL concentrations decline with age, cross-sectional

studies have indicated that mean HDL cholesterol concentrations remain the same, or even increase, with

aging (Wallace et al. 1992). Several other studies have suggested that these variations are gender-

dependent and that, in men, HDL concentrations remain unchanged with aging (Frishman et al. 1992;

Schaefer, Lamon-Fava, Cohn, et al. 1994; Wallace et al. 1992) while they decrease in women starting at

age 60 (Brown et al. 1993; Cheung et al. 2009; Kim et al. 2000). This decrease has been attributed to

hormonal changes in postmenopausal women (Wilson et al. 1994). Changes in the body mass index, in

addition to aging, may also be an important determinant of the decline in HDL concentrations in the elderly.

Some studies have reported that centenarians have HDL concentrations similar to those of middle-aged

healthy populations (Barbagallo et al. 1998). High HDL concentrations have even been reported as having a

positive effect on longevity (Landi et al. 2007). However, other studies have reported lower HDL

concentrations in centenarians (Baggio et al. 1998). This discrepancy has been attributed to differences in

sampling procedures and the nutritional and functional status of the centenarians (Arai et al. 2004).

Pharmacological intervention studies aimed at increasing HDL concentrations as well as studies on genetic

variants that influence HDL concentrations have raised considerable doubt about the relevance of the HDL

concentration hypothesis (Ishigami et al. 1994; Ohta et al. 1995; Yamashita et al. 1990). There is now

general agreement on the importance of HDL functionality (or quality) rather than HDL concentrations in

preventing cardiovascular diseases (Khera et al. 2011; Stock 2011). However, little is know about how HDL

functionality is affected during aging and what impact a decrease in functionality may have on the

development of atherosclerosis in the elderly.

Oxidative modifications of LDL and HDL may play an important role in the pathogenesis of

atherosclerosis. The oxidation of LDL contributes to the accumulation of cholesterol in macrophages,

resulting in their transformation into foam cells. The oxidation of LDL also initiates an inflammatory response

that exacerbates the atherosclerosis process (Ross 1999). We showed that the susceptibility of LDL to

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oxidation increases significantly with aging (Khalil et al. 1996). The increase in the susceptibility to oxidation

may be caused by biochemical changes that occur with aging, particularly a decrease in endogenous

vitamin E in LDL and an increase in total polyunsaturated fatty acids (Khalil et al. 1996; Reaven et al. 1999).

HDL from elderly subjects is more susceptible to oxidation and, more importantly, to a significant reduction

in their capacity to prevent LDL oxidation (Jaouad et al. 2006a; Khalil et al. 1998). The reduction in the

antioxidant activity of HDL has been attributed to a reduction in PON1 activity, which is approximately 43%

lower in the elderly (Milochevitch et al. 2001; Seres et al. 2004). Another study in our laboratory showed that

the anti-inflammatory activity of HDL also declines significantly with aging (Loued et al. 2013). Although we

have not clearly proven the involvement of PON1 in the reduction the anti-inflammatory activity of HDL,

some of our results support this hypothesis (Loued et al. 2013). We showed that enriching HDL with PON1

enhances the anti-inflammatory effect of HDL (Figure 2) and that stimulating PON1 paraoxonase activity

improves the anti-inflammatory potential of HDL (Loued et al. 2013).

The ability of HDL to mediate cholesterol efflux is considered the main anti-atherogenic activity of HDL

and is strongly and inversely associated with the severity of atherosclerosis, independently of HDL

cholesterol levels (Khera et al. 2011). The capacity to mediate cholesterol efflux from macrophages is a

metric of HDL functionality (Khera et al. 2011). We showed that HDL from elderly healthy subjects have a

lower capacity to mediate cholesterol efflux (Berrougui et al. 2007). Our results suggested that the alteration

of this HDL activity is due to oxidative modifications of apoA-1, which reduces its interaction with the ABCA1

transporter (Berrougui et al. 2007). However, the subsequent demonstration that PON1 is involved in the

stimulation of the cholesterol efflux capacity of HDL suggests that the decrease in PON1 activity during

aging may explain the alteration of the capacity of HDL from elderly subjects to mediate cholesterol efflux.

Our results from an animal study showed that plasma obtained from 24-month-old mice (aged mice) has a

significant lower capacity to mediate cholesterol efflux from J774 macrophages than plasma obtained from

4-month-old mice (young mice) (Figure 3). Interestingly, the overexpression of PON1 in mice significantly

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increases the ability of plasma from both in young and aged mice to mediate cholesterol efflux and also

reduces the age-related difference in the cholesterol efflux capacity of plasma (Figure 3). HDL3 have a

higher PON1 content than HDL2 (Rozek et al. 2005). Interestingly, the decrease with aging in the capacity of

HDL to mediate cholesterol efflux is more closely associated with HDL3 than with HDL2 (Berrougui et al.

2007). This provides additional support for the hypothesis that there is a relationship between PON1 activity

and the capacity of HDL to mediate cholesterol efflux. The reduction in the capacity of HDL to mediate

cholesterol efflux in acute coronary syndrome has also been associated with a reduction of PON1 activity

(Bounafaa et al. 2014).

Our results thus clearly showed that there is a decrease in the main anti-atherogenic activities of HDL in

the elderly, confirming that HDL functionality is altered with aging. The alteration of functionality is also

involved in several risk factors for CVD, in particular in patients with chronic inflammation disorders,

including diabetes mellitus (Morgantini et al. 2011) and atherosclerosis (Navab et al. 1997). Although

several factors may be involved the alteration of HDL functionality, a number of lines of evidence point to

the involvement of PON1 in the regulation of HDL functionality.

HDL functionality and diet

Dietary patterns have been shown to impair or improve HDL functionality. Nutrition studies on HDL

functionality have focused on the antioxidant, anti-inflammatory, antithrombotic, vasodilatory, and RCT

properties of HDL. We recently showed that consumption of extra virgin olive oil, one of the key components

of the Mediterranean diet, improves the antioxidant and anti-inflammatory activities of HDL as well as the

capacity of HDL to remove excess cholesterol from peripheral tissues. Interestingly, these improvements

are accompanied by an increase in PON1 activity (Helal et al. 2013). Other studies have reported that diets

rich in monounsaturated fatty acids (MUFA) are associated with an increase in PON1 activity (Ferretti et al.

2012). In terms of RCT, Treguier et al. reported that the efflux of cholesterol from macrophages to plasma

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as well as in vivo RCT are significantly impaired in hamsters fed a cholesterol-enriched diet (Treguier et al.

2011). Nishimoto et al. reported that C57B6/j mice fed fish oil displayed higher in vivo macrophage RCT and

ex vivo efflux to apoA-I than C57B6/j mice fed soybean or coconut oils (Nishimoto et al. 2009). In addition to

macronutrients, the effects of bioactive compounds on HDL functionality have been investigated. We

showed in a recent study that plant-sourced phenolic compounds enhance the anti-atherogenic properties

of HDL by reducing oxidative damage and improving the capacity to promote cholesterol efflux and the RCT

process (Berrougui et al. 2015). Fruit and vegetable intake also modulates HDL functionality. The

consumption of pomegranate juice increases serum PON1 activity and reduces HDL oxidation (Aviram et al.

2000). Other epidemiological studies have shown that vitamin E supplementation improves the RCT

function of HDL in type 2 diabetic individuals (Asleh et al. 2008). On the other hand, the supplementation of

the diets of healthy men with tomato or carrot juice has no effect on PON1 activity (Bub et al. 2005)

Conclusion

CVD is a major health problem, particularly among the elderly. The elderly account for almost 80% of

deaths due to CVD (Garcia-Palmieri 2006; Lakatta 2002). One epidemiological study showed that 0.025 M

step increases in HDL concentrations are associated with 2 to 3% step reductions in the incidence of CVD

(Toth 2005). However, pharmacological intervention studies aimed at increasing plasma HDL

concentrations have led to disappointing results with respect to cardiovascular protection. Given these

results, HDL functionality is increasingly being seen as important, if not more so, than HDL concentrations.

Understanding the parameters that regulate HDL functionality will make it possible to develop new

strategies to reduce the pathogenesis of atherosclerosis, especially in the presence of a risk factor such as

aging. There is some evidence to indicate that PON1 plays an important role in regulating HDL functionality.

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However, further studies are required to determine the mechanisms by which PON1 improves HDL

functionality and protects against CVD.

Acknowledgment

This work was supported by grants from the Canadian Institutes of Health Research (MOP-89912 and IAO-

134212).

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References:

Abbott, R. D., Garrison, R. J., Wilson, P. W., Epstein, F. H., Castelli, W. P., Feinleib, M. et al. 1983. Joint

distribution of lipoprotein cholesterol classes. The Framingham study. Arteriosclerosis. 3(3): 260-272.

Aharoni, S., Aviram, M., Fuhrman, B. 2013. Paraoxonase 1 (PON1) reduces macrophage inflammatory

responses. Atherosclerosis. 228(2): 353-361.

Ahmed, Z., Ravandi, A., Maguire, G. F., Emili, A., Draganov, D., La Du, B. N. et al. 2002. Multiple substrates

for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite. Biochem Biophys Res Commun.

290(1): 391-396.

Ahmed, Z., Babaei, S., Maguire, G. F., Draganov, D., Kuksis, A., La Du, B. N. et al. 2003. Paraoxonase-1

reduces monocyte chemotaxis and adhesion to endothelial cells due to oxidation of palmitoyl, linoleoyl

glycerophosphorylcholine. Cardiovasc Res. 57(1): 225-231.

Akita, H., Chiba, H., Tsuchihashi, K., Tsuji, M., Kumagai, M., Matsuno, K. et al. 1994. Cholesteryl ester

transfer protein gene: two common mutations and their effect on plasma high-density lipoprotein cholesterol

content. J Clin Endocrinol Metab. 79(6): 1615-1618.

Alexander, E. T., Tanaka, M., Kono, M., Saito, H., Rader, D. J., Phillips, M. C. 2009. Structural and

functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I. J lipid Res. 50(7):

1409-1419.

Ansell, B. J., Navab, M., Hama, S., Kamranpour, N., Fonarow, G., Hough, G. et al. 2003.

Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control

subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin

treatment. Circulation. 108(22): 2751-2756.

Arai, Y., Hirose, N. 2004. Aging and HDL metabolism in elderly people more than 100 years old. J

Atheroscler Thromb. 11(5): 246-252.

Page 20 of 37



Draft

21

Asleh, R., Blum, S., Kalet-Litman, S., Alshiek, J., Miller-Lotan, R., Asaf, R. et al. 2008. Correction of HDL

dysfunction in individuals with diabetes and the haptoglobin 2-2 genotype. Diabetes. 57(10): 2794-2800.

Asztalos, B. F., Schaefer, E. J., Horvath, K. V., Yamashita, S., Miller, M., Franceschini, G. et al. 2007. Role

of LCAT in HDL remodeling: investigation of LCAT deficiency states. J lipid Res. 48(3): 592-599.

Aviram, M., Billecke, S., Sorenson, R., Bisgaier, C., Newton, R., Rosenblat, M. et al. 1998. Paraoxonase

active site required for protection against LDL oxidation involves its free sulfhydryl group and is different

from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase

allozymes Q and R. Arterioscler Thromb Vasc Biol. 18(10): 1617-1624.

Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R. et al. 2000. Pomegranate

juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation:

studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr. 71(5): 1062-1076.

Baggio, G., Donazzan, S., Monti, D., Mari, D., Martini, S., Gabelli, C. et al. 1998. Lipoprotein(a) and

lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors. FASEB J. 12(6): 433-437.

Barbagallo, C. M., Averna, M. R., Frada, G., Noto, D., Cavera, G., Notarbartolo, A. 1998. Lipoprotein profile

and high-density lipoproteins: subfractions distribution in centenarians. Gerontology. 44(2): 106-110.

Barter, P. J., Caulfield, M., Eriksson, M., Grundy, S. M., Kastelein, J. J., Komajda, M. et al. 2007. Effects of

torcetrapib in patients at high risk for coronary events. N Engl J Med. 357(21): 2109-2122.

Berard, A. M., Foger, B., Remaley, A., Shamburek, R., Vaisman, B. L., Talley, G. et al. 1997. High plasma

HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-

cholesteryl acyltransferase. Nat Med. 3(7): 744-749.

Berard, A. M., Clerc, M., Brewer, B., Jr., Santamarina-Fojo, S. 2001. A normal rate of cellular cholesterol

removal can be mediated by plasma from a patient with familial lecithin-cholesterol acyltransferase (LCAT)

deficiency. Clin Chim Acta. 314(1-2): 131-139.

Page 21 of 37



Draft

22

Berrougui, H., Isabelle, M., Cloutier, M., Grenier, G., Khalil, A. 2007. Age-related impairment of HDL-

mediated cholesterol efflux. J Lipid Res. 48(2): 328-336.

Berrougui, H., Loued, S., Khalil, A. 2012. Purified human paraoxonase-1 interacts with plasma membrane

lipid rafts and mediates cholesterol efflux from macrophages. Free Radic Biol Med. 52(8): 1372-1381.

Berrougui, H., Ikhlef, S., Khalil, A. 2015. Extra Virgin Olive Oil Polyphenols Promote Cholesterol Efflux and

Improve HDL Functionality. Evid Based Complement Alternat Med. 2015: 208062.

Boekholdt, S. M., Thompson, J. F. 2003. Natural genetic variation as a tool in understanding the role of

CETP in lipid levels and disease. J Lipid Res. 44(6): 1080-1093.

Bonnefont-Rousselot, D., Khalil, A., Delattre, J., Jore, D., Gardes-Albert, M. 1997. Oxidation of human high-

density lipoproteins by .OH and .OH/O(.-)2 free radicals. Rad Res. 147(6): 721-728.

Bonnefont-Rousselot, D., Khalil, A., Gardes-Albert, M., Delattre, J. 1997. Reciprocal protection of LDL and

HDL oxidised by .OH free radicals in the presence of oxygen. FEBS Lett. 403(1): 70-74.

Bounafaa, A., Berrougui, H., Ikhlef, S., Essamadi, A., Nasser, B., Bennis, A. et al. 2014. Alteration of HDL

functionality and PON1 activities in acute coronary syndrome patients. Clin Biochem. 47(18): 318-325.

Brown, S. A., Hutchinson, R., Morrisett, J., Boerwinkle, E., Davis, C. E., Gotto, A. M., Jr. et al. 1993. Plasma

lipid, lipoprotein cholesterol, and apoprotein distributions in selected US communities. The Atherosclerosis

Risk in Communities (ARIC) Study. Arterioscler Thromb. 13(8): 1139-1158.

Brunham, L. R., Kruit, J. K., Iqbal, J., Fievet, C., Timmins, J. M., Pape, T. D. et al. 2006. Intestinal ABCA1

directly contributes to HDL biogenesis in vivo. J Clin Invest. 116(4): 1052-1062.

Brushia, R. J., Forte, T. M., Oda, M. N., La Du, B. N., Bielicki, J. K. 2001. Baculovirus-mediated expression

and purification of human serum paraoxonase 1A. J Lipid Res. 42(6): 951-958.

Bub, A., Barth, S. W., Watzl, B., Briviba, K., Rechkemmer, G. 2005. Paraoxonase 1 Q192R (PON1-192)

polymorphism is associated with reduced lipid peroxidation in healthy young men on a low-carotenoid diet

supplemented with tomato juice. Br J Nutr. 93(3): 291-297.

Page 22 of 37



Draft

23

Calabresi, L., Pisciotta, L., Costantin, A., Frigerio, I., Eberini, I., Alessandrini, P. et al. 2005. The molecular

basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and

biochemical findings in 13 unrelated Italian families. Arterioscler Thromb Vasc Biol. 25(9): 1972-1978.

Calabresi, L., Favari, E., Moleri, E., Adorni, M. P., Pedrelli, M., Costa, S. et al. 2009. Functional LCAT is not

required for macrophage cholesterol efflux to human serum. Atherosclerosis. 204(1): 141-146.

Calabresi, L., Simonelli, S., Gomaraschi, M., Franceschini, G. 2012. Genetic lecithin:cholesterol

acyltransferase deficiency and cardiovascular disease. Atherosclerosis. 222(2): 299-306.

Camps, J., Marsillach, J., Rull, A., Alonso-Villaverde, C., Joven, J. 2010. Interrelationships between

paraoxonase-1 and monocyte chemoattractant protein-1 in the regulation of hepatic inflammation. Adv Exp

Med Biol. 660: 5-18.

Cherki, M., Berrougui, H., Isabelle, M., Cloutier, M., Koumbadinga, G. A., Khalil, A. 2007. Effect of PON1

polymorphism on HDL antioxidant potential is blunted with aging. Exp Gerontol. 42(8): 815-824.

Cheung, B. M., Li, M., Ong, K. L., Wat, N. M., Tam, S., Pang, R. W. et al. 2009. High density lipoprotein-

cholesterol levels increase with age in American women but not in Hong Kong Chinese women. Clin

Endocrinol (Oxf). 70(4): 561-568.

Chroni, A., Liu, T., Gorshkova, I., Kan, H. Y., Uehara, Y., Von Eckardstein, A. et al. 2003. The central

helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino

acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein

formation in vivo. J Biol Chem. 278(9): 6719-6730.

Cohen, E., Aviram, M., Khatib, S., Artoul, F., Rabin, A., Mannheim, D. et al. 2014. Human carotid plaque

phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity, and enhances its uptake

by macrophage at the expense of its binding to HDL. Free Radic Biol Med. 76C: 14-24.

Connelly, P. W., Draganov, D., Maguire, G. F. 2005. Paraoxonase-1 does not reduce or modify oxidation of

phospholipids by peroxynitrite. Free Radic Biol Med. 38(2): 164-174.

Page 23 of 37



Draft

24

de Beer, M. C., Ji, A., Jahangiri, A., Vaughan, A. M., de Beer, F. C., van der Westhuyzen, D. R. et al. ATP

binding cassette G1-dependent cholesterol efflux during inflammation. J Lipid Res. 52(2): 345-353.

de Beer, M. C., Ji, A., Jahangiri, A., Vaughan, A. M., de Beer, F. C., van der Westhuyzen, D. R. et al. 2011.

ATP binding cassette G1-dependent cholesterol efflux during inflammation. J Lipid Res. 52(2): 345-353.

Elkhalil, L., Majd, Z., Bakir, R., Perez-Mendez, O., Castro, G., Poulain, P. et al. 1997. Fish-eye disease:

structural and in vivo metabolic abnormalities of high-density lipoproteins. Metabolism. 46(5): 474-483.

Esterbauer, H., Schmidt, R., Hayn, M. 1997. Relationships among oxidation of low-density lipoprotein,

antioxidant protection, and atherosclerosis. Adv Pharmacol. 38: 425-456.

Farid, A. S., Horii, Y. 2012. Modulation of paraoxonases during infectious diseases and its potential impact

on atherosclerosis. Lipids Health Dis. 11: 92.

Ferre, N., Marsillach, J., Camps, J., Mackness, B., Mackness, M., Riu, F. et al. 2006. Paraoxonase-1 is

associated with oxidative stress, fibrosis and FAS expression in chronic liver diseases. J Hepatol. 45(1): 51-

59.

Ferretti, G., Bacchetti, T. 2012. Effect of dietary lipids on paraoxonase-1 activity and gene expression. Nutr

Metab Cardiovasc Dis. 22(2): 88-94.

Frikke-Schmidt, R., Nordestgaard, B. G., Stene, M. C., Sethi, A. A., Remaley, A. T., Schnohr, P. et al. 2008.

Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels

and risk of ischemic heart disease. JAMA. 299(21): 2524-2532.

Frishman, W. H., Ooi, W. L., Derman, M. P., Eder, H. A., Gidez, L. I., Ben-Zeev, D. et al. 1992. Serum lipids

and lipoproteins in advanced age. Intraindividual changes. Ann Epidemiol. 2(1-2): 43-50.

Fuhrman, B., Gantman, A., Aviram, M. 2010. Paraoxonase 1 (PON1) deficiency in mice is associated with

reduced expression of macrophage SR-BI and consequently the loss of HDL cytoprotection against

apoptosis. Atherosclerosis. 211(1): 61-68.

Page 24 of 37



Draft

25

Garcia-Palmieri, M. R. 2006. Evidence based secondary prevention of coronary artery disease in the

elderly--2006. P R Health Sci J. 25(3): 229-239.

Gbandjaba, N. Y., Ghalim, N., Hassar, M., Berrougui, H., Labrazi, H., Taki, H. et al. 2012. Paraoxonase

activity in healthy, diabetic, and hemodialysis patients. Clin Biochem. 45(6): 470-474.

Glomset, J. A. 1968. The plasma lecithins:cholesterol acyltransferase reaction. J lipid Res. 9(2), 155-167.

Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B., Dawber, T. R. 1977. High density lipoprotein as

a protective factor against coronary heart disease. The Framingham Study. Am J Med. 62(5): 707-714.

Goyal, J., Wang, K., Liu, M., Subbaiah, P. V. 1997. Novel function of lecithin-cholesterol acyltransferase.

Hydrolysis of oxidized polar phospholipids generated during lipoprotein oxidation. J Biol Chem. 272(26):

16231-16239.

Gu, X., Huang, Y., Levison, B. S., Gerstenecker, G., DiDonato, A. J., Hazen, L. B. et al. 2016. Identification

of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction. J Biol Chem. 291(4):

1890-1904.

Haase, C. L., Tybjaerg-Hansen, A., Qayyum, A. A., Schou, J., Nordestgaard, B. G., Frikke-Schmidt, R.

2012. LCAT, HDL cholesterol and ischemic cardiovascular disease: a Mendelian randomization study of

HDL cholesterol in 54,500 individuals. J Clin Endocrinol Metab. 97(2): E248-256.

Haghpassand, M., Bourassa, P. A., Francone, O. L., Aiello, R. J. 2001. Monocyte/macrophage expression

of ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest. 108(9): 1315-1320.

Harel, M., Aharoni, A., Gaidukov, L., Brumshtein, B., Khersonsky, O., Meged, R. et al. 2004. Structure and

evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. Nat Struct Mol

Biol. 11(5): 412-419.

Heiss, G., Tamir, I., Davis, C. E., Tyroler, H. A., Rifkand, B. M., Schonfeld, G. et al. 1980. Lipoprotein-

cholesterol distributions in selected North American populations: the lipid research clinics program

prevalence study. Circulation. 61(2): 302-315.

Page 25 of 37



Draft

26

Helal, O., Berrougui, H., Loued, S., Khalil, A. 2013. Extra-virgin olive oil consumption improves the capacity

of HDL to mediate cholesterol efflux and increases ABCA1 and ABCG1 expression in human macrophages.

Br J Nutr. 109(10): 1844-1855.

Hiura, Y., Shen, C. S., Kokubo, Y., Okamura, T., Morisaki, T., Tomoike, H. et al. 2009. Identification of

genetic markers associated with high-density lipoprotein-cholesterol by genome-wide screening in a

Japanese population: the Suita study. Circ J. 73(6): 1119-1126.

Ikhlef, S., Berrougui, H., Kamtchueng Simo, O., Khalil, A. 2016. Paraoxonase 1-treated oxLDL promotes

cholesterol efflux from macrophages by stimulating the PPARgamma-LXRalpha-ABCA1 pathway. FEBS

Lett. 590: (11): 1614-1629.

Ikhlef, S., H, B., Khalil, A. 2017. Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol

efflux and reverse cholesterol transport. PLOS ONE. 12(3): e0173385

Inazu, A., Brown, M. L., Hesler, C. B., Agellon, L. B., Koizumi, J., Takata, K. et al. 1990. Increased high-

density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation. N Engl J

Med. 323(18): 1234-1238.

Ishigami, M., Yamashita, S., Sakai, N., Arai, T., Hirano, K., Hiraoka, H. et al. 1994. Large and cholesteryl

ester-rich high-density lipoproteins in cholesteryl ester transfer protein (CETP) deficiency can not protect

macrophages from cholesterol accumulation induced by acetylated low-density lipoproteins. J Biochem.

116(2): 257-262.

Jaouad, L., de Guise, C., Berrougui, H., Cloutier, M., Isabelle, M., Fulop, T. et al. 2006a. Age-related

decrease in high-density lipoproteins antioxidant activity is due to an alteration in the PON1's free sulfhydyl

groups. Atherosclerosis. 185(1): 191-200.

Jaouad, L., de Guise, C., Berrougui, H., Cloutier, M., Isabelle, M., Fulop, T. et al. 2006b. Age-related

decrease in high-density lipoproteins antioxidant activity is due to an alteration in the PON1's free sulfhydryl

groups. Atherosclerosis. 185(1): 191-200.

Page 26 of 37



Draft

27

Ji, A., Meyer, J. M., Cai, L., Akinmusire, A., de Beer, M. C., Webb, N. R. et al. 2011. Scavenger receptor

SR-BI in macrophage lipid metabolism. Atherosclerosis. 217(1): 106-112.

Johnson, W. J., Mahlberg, F. H., Rothblat, G. H., Phillips, M. C. 1991. Cholesterol transport between cells

and high-density lipoproteins. Biochim et biophys acta. 1085(3): 273-298.

Jonas, A. 2000. Lecithin cholesterol acyltransferase. Biochimica et biophysica acta. 1529: (1-3): 245-256.

Khalil, A., Wagner, J. R., Lacombe, G., Dangoisse, V., Fulop, T., Jr. 1996. Increased susceptibility of low-

density lipoprotein (LDL) to oxidation by gamma-radiolysis with age. FEBS Lett. 392(1): 45-48.

Khalil, A., Jay-Gerin, J. P., Fulop, T., Jr. 1998. Age-related increased susceptibility of high-density

lipoproteins (HDL) to in vitro oxidation induced by gamma-radiolysis of water. FEBS Lett. 435(2-3): 153-158.

Khera, A. V., Cuchel, M., de la Llera-Moya, M., Rodrigues, A., Burke, M. F., Jafri, K. et al. 2011. Cholesterol

efflux capacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med. 364(2): 127-135.

Kim, C. J., Kim, T. H., Ryu, W. S., Ryoo, U. H. 2000. Influence of menopause on high density lipoprotein-

cholesterol and lipids. J Korean Med Sci. 15(4): 380-386.

Koizumi, J., Mabuchi, H., Yoshimura, A., Michishita, I., Takeda, M., Itoh, H. et al. 1985. Deficiency of serum

cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia. Atherosclerosis. 58(1-

3): 175-186.

Lakatta, E. G. 2002. Age-associated cardiovascular changes in health: impact on cardiovascular disease in

older persons. Heart Fail Rev. 7(1), 29-49.

Lambert, G., Sakai, N., Vaisman, B. L., Neufeld, E. B., Marteyn, B., Chan, C. C. et al. 2001. Analysis of

glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice. J Biol Chem.

276(18): 15090-15098.

Landi, F., Russo, A., Pahor, M., Capoluongo, E., Liperoti, R., Cesari, M. et al. 2007. Serum High-Density

Lipoprotein Cholesterol Levels and Mortality in Frail, Community-Living Elderly. Gerontology. 54(2): 71-8.

Page 27 of 37



Draft

28

Loued, S., Isabelle, M., Berrougui, H., Khalil, A. 2012. The anti-inflammatory effect of paraoxonase 1

against oxidized lipids depends on its association with high density lipoproteins. Life Sciences. 90(1-2): 82-

88.

Loued, S., Berrougui, H., Componova, P., Ikhlef, S., Helal, O., Khalil, A. 2013. Extra-virgin olive oil

consumption reduces the age-related decrease in HDL and paraoxonase 1 anti-inflammatory activities. Br J

Nutr. 110(7): 1272-1284.

Mackness, B., Hunt, R., Durrington, P. N., Mackness, M. I. 1997. Increased immunolocalization of

paraoxonase, clusterin, and apolipoprotein A-I in the human artery wall with the progression of

atherosclerosis. Arterioscler Thromb Vasc Biol. 17(7): 1233-1238.

Mackness, B., Durrington, P. N., Mackness, M. I. 1998. Human serum paraoxonase. Gen Pharmacol. 31(3):

329-336.

Mackness, M. I., Arrol, S., Durrington, P. N. 1991. Paraoxonase prevents accumulation of lipoperoxides in

low-density lipoprotein. FEBS Lett. 286(1-2): 152-154.

Mehlum, A., Muri, M., Hagve, T. A., Solberg, L. A., Prydz, H. 1997. Mice overexpressing human lecithin:

cholesterol acyltransferase are not protected against diet-induced atherosclerosis. APMIS. 105(11): 861-

868.

Milochevitch, C., Khalil, A. 2001. Study of the paraoxonase and platelet-activating factor acetylhydrolase

activities with aging. Prostaglandins Leukot Essent Fatty Acids. 65(5-6): 241-246.

Morgantini, C., Natali, A., Boldrini, B., Imaizumi, S., Navab, M., Fogelman, A. M. et al. 2011. Anti-

inflammatory and Antioxidant Properties of HDLs Are Impaired in Type 2 Diabetes. Diabetes. 60(10): 2617-

2623.

Navab, M., Hama-Levy, S., Van Lenten, B. J., Fonarow, G. C., Cardinez, C. J., Castellani, L. W. et al. 1997.

Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio. J Clin Invest. 99(8): 2005-

2019.

Page 28 of 37



Draft

29

Ng, D. S., Chu, T., Esposito, B., Hui, P., Connelly, P. W., Gross, P. L. 2008. Paraoxonase-1 deficiency in

mice predisposes to vascular inflammation, oxidative stress, and thrombogenicity in the absence of

hyperlipidemia. Cardiovasc Pathol. 17(4): 226-232.

Nikkila, M., Heikkinen, J. 1990. High-density lipoprotein cholesterol and longevity. Age Ageing. 19(2): 119-

124.

Nikkila, M., Pitkajarvi, T., Koivula, T., Heikkinen, J. 1991. Elevated high-density-lipoprotein cholesterol and

normal triglycerides as markers of longevity. Klin Wochenschr. 69(17): 780-785.

Nishimoto, T., Pellizzon, M. A., Aihara, M., Stylianou, I. M., Billheimer, J. T., Rothblat, G. et al. 2009. Fish oil

promotes macrophage reverse cholesterol transport in mice. Arterioscler Thromb Vasc Biol. 29(10): 1502-

1508.

Ohta, T., Nakamura, R., Takata, K., Saito, Y., Yamashita, S., Horiuchi, S. et al. 1995. Structural and

functional differences of subspecies of apoA-I-containing lipoprotein in patients with plasma cholesteryl

ester transfer protein deficiency. J Lipid Res. 36(4): 696-704.

Perez-Jimenez, F., Lopez-Miranda, J. 2005. [University of Health Sciences: a blow of hope]. Med Clin

(Barc). 125(12): 477.

Rader, D. J. 2007. Illuminating HDL--is it still a viable therapeutic target? The New England journal of

medicine. 357(21): 2180-2183.

Reaven, P. D., Napoli, C., Merat, S., Witztumc, J. L. 1999. Lipoprotein modification and atherosclerosis in

aging. Exp Gerontol. 34(4): 527-537.

Remaley, A. T., Stonik, J. A., Demosky, S. J., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G. et al.

2001. Apolipoprotein specificity for lipid efflux by the human ABCAI transporter. Biochem Biophys Res

Commun. 280(3): 818-823.

Page 29 of 37



Draft

30

Roma, P., Gregg, R. E., Meng, M. S., Ronan, R., Zech, L. A., Franceschini, G. et al. 1993. In vivo

metabolism of a mutant form of apolipoprotein A-I, apo A-IMilano, associated with familial

hypoalphalipoproteinemia. J Clin Invest. 91(4): 1445-1452.

Romanchik, J. E., Morel, D. W., Harrison, E. H. 1995. Distributions of carotenoids and alpha-tocopherol

among lipoproteins do not change when human plasma is incubated in vitro. J Nutr. 125(10): 2610-2617.

Rosenblat, M., Gaidukov, L., Khersonsky, O., Vaya, J., Oren, R., Tawfik, D. S. et al. 2006. The catalytic

histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-

mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux. J

Biol Chem. 281(11): 7657-7665.

Ross, R. 1999. Atherosclerosis--an inflammatory disease. N Engl J Med. 340(2): 115-126.

Rozek, L. S., Hatsukami, T. S., Richter, R. J., Ranchalis, J., Nakayama, K., McKinstry, L. A. et al. 2005. The

correlation of paraoxonase (PON1) activity with lipid and lipoprotein levels differs with vascular disease

status. J Lipid Res. 46(9): 1888-1895.

Rozenberg, O., Shih, D. M., Aviram, M. 2003. Human serum paraoxonase 1 decreases macrophage

cholesterol biosynthesis: possible role for its phospholipase-A2-like activity and lysophosphatidylcholine

formation. Arterioscler Thromb Vasc Biol. 23(3): 461-467.

Schaefer, E. J., Moussa, P. B., Wilson, P. W., McGee, D., Dallal, G., Castelli, W. P. 1989. Plasma

lipoproteins in healthy octogenarians: lack of reduced high density lipoprotein cholesterol levels: results

from the Framingham Heart Study. Metabolism: clinical and experimental. 38(4): 293-296.

Schaefer, E. J., Lamon-Fava, S., Cohn, S. D., Schaefer, M. M., Ordovas, J. M., Castelli, W. P. et al. 1994.

Effects of age, gender, and menopausal status on plasma low density lipoprotein cholesterol and

apolipoprotein B levels in the Framingham Offspring Study. J Lipid Res. 35: (5): 779-792.

Page 30 of 37



Draft

31

Schaefer, E. J., Lamon-Fava, S., Johnson, S., Ordovas, J. M., Schaefer, M. M., Castelli, W. P. et al. 1994.

Effects of gender and menopausal status on the association of apolipoprotein E phenotype with plasma

lipoprotein levels. Results from the Framingham Offspring Study. Arterioscler Thromb. 14(7): 1105-1113.

Schilling, T., Eder, C. 2009. Lysophosphatidylcholine- and MCP-1-induced chemotaxis of monocytes

requires potassium channel activity. Pflugers Arch. 459(1): 71-77.

Seres, I., Freyss-Beguin, M., Mohacsi, A., Kozlovsky, B., Simon, J., Devynck, M. A. et al. 1996. Alteration of

lymphocyte membrane phospholipids and intracellular free calcium concentrations in hyperlipidemic

subjects. Atherosclerosis. 121(2): 175-183.

Seres, I., Paragh, G., Deschene, E., Fulop, T., Jr., Khalil, A. 2004. Study of factors influencing the

decreased HDL associated PON1 activity with aging. Exp Gerontol. 39(1): 59-66.

Shih, D. M., Gu, L., Xia, Y. R., Navab, M., Li, W. F., Hama, S. et al. 1998. Mice lacking serum paraoxonase

are susceptible to organophosphate toxicity and atherosclerosis. Nature. 394: (6690), 284-287.

Stock, J. 2011. Importance of HDL functionality to cardiovascular risk. Atherosclerosis. 218(1): 19-20.

Subbaiah, P. V., Liu, M. 1996. Comparative studies on the substrate specificity of lecithin:cholesterol

acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates. J

Lipid Res. 37(1): 113-122.

Sviridov, D., Nestel, P. 2002. Dynamics of reverse cholesterol transport: protection against atherosclerosis.

Atherosclerosis. 161(2): 245-254.

Sviridov, D., Mukhamedova, N., Remaley, A. T., Chin-Dusting, J., Nestel, P. 2008. Antiatherogenic

functionality of high density lipoprotein: how much versus how good. J Atheroscler Thromb. 15(2): 52-62.

Tall, A. R. 1993. Plasma cholesteryl ester transfer protein. J Lipid Res. 34(8): 1255-1274.

Tanigawa, H., Billheimer, J. T., Tohyama, J., Fuki, I. V., Ng, D. S., Rothblat, G. H. et al. 2009. Lecithin:

cholesterol acyltransferase expression has minimal effects on macrophage reverse cholesterol transport in

vivo. Circulation. 120(2): 160-169.

Page 31 of 37



Draft

32

Teiber, J. F., Draganov, D. I., La Du, B. N. 2004. Purified human serum PON1 does not protect LDL against

oxidation in the in vitro assays initiated with copper or AAPH. J Lipid Res. 45(12): 2260-2268.

Timmins, J. M., Lee, J. Y., Boudyguina, E., Kluckman, K. D., Brunham, L. R., Mulya, A. et al. 2005.

Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney

hypercatabolism of apoA-I. J Clin Invest. 115(5): 1333-1342.

Toth, P. P. 2005. High-density lipoprotein as a therapeutic target: clinical evidence and treatment strategies.

Am J Cardiol. 96(9A): 50K-58K.

Treguier, M., Briand, F., Boubacar, A., Andre, A., Magot, T., Nguyen, P. et al. 2011. Diet-induced

dyslipidemia impairs reverse cholesterol transport in hamsters. Eur J Clin Invest. 41(9): 921-928.

Tsompanidi, E. M., Brinkmeier, M. S., Fotiadou, E. H., Giakoumi, S. M., Kypreos, K. E. 2010. HDL

biogenesis and functions: role of HDL quality and quantity in atherosclerosis. Atherosclerosis. 208(1): 3-9.

Tward, A., Xia, Y. R., Wang, X. P., Shi, Y. S., Park, C., Castellani, L. W. et al. 2002. Decreased

atherosclerotic lesion formation in human serum paraoxonase transgenic mice. Circulation. 106(4): 484-

490.

Wallace, R. B., Colsher, P. L. 1992. Blood lipid distributions in older persons. Prevalence and correlates of

hyperlipidemia. Ann Epidemiol. 2(1-2): 15-21.

Wang, N., Silver, D. L., Costet, P., Tall, A. R. 2000. Specific binding of ApoA-I, enhanced cholesterol efflux,

and altered plasma membrane morphology in cells expressing ABC1. The Journal of biological chemistry.

275(42): 33053-33058.

Wang, X., Rader, D. J. 2007. Molecular regulation of macrophage reverse cholesterol transport. Curr Opin

Cardiol. 22(4): 368-372.

Wilson, P. W., Anderson, K. M., Harris, T., Kannel, W. B., Castelli, W. P. 1994. Determinants of change in

total cholesterol and HDL-C with age: the Framingham Study. J Gerontol. 49(6): M252-257.

Page 32 of 37



Draft

33

Xie, Q., Zhao, S. 2011. [Effect of apolipoprotein A-I mimetic peptides on cholesterol efflux in RAW264.7

cells]. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 36(1): 51-57.

Yamashita, S., Sprecher, D. L., Sakai, N., Matsuzawa, Y., Tarui, S., Hui, D. Y. 1990. Accumulation of

apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma

cholesteryl ester transfer protein deficiency. J Clin Invest. 86(3): 688-695.

Yancey, P. G., Bortnick, A. E., Kellner-Weibel, G., de la Llera-Moya, M., Phillips, M. C., Rothblat, G. H.

2003. Importance of different pathways of cellular cholesterol efflux. Arterioscler Thromb Vasc Biol. 23(5):

712-719.

Yvan-Charvet, L., Matsuura, F., Wang, N., Bamberger, M. J., Nguyen, T., Rinninger, F. et al. 2007.

Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol

efflux to HDL. Arterioscler Thromb Vasc Biol. 27(5): 1132-1138.

Yvan-Charvet, L., Wang, N., Tall, A. R. 2010. Role of HDL, ABCA1, and ABCG1 transporters in cholesterol

efflux and immune responses. Arterioscler Thromb Vasc Biol. 30(2): 139-143.

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Fig. 1: Summary of the anti-atherogenic activities of HDL

Fig. 2. Assessment of the anti-inflammatory effect of PON1 on Ea.hy926 endothelial cells and the

interaction of PON1 with LCAT

Oxidized HDL (oxHDL) were generated by exposing HDL to oxygen free radicals produced by the gamma

radiolysis of water. The oxHDL were pre-incubated with PON1 or with PON1 and LCAT for 4 h. They were

then incubated with Ea.hy926 endothelial cells. PON1 was used at a concentration of 50 µg/mL and LCAT

at a concentration of 5 µg/mL. Data are expressed as means ± SEM. Mean values were compared using

one way ANOVA followed by Bonferroni's multiple comparison post test. *** p < 0.001 with respect to

oxHDL alone.

Fig. 3. PON1 overexpression in mice increases the capacity plasma to mediate cholesterol efflux

from macrophages

J774 macrophages (1 x 106 cells/mL) were loaded with [3H]-cholesterol (2 µCi/mL) for 24 h. Plasma

samples obtained from WT and PON1-Tg mice were treated with polyethylene glycol to precipitate ApoB-

100-containing particles and were then incubated with J774 macrophages for 24 h. Cholesterol efflux

(radiolabeled cholesterol released from cells) was calculated using the following formula: (radioactivity

(cpm) in supernatant/radioactivity (cpm) in cells + medium) × 100. Data are expressed as means ± SEM.

*p<0.03 and ***p<0.003.

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Figure 1

.

Antioxidant

Reverse cholesterol transport

An#-‐thrombo#c

Regula#on of endothelial func#on

Immuno-‐modula#on

An#-‐inflammatory

An#-‐apopto#c

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Figure 2

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