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Cardiovascular Research 2003 57(1):225-231; doi:10.1016/S0008-6363(02)00659-4
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Copyright © 2003, European Society of Cardiology

Paraoxonase-1 reduces monocyte chemotaxis and adhesion to endothelial cells due to oxidation of palmitoyl, linoleoyl glycerophosphorylcholine

Zakaria Ahmedh, Saeid Babaeic, Graham F. Maguirea, Dragomir Draganovg, Arnis Kuksisd, Bert N. La Dug and Philip W. Connellya,b,e,f,*

aJ. Alick Little Lipid Research Laboratory, St. Michael's Hospital, Room 1004 WA, 38 Shuter Street, Toronto, Ont., Canada M5B 1A6
bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada
cTerrence Donnelly Vascular Biology Laboratory, St. Michael's Hospital, Toronto, Ont., Canada
dBanting and Best Department of Medical Research, University of Toronto, Toronto, Ont., Canada
eDepartment of Biochemistry, University of Toronto, Toronto, Ont., Canada
fDepartment of Medicine, University of Toronto, Toronto, Ont., Canada
gDepartment of Pharmacology, University of Michigan, Ann Arbor, MI, USA
hDepartment of Pathology and Laboratory Medicine, University of Louisville Medical Center, Louisville, KY 40202, USA

* Corresponding author. Present address: J. Alick Little Lipid Research Laboratory, St. Michael's Hospital, Room 1004 WA, 38 Shuter Street, Toronto, Ont., Canada M5B 1A6. Tel.: +1-416-864-6023; fax: +1-416-864-5870. connellyp{at}smh.toronto.on.ca

Received 27 June 2002; accepted 30 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: High-density lipoprotein (HDL) is postulated to protect against the development of atherosclerosis, in part, by inhibiting the oxidation of low density lipoprotein (LDL) in the sub-endothelial space and thus inhibiting activation of the endothelium. The HDL-associated enzyme, paraoxonase-1, is proposed to be a major protective factor. However, HDL is also prone to oxidation when exposed to peroxynitrite and may therefore, once oxidized, have properties similar to oxidized LDL. Methods and results: We exposed human HDL to the peroxynitrite donor 3-morpholinosydnonimine and incubated oxidized HDL with human umbilical vein endothelial cells (HUVECs). Oxidized HDL increased monocyte binding (P<0.001) and enhanced chemotaxis (P<0.001). The major oxidized phospholipids were 1-palmitoyl (stearoyl)-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, derived from linoleate-containing phosphatidylcholines, and 1-palmitoyl(stearoyl)-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine, derived from arachidonate-containing phosphatidylcholines. Incubation of HUVECs with synthetically prepared 1-palmitoyl-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, or 1-palmitoyl-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine increased binding of monocytes (P<0.001) and chemotaxis (P<0.001). Purified paraoxonase-1 reduced monocyte adhesion and chemotaxis (P<0.001). Conclusions: (i) HDL can be a source of oxidatively-derived bioactive phospholipids; (ii) the fragmented phospholipids with a 9-carbon aldehyde or acid are as effective as a 5-carbon aldehyde or acid at inducing monocyte adhesion and chemotaxis; and (iii) paraoxonase-1 is effective at reducing the activity of these phospholipid oxidation products.

KEYWORDS apoA-I, apolipoprotein A-I; HDL, high density lipoproteins; HPF, high powered field; HUVECs, human umbilical vein endothelial cells; LC–ESI-MS, liquid chromatography–electrospray ionization-mass spectrometry; LDL, low density lipoproteins; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PAzPC, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; PGPC, 1-palmitoyl-2-(glutaroyl)-sn-glycero-phosphocholine; PLPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; PON-1, paraoxonase-1; PONPC, 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine; POVPC, 1-palmitoyl-2-[5-oxo]valeroyl-sn-glycero-phosphocholine; SGPC, 1-stearoyl-2-(glutaroyl)-sn-glycero-phosphocholine; SIN-1, 3-morpholinosydnonimine; SOVPC, 1-stearoyl-2-[5-oxo]valeroyl-sn-glycero-phosphocholine


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The cellular events in early atherosclerosis and in mature atherosclerotic plaques are characterized by activation of the vascular endothelium and increased monocyte binding [1,2]. These events are initiated by inflammatory cytokines that induce the synthesis of adhesion molecules and subsequently increase monocyte adhesion and chemotaxis [3]. Minimally oxidized low density lipoprotein (LDL) has been shown to induce endothelial changes via synthesis and secretion of the chemoattractant, monocyte chemoattractant protein-1, and expression of vascular cell adhesion molecule-1 [4]. Oxidized phospholipids in oxidized LDL induce expression of endothelial growth factors [5], and increase monocyte chemotaxis and adherence to endothelial cells [6–8]. Oxidation products of arachidonate-containing phosphatidylcholines (PC), identified as 1-palmitoyl(stearoyl)-2-[5-oxo]valeroyl-sn-glycero-phosphocholine (P(S)OVPC), 1-palmitoyl(stearoyl)-2-(glutaroyl)-sn-glycero-phosphocholine (P(S)GPC) and epoxyisoprostane PC are involved in these changes [9]. Therefore, oxidized LDL has been proposed to promote atherogenesis, at least in part, via the oxidation products of arachidonate-containing PC. The effect of oxidation products, generated from linoleate-containing PC of LDL, or PC oxidation in high density lipoprotein (HDL), has received little attention. Oxidation products of linoleate-containing PC have been shown to have biological activity [10], but the identity of the product(s) is unknown.

Epidemiological studies have shown a strong inverse correlation between the concentration of plasma HDL and the incidence of cardiovascular disease [11]. There is significant evidence that HDL protects against cardiovascular disease by a mechanism distinct from reverse cholesterol transport [12]. Hypercholesterolemia can result in the inflammatory activation of the vessel wall [3]. In vitro, HDL inhibits the effect of the oxidized phospholipids that accumulate in mildly oxidized LDL and subsequently prevents activation of the endothelium [12–15]. The ability of HDL to infiltrate more readily into the arterial wall and atherosclerotic plaques, is postulated to be critical to the protection of the endothelium against oxidized LDL. The HDL proteins apolipoprotein A-I (apoA-I) [16], platelet activating factor acetyl hydrolase and paraoxonase-1 (PON-1), are thought to be mechanistically important in this process [17]. Low HDL or HDL isolated from subjects with cardiovascular disease does not protect LDL against oxidation and does not protect the endothelium in vitro [18]. Exposure of HDL to oxidants may also be important. We have shown that oxidation of HDL with peroxynitrite results in the production of a range of PC oxidation products derived from arachidonate and linoleate-containing PCs. HDL-associated PON-1 may protect against the bioactivity of these products by hydrolyzing them to lysoPC [19]. HDL is rich in unsaturated phospholipids and therefore, is sensitive to oxidation [20]. Oxidation is thought to impair the ability of HDL to efflux cellular cholesterol [21] and impair the ability of HDL to protect LDL against oxidation [22]. However, the effect of oxidized phospholipids that accumulate in oxidized HDL on the endothelium is not known.

In this study we compared the effect of oxidized HDL and specific arachidonate and linoleate-derived oxidized PCs on human umbilical vein endothelial cells (HUVECs). We report that, in contrast to native HDL, HDL oxidized by exposure to 3-morpholinosydnonimine (SIN-1) increases monocyte binding to HUVECs and enhances monocyte chemotaxis. We demonstrate that oxidized PCs containing aldehydes and carboxylic acids, produced from both arachidonoyl- and linoleoyl PCs, activated HUVECs, while PON-1 reduced this activity.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) were obtained from Avanti-Polar Lipids (Alabaster, USA). Dipentadecanoyl-sn-glycero-3-phosphocholine, 3-morpholinosydnonimine (SIN-1), diethylenetriamine pentacetic acid, dimethylsulphoxide, and lipoprotein deficient serum were purchased from Sigma (St. Louis, MO). Choline phospholipids were measured using the choline phospholipid enzymatic kit from Boehringer Mannheim. All solvents used in liquid chromatography–mass spectrometry (LC–MS) were HPLC grade. Other solvents and chemicals were of reagent grade, provided by local suppliers. PON-1 (type Q192) was purified from outdated human plasma as described [23].

2.2 Isolation and oxidation of lipoproteins
LDL and HDL were isolated from serum, taken from subjects fasted for 12–14 h, by ultracentrifugation between densities 1.019–1.063 and 1.063–1.21 g/ml as described [24]. HDL protein concentration was measured with the Lowry method [25], and PC was measured with a choline-based assay (Boehringer Mannheim). Oxidation of HDL by SIN-1 was stopped by the addition of 100 µM butylated hydroxytoluene [26]. Lipid-soluble oxidation products were extracted with chloroform/methanol 2:1 (v/v) and analyzed by liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) as described [23].

HDL and oxidized HDL phospholipids were separated from total lipid extracts by solid phase extraction using a silica Sep-Pak cartridge (Waters, MA, USA) pre-washed with hexane/ether (98:2, v/v). Phospholipids were eluted after first removing neutral lipids (cholesterol ester and triglycerides) as described [27]. Separated phospholipids were monitored by normal phase LC–ESI-MS as described [23].

2.3 Preparation of phosphatidylcholine core aldehydes and carboxylates and separation of phospholipids
1-Palmitoyl-2(5-oxo)valeroyl-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) or 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine (PONPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PAzPC) were prepared in the laboratory by reductive ozonization of PAPC and PLPC, respectively, as described [28]. Liposomes of oxidized phospholipids were prepared by bath sonication as described previously [23].

2.4 Cell culture
THP-1, a human monocyte cell line, was obtained from the American Type Culture Collection (TIB 202) and propagated in RPMI 1640, at a density of 1x106 cells/ml supplemented with 10% fetal calf serum, containing penicillin (100 U/ml) and streptomycin (100 µg/ml), at 37 °C in 5% CO2. The cells were then washed extensively with serum-free RPMI 1640 medium, diluted to the appropriate density, and used in experiments as indicated. Cell viability exceeded 90%, as determined by trypan blue exclusion [29].

HUVECs were purchased from Clontech (San Diego, CA) and cultured as previously described [30]. Cells were cultured on gelatin-coated culture flasks, in F-12 medium supplemented with 15% fetal calf serum (Gibco BRL), containing 20 mmol/l HEPES, 2 mmol/l glutamine, 1 mmol/l sodium pyruvate, non-essential amino acids, penicillin, streptomycin, 50 µg/ml endothelial growth supplement (Collaborative Research), and 50 µg/ml heparin (Sigma). Confluent preparations of passage levels of 2, 3, or 4 were washed with Hanks balanced salt solution, trypsinized, resuspended in complete media (as described above) and plated onto 24-well gelatin-coated plates at a density of 1.5x106 cells/ml (1 ml/well). Cells were treated overnight with media containing 0.8 mg/ml lipoprotein-deficient serum. All incubations were performed in lipoprotein-deficient serum to minimize basal HUVEC activation. Agents to be investigated included: native LDL, oxidized LDL, native HDL, oxidized HDL, native and oxidized phospholipids, separated from oxidized HDL, and the PC core aldehydes and acids, POVPC and PGPC, PONPC and PAzPC, that were prepared synthetically. These were also incubated in the presence of purified PON-1.

2.5 Monocyte chemotaxis assay
Assays were performed in chemotaxis chambers (Corning-Costar) with a polycarbonate filter (Poretics, Livermore, CA) of 5-µm pore size placed in 24-well tissue culture plates as previously described with minor modifications [30]. The lower compartments were filled with 250 µl of the conditioned media from the oxidation experiments or other conditions as described above. Next, ~45 000 THP-1 cells, suspended in 0.5 ml of serum-free medium, were placed in the upper chamber of the plates and incubated for 5 h at 37 °C in a 5% CO2 incubator [10]. At the end of the incubation period, the filters were fixed and stained with the Diff-Quick staining kit (VWR, Ont., Canada). The results are expressed as the number of migrated cells counted by light microscopy per high power field (HPF, 100x). The results of at least four to six wells were averaged for each experiment.

2.6 Monocyte adhesion assay
Native HDL, oxidized HDL, oxidized HDL phospholipids, and synthetically prepared PC core aldehydes and acids were incubated with confluent HUVECs cultured in 24-well plates. Cells were then rinsed twice with fresh medium, and the binding of THP-1 monocytes to HUVECs was performed as described [10,31]. Monocytes were added to the HUVECs at 45 000 cells/well, and the plates were incubated for 20 min at 37 °C. The suspension was removed, and the cells were vigorously washed (at least three times) to remove all but the firmly adherent monocytes. The number of adherent THP-1 cells was determined in four high power fields per well and the results of three to four separate wells were averaged for each experiment.

2.7 Liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS)
Lipid-soluble HDL oxidation products were extracted and phospholipid classes were determined by relative retention time and molecular mass, using a PC standard. Analysis was performed using a normal phase silica column (2.1x25 mm, Supelco, Bellefonte, PA), in a Hewlett-Packard model 1050 liquid chromatograph (LC), connected to a Hewlett-Packard model 5989A quadrupole mass spectrometer (MS), equipped with a nebulizer-assisted electrospray ionization (ESI) interface (HP 59987A). Determination and analysis of phospholipids and oxidized phospholipids were performed as previously described [23].

2.8 Statistical analysis
Because cell number follows a Poisson distribution, counts were first transformed as the square root of the counts plus 0.5, to better approximate a Gaussian distribution. Transformed values were analysed by one-way analysis of variance. Duncan's and Tukey's post-test were used to examine statistical differences for all a posteriori comparisons. All statistical analyses were performed using Graphpad Prism (San Diego, CA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Formation of oxidatively fragmented PC during exposure of HDL to SIN-1
Exposure of HDL to SIN-1 significantly increases formation of oxidatively fragmented PC compared to polyoxygenated PC derivatives [23]. The oxidized phospholipids are the same as the bioactive phospholipids formed in oxidized LDL. We observed that the major products of PC oxidation were derived from arachidonoyl and linoleoyl containing species. These were the 5-carbon aldehyde and acid derived from arachidonate, in combination with palmitate or stearate, palmitoyl (or stearoyl) oxovaleroyl PC (P(S)OVPC) and palmitoyl(or stearoyl)glutaroyl PC (P(S)GPC), and the 9-carbon aldehyde and acid derived from linoleate, palmitoyl(or stearoyl)oxononanoyl PC (P(S)ONPC) and palmitoyl(or stearoyl)azelaoyl PC (P(S)AzPC). Characterization of these products was reported previously [23]. The formation of linoleate-derived oxidized PCs was more than double compared to that formed by arachidonate-derived PCs (P=0.028) (Fig. 1).


Figure 1
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  		Fig. 1 Phosphatidylcholine core aldehydes and their corresponding carboxylates in native HDL (open bar) and HDL exposed to SIN-1 for 6 h (closed bar). Determination of the oxidation products and details of LC–ESI-MS are described in the Methods section. The results represent the mean±S.D. of three independent experiments. Results of a one-tailed t-test with Welch's correction for unequal variance for 6 vs. 0 h: POVPC, P=0.006; PGPC, P=0.09; PONPC, P=0.013; PAzPC, P=0.028. *P<0.05.

 
3.2 Effect of oxidized HDL and oxidized phospholipids on endothelial cells
We incubated native LDL, HDL, oxidized LDL, oxidized HDL and native and oxidized HDL phospholipids with HUVECs at 37 °C for 4 h, removed the media, and then added THP-1 cells to study monocyte adhesion. TNF-{alpha} treatment of HUVECs was used as a positive control for the activation of monocyte adhesion to HUVECs. Oxidized LDL, oxidized HDL and the phospholipids from oxidized HDL increased monocyte adhesion compared to control (Fig. 2, panel A) (P<0.001). Oxidized LDL increased adhesion significantly more than either oxidized HDL or the phospholipids from oxidized HDL (Fig. 2, panel A) (P<0.001). There was a modest, but statistically significant, increase in monocyte adhesion in the presence of native LDL (P<0.05) and native HDL (P<0.01). Next, we used the conditioned medium from the HUVEC experiments to study monocyte chemotaxis. Oxidized LDL, oxidized HDL and the phospholipids from oxidized HDL increased chemotaxis compared to control (Fig. 2, panel B) (P<0.001). Native LDL, HDL and HDL phospholipid preparations had a modest, but statistically significant effect, increasing THP-1 cell chemotaxis compared with the control (Fig. 2, panel B) (P<0.01). In contrast to the effects on adhesion, oxidized HDL was equally effective compared to oxidized LDL in the stimulation of chemotaxis.


Figure 2
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  		Fig. 2 Stimulation of monocyte adhesion (panel A) and chemotaxis (panel B) by oxidized HDL. Monocyte adhesion and chemotaxis assays were performed as described in the Methods section. The mean±S.D. values from three different experiments are shown. **P<0.001, *P<0.01, treatment compared to control condition by Dunnett's multiple comparison test. The amounts used were: 50 µg HDL or oxidized HDL protein (24 µg phospholipid), 50 µg LDL protein (20 µg phospholipid), or 20 µg phospholipid isolated from HDL or oxidized HDL. HPF, high-powered field.

 
Liposomes were made consisting of 10 µg/ml PONPC and 2 µg/ml PAzPC (referred to as oxidized PLPC) or 6 µg/ml POVPC and 1 µg/ml PGPC (referred to as oxidized PAPC), to mimic the concentration of oxidized phospholipid observed in oxidized HDL. Oxidized PLPC increased monocyte adhesion (Fig. 3, panel A) (P<0.001) and chemotaxis (Fig. 3, panel B) (P<0.001). This effect was similar to the effect of oxidized PAPC on THP-1 cell adhesion (Fig. 3, panel A) (P<0.001) and chemotaxis (Fig. 3, panel B) (P<0.001).


Figure 3
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  		Fig. 3 The effect of phosphatidylcholine core aldehydes and carboxylates on monocyte adhesion (panel A) and chemotaxis (panel B) in the absence and presence of purified PON-1. Adhesion assays (panel A) were performed after 20-min incubation with HUVEC at 37 °C as described in the Methods section. The number of adherent THP-1 cells was determined in five high-power fields per well and expressed as cells per high-powered field. Chemotaxis assays (panel B) were performed after a 4-h incubation with HUVECs at 37 °C as described in the Methods section. The mean±S.D. for three separate experiments are shown for both assays. **P<0.001. Liposomes were used at concentrations of 10 µg/ml PONPC/2 µg/ml PAzPC (referred to in the figure as PONPC) or 6 µg/ml POVPC/1 µg/ml PGPC (referred to in the figure as POVPC). These concentrations were equivalent to the concentration of oxidized phosphatidycholines present when oxidized HDL was used in Fig. 2. Abbreviations as in Fig. 2.

 
We then tested the effect of purified PON-1 (30 arylesterase U) on the stimulation of adhesion and chemotaxis by oxidized PCs. PON-1 resulted in a significant reduction in monocyte adhesion due to oxidized PLPC (Fig. 3, panel A) (P<0.001), and significantly reduced monocyte adhesion due to oxidized PAPC (Fig. 3, panel A) (P<0.001). PON-1 significantly reduced chemotaxis due to both oxidized PLPC and oxidized PAPC (Fig. 3, panel B) (P<0.001).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrated that exposure of HDL to peroxynitrite increased monocyte adhesion and chemotaxis. This activity could be attributed to the formation of oxidized PCs. Previous reports have shown that the increased formation of oxidatively fragmented PCs in minimally oxidized LDL was paralleled with increased monocyte adhesion and chemotaxis to human aortic endothelial cells [16,32]. Indeed, oxidized phospholipids constitute the major portion of oxidized lipoprotein bioactivity formed in each class of oxidized apoB-containing lipoprotein [32].

Analysis of oxidized phospholipids in HDL showed the major species were PC core aldehydes and core acids, generated from linoleate and arachidonate derivatives of phosphatidylcholine. These diacyl species are distinct in structure and function from the alkyl, acyl species identified by Davies et al. [33] as a ligand for PPAR{gamma}. The concentration of PC linoleate in lipoproteins is greater than that of PC arachidonate; therefore during oxidation of HDL, the formation of PONPC and PAzPC were more than two-fold higher compared to the formation of POVPC and PGPC.

The effect of oxidized LDL to induce expression of adhesion molecules by human aortic endothelial cells has been investigated [32,34]. These activities have been attributed in major part to the fragmented phospholipids POVPC and PGPC [34]. They are short fatty chain derivatives with different chemical groups at the sn-2 position, but their activity mimics platelet activating factor. Their actions have been shown to be blocked by platelet activating factor-receptor antagonists [14,35]. Lee et al. [10] demonstrated that oxidation of liposomes enriched with linoleate PC generated oxidized products capable of stimulating monocyte chemotaxis and adhesion. Interestingly, without knowledge of the nature of these products, it was shown that their function was also inhibited by a platelet activating factor-receptor antagonist [10]. The formation of oxidation products of linoleate PC in oxidized LDL has been overlooked and subsequently their effect on the endothelium was not known. In our study, the core aldehyde and acid derived from PLPC stimulated chemotaxis as effectively as the core aldehyde and acid derived from PAPC, when used at levels similar to those produced during HDL oxidation. Our observations provide a mechanism to support the report of Ehara et al. [36] that the concentration of ‘oxidized’ LDL was significantly higher in patients with acute myocardial infarction and unstable angina, when measured using a PONPC-specific monoclonal antibody.

HDL has been reported to inhibit TNF-{alpha}-induced expression of adhesion molecules by endothelial cells [37,38]. This effect has been attributed to PAPC and PLPC [39]. Therefore oxidation of HDL may alter its biological function, resulting in the loss of its ability to inhibit LDL oxidation or to protect the endothelium.

HDL can acquire pro-atherogenic characteristics during inflammation and this is often associated with low PON-1 [40,41]. This effect has been demonstrated in mouse models that exhibit susceptibility to atherosclerosis or acute infection [42,43]. Although the change in characteristics of HDL during acute phase could be initiated in part by LDL-derived oxidized lipids, and aggravated by infection and stress [44], oxidation of HDL itself could be implicated. It has been shown that during oxidative stress, associated with the progression of atherosclerosis, there is an increase in immunoreactivity in the aortic samples against PON-1, apoAI, and clusterin [45]. The presence of these products in macrophage and smooth muscle cells in human atherosclerotic tissues may provide further evidence for HDL oxidation in vivo.

Independent of apoAI, we found purified PON-1 reduced the ability of 5-carbon and 9-carbon PC core aldehydes and acids to increase monocyte binding and chemotaxis. Watson et al. [17] showed that addition of PON-1 to oxidized LDL inhibited activation of endothelial cells by oxidized LDL alone. Subbanagounder et al. [34] demonstrated that addition of phospholipase A2 to oxidized phospholipids abolished the activation of endothelial cells. HDL has the capacity to hydrolyze oxophospholipids [23] and we have recently shown that PON-1 hydrolyses these oxophospholipids [19]. In the present study, PON-1 significantly reduced the effectiveness of oxidized phospholipids on endothelial cells. Given the fact that linoleoyl PC is the predominant polyunsaturated PC present in lipoproteins and cell membranes, the activity of its oxidation products may provide a new understanding of the proinflamatory effects of oxidation and their effects on the arterial wall.

It is thought that, under normal conditions, the concentration of HDL is greater than the concentration of LDL in the interstitial fluid or in the arterial wall. Our results suggest that the oxidation hypothesis should be revised to consider the possibility that, under conditions of acute oxidative stress, native HDL could be oxidized and become a source of bioactive phospholipids in the arterial wall. The bioactive phospholipids generated from HDL would then prime the endothelial cells to promote monocyte adhesion and chemotaxis. Development of atherosclerosis would require the retention and oxidation of LDL. Our results also suggest that the qualitative changes, such as lower PON1, that occur to HDL under conditions of chronic inflammation, would favor the accumulation of HDL-derived bioactive phospholipids before or coincident with the accumulation of LDL-derived bioactive phospholipids.

We conclude that HDL can be a source of oxidized phospholipids that cause activation of the endothelium. In addition PC linoleate oxidation products, PONPC and PAzPC contribute effectively to activation of endothelial cells, increasing binding and chemotaxis of monocytes. Finally, the enzyme PON-1 is effective at reducing the effects of both linoleoyl PC and arachidonoyl PC oxidation products.

Time for primary review 19 days.


   Acknowledgements
 
This work was supported by grant-in-aid #T4027 from the Heart and Stroke Foundation of Ontario (P.W.C.).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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