Lipoprotein-Associated Phospholipase A2, Platelet-Activating Factor Acetylhydrolase, Is Expressed by Macrophages in Human and Rabbit Atherosclerotic Lesions
Abstract—We studied the expression of lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA2 activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA2 mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase—polymerase chain reaction analysis indicated an increased expression of Lp-PLA2 mRNA in human atherosclerotic lesions. In addition, ≈6-fold higher Lp-PLA2 activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA2, which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA2 activity could lead to antiatherogenic effects in the vessel wall.
- platelet-activating factor
- oxidized LDL
- real-time fluorescence polymerase chain reaction
Presented in part as preliminary results at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10–13, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-585).
- Received September 28, 1998.
- Accepted May 28, 1999.
Oxidative modification of LDL, monocyte migration into the vessel wall, subsequent macrophage activation, and foam cell formation are key events in the pathogenesis of atherosclerosis.1 2 Several studies have demonstrated that one of the earliest events in LDL oxidation is the hydrolysis of oxidatively modified phosphatidylcholines, which generates lysophosphatidylcholine (lyso-PC) and oxidized fatty acids.3 4 5 6 7 8 9 This hydrolysis of oxidized phosphatidylcholines within LDL is mediated by the lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as a platelet-activating factor (PAF) acetylhydrolase, which has been cloned and characterized previously.3 6 9 The arterial wall also contains other types of secreted group II phospholipase A2, which may play a role in this process.10 11 12 13 14 Although lyso-PC itself is a potent biological effector molecule able to stimulate monocyte5 and T-lymphocyte chemotaxis,15 induce adhesion molecules7 16 and various growth factors,17 and impair vascular relaxation,18 19 the oxidized fatty acids liberated together with lyso-PC may also possess relevant biological activity. Given its many biological properties, lyso-PC, together with the enzyme responsible for its generation, Lp-PLA2, has been postulated to play a causal role in inflammation20 and atherosclerosis.21 In addition to lyso-PC formation, it has been established that biologically active PAF-like polar phospholipids are formed during the LDL oxidation process.22 23 On the other hand, the transient appearance of these PAF-like phospholipids has been postulated to be due to their hydrolysis and subsequent inactivation by Lp-PLA2.24 Thus, in the context of atherogenesis, the enzyme Lp-PLA2 would appear to have a dual role, one that is proinflammatory (generation of lyso-PC) and another that is anti-inflammatory (degradation of PAF-like phospholipids).
To explore further the role of Lp-PLA2 in atherogenesis, we have investigated whether the enzyme is expressed in human and rabbit atherosclerotic lesions. Previous work has shown that in addition to its being distributed among plasma lipoprotein fractions (predominantly LDL in humans),25 an important cellular source appears to be macrophages.26 The results of the present study show that lesion macrophages express Lp-PLA2 mRNA and protein and that Lp-PLA2 enzyme activity is increased in rabbit atherosclerotic lesions.
Human aortic samples were obtained from 8 medicolegal autopsies (4 males and 4 females, aged 29 to 73 years; cause of death, traffic accidents, suicide, and gun shot wounds) 4 to 12 hours postmortem. Rabbit aortic samples were dissected from aortic arch and thoracic aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits (aged 6 to 12 months) maintained on normal chow and New Zealand White (NZW) rabbits (aged 4 to 6 months) fed a 0.5% cholesterol diet for 8 weeks (Table⇓). Plasma cholesterol levels for the WHHL and NZW rabbits were 20 to 23 and 11 to 17 mmol/L, respectively. The animals were killed under intravenous fentanyl-fluanisone (0.3 mL/kg, Hypnorm, Jansen Pharmaceuticals) and midazolam (1 mg/kg, Dormicum, Hoffman-La Roche) anesthesia. Tissue samples were removed, immersion-fixed for 4 hours in formal sucrose (4% paraformaldehyde and 15% sucrose containing 50 μmol/L BHT and 1 mmol/L EDTA), and rinsed in 15% sucrose/50 μmol/L BHT/1 mmol/L EDTA for 12 hours.27 Serial paraffin-embedded 7- to 10-μm sections were used for the assays. Atherosclerotic lesions were classified according to Stary et al28 into normal areas, type I (initial lesions), type II (fatty streaks), type III (intermediate lesions), type IV (atheroma), and type V (fibroatheroma, calcified and smooth muscle cell–rich plaques) lesions. All human studies were approved by the Ethics Committee of the University of Kuopio, and animal studies were approved by the Experimental Animal Committee of the University of Kuopio.
In Situ Hybridization and Immunocytochemistry
Whole-length 1.4-kb human Lp-PLA2 antisense and sense riboprobes were synthesized using T7 and T3 polymerase in the presence of [33P]UTP (NEN Life Science Products) from a pBluescript II KS plasmid (Stratagene). In situ hybridizations were performed on pretreated tissue sections (1×106 cpm per section) as described.27 The final wash was with 0.1× SSC at 53°C for 30 minutes. The slides were dipped in Kodak NTB-2 nuclear track emulsion (Eastman-Kodak) and exposed for 4 weeks. Nonhybridizing sense probes were used as controls.27 29
Serial paraffin-embedded sections were used for immunocytochemistry with the following antibodies: mouse monoclonal antibody (mAb) against human macrophages (CD68, dilution 1:150; Dako); mouse mAb against rabbit macrophages (RAM-11, dilution 1:50; Dako), mouse mAb against muscle α- and γ-actin (HHF35, dilution 1:50; Enzo Diagnostics), guinea pig polyclonal antisera against malondialdehyde-modified LDL (MAL-2, dilution 1:1000),30 and highly specific mAbs (2C10 and 3H2, dilution 1:50) against human Lp-PLA2 purified to homogeneity.9 The preparation and specificity of these monoclonal antibodies has been described in detail elsewhere.30A Briefly, no cross-reactivity was noted with 3 different varieties of human recombinant PLA2: 14-kDa PLA2, 85-kDa PLA2, and a recently described related serine-dependent PLA2. Both 14-kDa PLA2 and 85-kDa PLA2 are calcium-dependent arachidonic acid–selective enzymes,31 whereas the serine-dependent enzyme is calcium independent and has 40% amino acid identity with Lp-PLA2.32
An avidin-biotin-horseradish peroxidase system (Histostain-Plus Kit, Zymed Laboratories) was used for signal detection according to manufacturer’s instructions with either diaminobenzidine or aminoethyl carbazole as color substrates. When immunocytochemistry was combined with in situ hybridization to facilitate the simultaneous detection of Lp-PLA2 mRNA and protein on the same section, the immunostaining step was performed after the in situ hybridization.29 33 Irrelevant class- and species-matched immunoglobulins and incubations without the primary antibody were used as controls for the immunostainings.27
Micrographs were taken by a digital camera (SenSys KAF1400-G2, Photometrics Ltd), processed with digital image–processing software (Image-Pro Plus, Media Cybernetics), and printed using a sublimation printer (Kodak DS 8650, Eastman-Kodak).
Reverse Transcriptase—Polymerase Chain Reaction
Human atherosclerotic plaque mRNAs were isolated from pooled human samples consisting of type II lesions, type IV lesions, and type V lesions by use of the Fast Track mRNA isolation kit (Invitrogen) and reverse-transcribed by use of the cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions. All other RNAs were isolated by use of Trizol reagent (GIBCO BRL) and were reverse-transcribed from DNase I (GIBCO BRL)–treated total RNA by use of Superscript II and random hexamer primers (GIBCO BRL) according to the manufacturer’s instructions. A series of standards were also prepared by performing a 4-fold serial dilution of total RNA from a 6-day primary culture of human monocyte–derived macrophages in the range 2 μg to 0.12 ng RNA per reverse transcriptase (RT) reaction.
cDNA samples (5 μL of each) were analyzed for expression of Lp-PLA2 and the housekeeping gene GAPDH by a real-time quantitative reverse transcriptase—polymerase chain reaction (RT-PCR) by use of the fluorescent TaqMan 5′ nuclease assay. TaqMan assay oligonucleotide primers and probes were designed using Primer Express software, version 1.0 (PE Biosystems). Each TaqMan hydrolysis probe consisted of the fluorescent reporter dye 6-carboxyfluorescein (FAM), covalently linked to the 5′ end of the oligonucleotide, and the quencher dye 6-carboxytetramethylrhodamine (TAMRA), attached to the 3′ end via a linker group (PE Biosystems).
PCRs (5′>3′ nuclease assay) were performed in MicroAmp Optical 96-Well Reaction Plates with Optical Caps (PE Biosystems) by use of the ABI PRISM 7700 Sequence Detection System for thermal cycling and real-time fluorescence measurements (PE Biosystems). Each 25-μL reaction consisted of 1× TaqMan Universal PCR Master Mix (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, 10 mmol/L EDTA, 60 nmol/L passive reference dye 1 [6-carboxy-X-rhodamine], 0.2 mmol/L dATP, 0.2 mmol/L dCTP, 0.2 mmol/L dGTP, 0.4 mmol/L dUTP, 5.5 mmol/L MgCl2, 8% glycerol, 0.625 U AmpliTaq Gold DNA polymerase, and 0.25 U AmpErase uracil N-glycosylase), 300 nmol/L forward primer, 300 nmol/L reverse primer, 100 nmol/L TaqMan quantification probe, and 5 μL template with a 20 μL mineral oil overlay (Promega). The forward and reverse primers for Lp-PLA2 were 5′-CCACCCAAATTGC-ATGTGC-3′ and 5′-GCCAGTCAAAAGGATAAACCACAG-3′, respectively. The forward and reverse primers for GAPDH were 5′-GCCAAGGTCATCCATGACAAC-3′ and 5′-GGGGCCATC-CACAGTCTTC-3′, respectively. The TaqMan probe sequences (FAM-5′>3′-TAMRA) for Lp-PLA2 and GAPDH were 5′-TTCTGCCTCTGCGGCTGCCTG-3′ and 5′-CTCATGACCACA-GTCCATGCCATCACT-3′, respectively. Reaction conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Emitted fluorescence for each reaction well was measured every cycle during both the denaturation and annealing/extension phases, and amplification plots were constructed using the ABI PRISM 7700 Sequence Detection System software, version 1.6 (PE Biosystems).
Subsequent analysis was performed on the data output from the Sequence detector software by use of Microsoft Excel. In brief, the arbitrary quantity values generated for Lp-PLA2 expression by Sequence Detector (values were generated by comparison of the fluorescence generated by each sample with a standard curve of known quantities) were divided by those obtained for each sample for GAPDH. This gave a normalized value for the expression level of Lp-PLA2 in each sample. These values were then divided by the lowest value obtained (that of the aortic smooth muscle cells, which was set to 1) to give a fold increase value for each sample.
As additional controls, monocytes and lymphocytes were included in the RT-PCR analysis. Monocytes and lymphocytes were isolated from human blood by countercurrent centrifugal elution with a minor modification.34 Monocytes were obtained at 95% purity; lymphocytes, at 100% purity. The macrophage sample was generated by culturing monocytes for 4 days in RPMI supplemented with 2% human serum and 2 mM glutamine. The monocyte, macrophage, and lymphocyte samples used in the analysis were from the same donor. Aortic smooth muscle cells were also included in the assay. The cells were primary smooth muscle cells from a human donor that were made quiescent in SmGM-2 medium (Clonetics) over a 2-day period.
Analysis of Rabbit Aortic Lp-PLA2 Activity
WHHL rabbits with a Half-Lop (H/LOP) background (Froxfield Farms Ltd, Hampshire, UK) were used to investigate Lp-PLA2 activity in aortic atherosclerotic lesions. Male WHHL rabbits were compared with sex- and age-matched nondiseased control rabbits, which were either H/LOP or NZW rabbits maintained on normal chow. Rabbits were killed with an overdose of anesthetic, and aortas were immediately removed. Aortic samples were washed at 4°C in a homogenizing buffer (mmol/L: Tris 50 [pH 8], CHAPS 10, EGTA 2, and EDTA 2, along with 1 μg/mL each of leupeptin, antipain, and pepstatin-A), and 3×0.5-cm sections at the very beginning of the ascending aorta were removed, frozen in liquid nitrogen, and stored at −70°C until analyzed. To measure aortic PLA2 activity, each slice of aorta was first homogenized in 1 mL of homogenization buffer by use of a mortar and pestle on ice. The homogenate was then removed to Eppendorf tubes and microfuged for 20 minutes at 4°C. Supernatants (20 μL), which contained all the PLA2 activity (data not shown), were then assayed using 50 μmol/L PAF as a substrate exactly as outlined previously.9 Assays were repeated in the presence of 300 nmol/L SB-222657, a potent and selective Lp-PLA2 inhibitor,35 to demonstrate what proportion of the total activity was attributable to Lp-PLA2. Activities obtained from the 3 sections were averaged for each rabbit, and protein content was determined by a modified Lowry method.36
In situ hybridization analysis with human Lp-PLA2 antisense riboprobe showed that Lp-PLA2 mRNA expression was localized over the macrophage-rich regions in all types of human atherosclerotic lesions (type I to V lesions according to the classification of Stary et al28 ). Two representative examples of Lp-PLA2 mRNA expression in human type I and II lesions (diffuse intimal thickening and fatty streak, respectively) are seen in Figures 1C⇓ and 2A⇓. In the type I lesion, the thickened intima consists mostly of smooth muscle cells (Figure 1B⇓) and connective tissue, among which are scattered solitary macrophages showing a strong signal (Figure 1C⇓). In type II lesion intima there are, among smooth muscle cells (Figure 2D⇓), a streak of macrophage foam cells (Figure 2C⇓), which show a positive hybridization signal (Figure 2A⇓). An example of Lp-PLA2 mRNA expression in an advanced type IV lesion (atheroma) is seen in Figure 3A⇓. This lesion has a macrophage foam cell–rich cap. The micrograph shows the shoulder area of the lesion with scattered macrophage foam cells expressing Lp-PLA2. No hybridization signal was seen with the corresponding sense probe (Figure 2B⇓ and 3B⇓).
Immunostaining of serial sections with monoclonal antibody 2C10 against Lp-PLA2 protein showed positive staining in the same areas where Lp-PLA2 mRNA was detected (Figures 1D⇑, 2F⇑, and 3E⇑ and the Table⇑). By use of a combined in situ hybridization and immunocytochemical analysis, macrophages (CD68 mAbs) were clearly identified as the source of Lp-PLA2 mRNA (Figure 1C⇑). Immunostainings with an antibody for oxidized LDL (MAL-2) showed a positive signal in the same areas that were positive for the antibody 2C10 (results not shown). Medial smooth muscle cells showed no detectable hybridization signal or immunostaining for Lp-PLA2.
RT-PCR analysis from human type II, type IV, and type V lesions confirmed the induced expression of Lp-PLA2 mRNA in atherosclerotic lesions (Figure 4⇓). It is also clear from the RT-PCR analysis that macrophages and lymphocytes are the major source of Lp-PLA2 expression, in view of the fact that only a very low level of expression was found in human aortic smooth muscle cells (Figure 4⇓).
Positive in situ hybridization (data not shown) and immunocytochemistry for Lp-PLA2 were detected in WHHL rabbit and NZW rabbit atherosclerotic lesions. Examples of advanced macrophage-rich atherosclerotic plaques from rabbit aorta are seen in Figure 5⇓. Immunostainings for Lp-PLA2 protein with monoclonal antibody 3H2 (Figure 5⇓, panels B and F) colocalized with macrophages (Figure 5⇓, panels A and E). As with the human lesions, no positive Lp-PLA2 signal was detected in medial smooth muscle areas (Figure 5⇓, panels C and G).
Compared with aortas from age- and sex-matched control rabbits, extracts from diseased aortas of the WHHL rabbits were shown to contain increased PLA2 activity (Figure 6⇓). The identity of the increased PLA2 activity was confirmed as Lp-PLA2, in view of the fact that all of the elevated PLA2 activity could be inhibited by preincubation of the extract with Lp-PLA2–specific inhibitor SB-222657. From these findings, it was also demonstrated that whereas in control rabbits ≈60% of the aortic PAF-hydrolyzing activity could be attributed to Lp-PLA2, this proportion was increased to 90% in aortas from diseased rabbits (Figure 6⇓). This actually represents a >6-fold increase in Lp-PLA2 activity in atherosclerotic lesions from WHHL rabbits compared with aortas from the control rabbits.
Lp-PLA2 is found to be predominantly associated with LDL in human plasma,25 37 and it is also known that macrophages secrete Lp-PLA2 activity in cell culture.20 26 How much macrophages contribute to blood levels of Lp-PLA2 is presently unknown. More recently, it has been speculated that the level of Lp-PLA2 in the blood is completely dependent on the rate of lipoprotein clearance.38 In this model, blood Lp-PLA2 levels will be lower when the rate of lipoprotein, particularly LDL, removal is high, and the opposite is true when the clearance rate of lipoproteins is low. Consistent with this notion is the observation that small dense LDL, a lipoprotein pool that is slowly metabolized and very atherogenic,39 is actually enriched with Lp-PLA2.40
Lp-PLA2 would appear to play an important role in inflammatory reactions. On one hand, this enzyme is capable of hydrolyzing and inactivating PAF and related oxidized or polar phospholipids, whereas on the other hand, it has the capacity for generating large quantities of 2 proinflammatory lipid mediators, lyso-PC and free oxidized fatty acids, after the hydrolysis of oxidized phosphatidylcholines. Which of these activities predominate in atherogenesis remains unknown.21 23 In the present study, we show that Lp-PLA2 is expressed in lesion macrophages and that Lp-PLA2 enzyme activity is 6-fold higher in WHHL rabbit atherosclerotic arteries than in control rabbit arteries. Thus, the expression and enzyme activity of Lp-PLA2 are increased in atherogenesis, which is characterized by a microenvironment of high oxidative stress and the presence of oxidized LDL.41 In situ hybridization and RT-PCR were used to confirm arterial expression of Lp-PLA2, which cannot be distinguished from LDL or plasma-derived Lp-PLA2 on the basis of immunocytochemistry or enzyme activity analyses. Also, simultaneous in situ hybridization and cell typing by immunocytochemistry were used to confirm that macrophages are the source of the enzyme in atherosclerotic lesions.
Oxidized LDL plays an important role in the pathogenesis of atherosclerosis.2 Oxidized LDL is present in atherosclerotic lesions in vivo,41 and at least part of the proinflammatory effects of oxidized LDL are mediated by lyso-PC.3 4 5 6 7 Indeed, several studies have indicated that elevated levels of lyso-PC are found in atherosclerotic lesions.42 43 Lp-PLA2 may be a key enzyme responsible for the increased formation of lyso-PC in atherosclerotic lesions, in view of the fact that oxidative modification of LDL generates substrates for the enzyme. Thus, expression of Lp-PLA2 in activated macrophages will probably lead to the release in atherosclerotic lesions of lyso-PC and free oxidatively modified fatty acids in potentially large quantities. Several biological activities have been assigned to increased lyso-PC content, such as chemoattractant activity for human monocytes,5 endothelial dysfunction,18 44 induction of the expression of endothelial leukocyte adhesion molecules,7 and increased expression of platelet-derived growth factor and heparin-binding epidermal growth factor–like proteins.17 Thus, although preventing the proposed biological activities of PAF-like substances, Lp-PLA2 could augment the atherosclerotic process by releasing into the microenvironment increased concentrations of lyso-PC and oxidatively modified free fatty acids from oxidized LDL.
It has been shown previously that Lp-PLA2 is able to inhibit LDL oxidation in vitro.8 On the other hand, others have not been able to confirm these observations.6 9 32 44 Whether Lp-PLA2 activity is primarily proatherogenic or antiatherogenic remains to be elucidated. The final test will come from evaluating potent and selective inhibitors of the enzyme in animal models of atherosclerosis. It appears that Lp-PLA2 expression is clearly derived from monocyte/macrophages and lymphocytes, whereas group II secretory phospholipase A2 is highly expressed in smooth muscle cells in both normal and atherosclerotic arteries.13 Also, group II phospholipase A2 can cleave normal unmodified LDL phospholipids, whereas Lp-PLA2 requires oxidation to generate a substrate.32 Thus, Lp-PLA2 is closely associated to the inflammatory aspects of atherogenesis and oxidation of LDL. Even though Lp-PLA2 can be anti-inflammatory under certain conditions, such as in a rat foot pad model after exogenous PAF application,20 lyso-PC and free oxidized fatty acids in atherosclerotic lesions can substantially amplify the pathological process and cause chronic monocyte/macrophage-dominated inflammation, which is typical of atherosclerosis, in view of the fact that the arterial wall contains much higher concentrations of LDL than most other physiological compartments.45 46 Increased expression of Lp-PLA2 in lesion macrophages suggests that modulation of the enzyme activity could become a potential target for the development of antiatherogenic therapy in the vessel wall.
This study was supported by grants from the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, and the Finnish Academy. The authors also thank Mervi Nieminen for excellent technical assistance, Dr Theresa Reape for the supply of smooth muscle cells, and Marja Poikolainen for preparing the manuscript.
Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785–1792.
Parthasarathy S, Steinbrecher UP, Barnett J, Witztum JL, Steinberg D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein. Proc Natl Acad Sci U S A. 1985;82:3000–3004.
Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 1987;84:2995–2998.
Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc Natl Acad Sci U S A. 1988;85:2805–2809.
Steinbrecher UP, Pritchard PH. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet activating factor acetylhydrolase. J Lipid Res. 1989;30:305–315.
Kume N, Cybulsky MI, Gimbrone MA. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138–1144.
Stafforini DM, Zimmerman GA, McIntyre TM, Prescott SM. The platelet activating factor acetylhydrolase from human plasma prevents oxidative modification of low density lipoprotein. Trans Assoc Am Phys. 1993;105:44–63.
Tew DG, Southan C, Rice SQJ, Lawrence MP, Li H, Saul HF, Moores K, Gloger IS, Macphee CH. Purification, properties, sequencing and cloning of a lipoprotein associated, serine dependent phospholipase which is involved in the oxidative modification of low density lipoproteins. Arterioscler Thromb Vasc Biol. 1996;16:591–599.
Hurt-Camejo E, Andersen S, Standal R, Rosengren B, Sartipy P, Stadberg E, Johansen B. Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries. Arterioscler Thromb Vasc Biol. 1997;17:300–309.
Elinder LS, Dumitrescu A, Larsson P, Hedin U, Frostegård J, Claesson H-E. Expression of phospholipase A2 isoforms in human normal and atherosclerotic arterial wall. Arterioscler Thromb Vasc Biol. 1997;17:2257–2263.
McMurray HF, Parthasarathy S, Steinberg, D. Oxidatively modified low density lipoprotein is a chemoattractant for human T lymphocytes. J Clin Invest. 1993;92:1004–1008.
Nakano T, Raines EW, Abraham JA, Klagsbrun M, Ross R. Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes. Proc Natl Acad Sci U S A. 1994;91:1069–1073.
Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907–911.
Murohara T, Kugiyama K, Ohgushi M, Sugiyama S, Ohta Y, Yasue H. LPC in oxidized LDL elicits vasoconstriction and inhibits endothelium-dependent relaxation. Am J Physiol. 1994;267:H2441–H2449.
Macphee CH, Milliner K, Moores K, Tew DG. The involvement of LDL-associated phospholipase A2 in atherogenesis. Pharmacol Rev Commun. 1996;8:309–315.
Heery JM, Kozak M, Stafforini DM, Jones DA, Zimmerman GA, McIntyre TM, Prescott SM. Oxidatively modified LDL contains phospholipids with platelet activating factor-like activity and stimulates the growth of smooth muscle cells. J Clin Invest. 1995;96:2322–2330.
Watson AD, Navab M, Hama SY, Sevanian A, Prescott SM, Stafforini DM, McIntyre TM, La Du BN, Fogelman AM, Berliner JA. Effect of platelet-activating factor acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J Clin Invest. 1995;95:774–782.
Stafforini DM, Prescott SM, McIntyre TM. Human plasma platelet-activating factor acetylhydrolase: purification and properties. J Biol Chem. 1987;262:4223–4230.
Stafforini DM, Elstad MR, McIntyre TM, Zimmerman GA, Prescott SM. Human macrophages secrete platelet-activating factor acetylhydrolase. J Biol Chem. 1990;265:9682–9687.
Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, Glass CK, Sigal E, Witztum JL, Steinberg D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A. 1990;87:6959–6963.
Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. Circulation. 1995;92:1355–1374.
Palinski W, Ylä-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990;10:325–335.
Pulinski W, Ylä-Hertuala S, Rosenfeld ME, Bulter SW, Socher SA, Parthasarathy S, Curtiss KL, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Atherosclerosis. In press.
Rice S, Southan C, Boyd H, Terrett J, Macphee CH, Moores K, Gloger I, Tew D. Expression, purification, and characterisation of a human serine dependent phospholipase with high specificity for oxidised phospholipids and platelet activating factor. Biochem J. 1998;330:1309–1315.
Luoma JS, Strålin P, Marklund SL, Hiltunen TP, Särkioja T, Ylä-Herttuala S. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1998;18:157–167.
Stevenson HC, Millar P, Akiyama Y, Favilla T, Beman JA, Herberman R, Stull H, Thurman G, Maluish A, Oldham R. A system for obtaining large numbers of cryopreserved human monocytes purified by leukapheresis and counter-current centrifugation elutriator. J Immunol Methods. 1983;62:353–363.
Macphee CH, Moores KE, Boyd HF, Dhanak D, Ife RJ, Leach CA, Leake DS, Milliner KJ, Patterson RA, Suckling KE, Tew DG, Hickey DMB. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low density lipoprotein: use of a novel inhibitor. Biochem J. 1999;338:479–487.
Tsoukatos DC, Arborati M, Liapikos T, Clay KL, Murphy RC, Chapman MJ, Ninio E. Copper-catalyzed oxidation mediates PAF formation in human LDL subspecies: protective role of PAF: acetylhydrolase in dense LDL. Arterioscler Thromb Vasc Biol. 1997;17:3505–3512.
Guerra R, Zhao B, Mooser V, Stafforini D, Johnston JM, Cohen JC. Determinants of plasma platelet-activating factor acetylhydrolase: heritability and relationship to plasma lipoproteins. J Lipid Res. 1997;38:2281–2288.
Chapman MJ, Guerin M, Bruckert E. Atherogenic, dense low-density lipoproteins: pathophysiology and new therapeutic approaches. Eur Heart J. 1998;19:A24–A30.
Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
Portman OW, Alexander M. Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis. J Lipid Res.. 1969;10:158–165.
Keaney JF Jr, Xu A, Cunningham D, Jackson T, Frei B, Vita JA. Dietary probucol preserves endothelial function in cholesterol-fed rabbits by limiting vascular oxidative stress and superoxide generation. J Clin Invest. 1995;95:2520–2529.
Hoff HF, Heideman CL, Gotto AM Jr, Gaubatz JW. Apolipoprotein B retention in the grossly normal and atherosclerotic human aorta. Circ Res. 1977;41:684–690.
Ylä-Herttuala S, Solakivi T, Hirvonen J, Laaksonen H, Möttönen M, Pesonen E, Raekallio J, Åkerblom HK, Nikkari T. Glycosaminoglycans and apolipoproteins B and A-I in human aortas: chemical and immunological analysis of lesion-free aortas from children and adults. Arteriosclerosis. 1987;7:333–340.