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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1284-1290.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Role of Group II Secretory Phospholipase A₂ in Atherosclerosis

1. Increased Atherogenesis and Altered Lipoproteins in Transgenic Mice Expressing Group IIa Phospholipase A₂

Boris Ivandic; Lawrence W. Castellani; Xu-Ping Wang; Jian-Hua Qiao; Margarete Mehrabian; Mohamad Navab; Alan M. Fogelman; David S. Grass; Mark E. Swanson; Maria C. de Beer; Frederick de Beer; Aldons J. Lusis

From the Department of Microbiology and Molecular Genetics and Molecular Biology Institute (B.I., L.C., X.-P.W., J.-H.Q., M.M., A.J.L.), and the Department of Medicine, Division of Cardiology (B.I., L.C., X.-P.W., J.-H.Q., M.M., M.N., A.M.F., A.J.L.), University of California, Los Angeles; Chrysalis DNX Transgenic Sciences (D.S.G., M.E.S.), Princeton, New Jersey; and the Department of Internal Medicine and Biochemistry (M.C.d.B., F.d.B.), University of Kentucky, Lexington.

Correspondence to Aldons J. Lusis, Department Medicine, UCLA School of Medicine, Los Angeles, CA 90095.

	Abstract

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Abstract—Some observations have suggested that the extracellular group IIa phospholipase A₂ (sPLA₂), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA₂ was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA₂ may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.

Key Words: inflammation • lipoproteins • paraoxonase

	Introduction

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Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 fatty acyl ester bonds of phospholipids to produce a free fatty acid, often arachidonic acid, and a lysophospholipid.^{1 2} Several different species of PLA₂ have been recognized in mammals.³ Most extensively studied are the pancreatic enzymes functioning in digestion (group I) and a low molecular weight (14 kDa) soluble species of PLA₂ widely expressed in different tissues (group II). The release of arachidonic acid, by both cellular and extracellular PLA₂s, is rate limiting in the generation of various oxidation products of arachidonic acid, including prostaglandins, thromboxanes, and leukotrienes.^{4 5} The secreted group IIa PLA₂ (sPLA₂) has been shown to stimulate oxidation of LDL by lipoxygenase.⁶ Lysophospholipids, on the other hand, can influence cellular functions⁷ and can be further processed to produce platelet-activating factor.⁶

Grass et al⁸ previously reported the development of lines of transgenic mice expressing the entire human group IIa sPLA₂ gene. The pattern of expression of the enzyme resembled that of the human enzyme, with expression in almost all tissues examined and highest levels of expression in liver, lung, skin, and kidney. The sPLA₂ activity in serum was elevated 8-fold compared with nontransgenic mice. The transgenic mice did not exhibit inflammatory infiltrates in various tissues examined, although they exhibited epidermal hyperplasia.

Because sPLA₂ is found in human atherosclerotic lesions^{9 10} and a variety of in vitro studies suggest that the enzyme can promote inflammation,² we have examined the sPLA₂ transgenic mice for susceptibility to diet-induced atherogenesis. For these studies, the transgene was transferred by repeated backcrossing to the atherosclerosis-susceptible C57BL/6J inbred strain. This strain exhibits a naturally occurring null mutation of the group IIa sPLA₂ gene, providing a clear contrast for the effects of the transgene.¹¹ Our results reveal that the transgenic mice exhibit dramatically increased atherosclerosis when fed an atherogenic diet. The lipoprotein profiles of the transgenic mice appeared to be more pro-atherogenic than those of nontransgenic littermates, and the transgenic mice exhibited markedly reduced levels of paraoxonase, an enzyme associated with HDL that protects against LDL oxidation.

	Methods

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Animals
Mice were obtained from Chrysalis DNX Transgenic sciences (Princeton, NJ). The sPLA₂ transgenic mice were produced by microinjection of a 6.2-kb HindIII restriction fragment containing the human secretory PLA₂ gene into (B6xSJL)F2 hybrid embryos, which were transferred to strain ICR recipients and developed to term according to standard protocols.⁸ The transgene contained the complete gene with 1.6 kb of 5'-flanking and 0.35 kb of 3'-flanking genomic sequence, permitting the regulation of the transgene by its natural promoter.⁸ The sPLA₂ transgenic mice were backcrossed repeatedly (for at least 4 generations) to C57BL/6J mice. The genomes of the resulting experimental mice, both transgenic and nontransgenic littermates, were thus derived primarily (>90% on average) from the parental C57BL/6J mice. Transfer of lines onto the C57BL/6J background had the advantage of increasing the susceptibility to diet-induced atherosclerosis in these mice. Also, C57BL/6J mice have a natural null mutation for mouse sPLA₂, which facilitates studies of the effects of the human transgene.¹¹ All animals were maintained in a barrier facility with equal light/dark cycle and free access to water and food. At 4 to 6 months of age some animals were maintained for 12 weeks on an atherogenic diet containing 1.25% cholesterol, 15.75% fat, and 0.5% sodium cholate (TD 90221, Harlan-Teklad, Madison, WI). Both before and after the high-fat diet challenge, blood samples were collected from the overnight-fasted mice by bleeding through the retro-orbital sinus after anesthesia with isofluorane, using heparin (3.0 U/mL blood) as an anticoagulant. At the end of the experimental period, mice were anesthetized with isoflurane and euthanized by cervical dislocation. All animal procedures were carried out according to the regulations of the University of California Animal Research Committee.

Histological Analyses
After the mice were killed, the heart and proximal aorta were excised and washed in PBS. The apex and lower half of the ventricle were cut off. The remaining specimen was embedded in Tissue-Tek (Miles), frozen on dry ice, and stored at -70°C until sectioning. Serial cryosections were prepared through the ventricle until the aortic valves appeared. From then on, every other 10-µm section was collected on poly-D-lysine–coated slides until the aortic sinus was completely sectioned. Sections were stained with hematoxylin and oil red O, which specifically stains lipids.¹² Slides were examined by light microscopy. The average fatty streak lesion area was quantified throughout the aortic sinus using an ocular with a µm² grid and was normalized to 40 sections according to protocols previously published.¹² Immunohistochemical staining was performed as described previously¹² using an avidin-biotinylated peroxidase system (ABC kit, Vector and AEC kit; Biomeda Corp). We used a monospecific polyclonal rabbit anti-human sPLA₂ antiserum (F.C. de Beer, unpublished) as the primary antibody. This antibody was a gift from Dr Jeff Browning, Biogen Inc, Cambridge, MA, and was prepared using recombinant human sPLA₂ expressed in CHO cells. As judged by Western blotting, it was monospecific. For example, a single 14 kDa band was observed by Western blotting of extracts of CHO cells expressing recombinant group II sPLA₂, whereas no signal was obtained with control CHO cells (data not shown). The optimal dilution used for the antiserum to sPLA₂ was 1:200. Controls included the omission of primary antibody and the use of frozen sections of aortic tissues from nontransgenic littermates fed the atherogenic diet.

Lipids and Lipoproteins
Plasma esterified and unesterified cholesterol and plasma triglycerides, glycerol, and free fatty acid concentrations were measured using colorimetric assays as described previously.¹³ LDL (d=1.019 to 1.063 g/mL) and HDL (d=1.063 to 1.210 g/mL) were isolated from plasma by sequential density ultracentrifugation^{13 14} in the absence of EDTA to avoid inactivation of paraoxonase. These preparations were judged to be at least 95% pure as determined by gel electrophoresis. In addition, mouse HDL-containing fractions were isolated from pooled plasma by fast protein liquid chromatography (FPLC).¹³ Protein content was measured using a microtiter plate assay.^{15 16}

Paraoxonase Activity
Pooled plasma samples and FPLC fractions were assayed for paraoxonase activity using paraoxon as substrate.^{17 18} The cuvette contained 1.0 mmol/L paraoxon in 20 mmol/L Tris-HCl, pH 8.0. The reaction was initiated by the addition of the lipoprotein sample and the increase in the absorbance at 405 nm was recorded for 90 seconds. Blanks were included to correct for the spontaneous hydrolysis of paraoxon. Enzymatic activity was calculated from the molar extinction coefficient 13 100 mol · L^-1 · cm^-1. A unit of paraoxonase activity is defined as 1 nmol of 4-nitrophenol formed per minute under the above assay conditions.¹⁷

Statistical Methods
Data analysis (Mann-Whitney U Test) was performed using the StatView (Abacus Concepts Inc) program. Significance levels were set to P<0.05 and not corrected for multiple comparisons. All measurements were done at least in triplicate, unless indicated otherwise.

	Results

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sPLA₂ Expression in Transgenic Mice
Grass et al⁸ previously examined the expression of sPLA₂ in transgenic mice and observed a pattern similar to human sPLA₂, with expression in most tissues. The background C57BL/6J strain mice have a naturally occurring null mutation of the group IIa sPLA₂ and, therefore, lack enzyme activity. We utilized immunohistochemical staining with a rabbit polyclonal antibody to examine sPLA₂ presence in certain tissues relevant to our studies, including arterial vessels and liver, on both chow and high-fat diets. As shown in Figure 1, positive staining for sPLA₂ was observed in atherosclerotic lesions of the aorta of transgenic mice but not in the lesions of nontransgenic controls. Little or no staining of sPLA₂ was observed in normal arteries of either transgenic or nontransgenic mice (data not shown).

View larger version (141K): [in this window] [in a new window]   Figure 1. sPLA2 is present in atherosclerotic lesions of the aorta. The photomicrographs (x200 magnification) show immunohistochemical staining for sPLA2 (reddish-brown color) in aortic lesions of sPLA2 transgenic mice (B) but not in aortic lesions of nontransgenic littermates (A). The slides were counterstained with hematoxylin (Reference 12). All mice were females maintained on an atherogenic diet for 12 weeks. Immunohistochemical staining was performed as described in Methods.

Increased Aortic Fatty Streak Lesions in sPLA₂ Transgenic Mice Maintained on a High-Fat Diet
Adult sPLA₂ transgenic mice and their nontransgenic littermates, both of a strain C57BL/6J genetic background, were challenged for 12 weeks with a high-fat, high-cholesterol "atherogenic" diet. After euthanasia, the hearts, along with the proximal aorta, were dissected, sectioned, and stained as previously described.¹² Fatty streak lesions were observed as intimal lipid deposits appearing red after staining with oil red O (Figure 2). More advanced lesion stages of a fibroproliferative type or extending beyond the aortic sinus were not observed. The total lesion areas of male transgenic animals were 6-fold greater than those of nontransgenic male littermates and the lesion areas of female transgenic mice were about twice those of female littermates (Figure 3). The absolute increase in lesion size resulting from the transgene was similar in males and females (Figure 3). These differences were significant in transgenic mice compared with control mice irrespective of gender (P=0.0006 in all, P=0.0011 in male, and P=0.0046 in female mice, Figure 2). The average lesion area in the female mice was significantly greater than in male mice (P=0.0046 in transgenic and P<0.0001 in nontransgenic littermates), a phenomenon observed in most mouse models, including apolipoprotein E knockout mice (see Discussion).

View larger version (171K): [in this window] [in a new window]   Figure 2. Photomicrographs (x100 magnification) depicting sections of the proximal ascending aortic arch of sPLA2 transgenic mice (B, D) and nontransgenic littermates (A, C) after chow (A, B) and a 12-week atherogenic diet (C, D). Atherosclerotic lesions were characterized by intimal lipid deposits, stained red by oil-red-O, which were more severe in transgenic animals (B, D) compared with controls (A, C). The arrows in Panel C indicate a small lesion in the aortic root.

View larger version (21K): [in this window] [in a new window]   Figure 3. Atherosclerotic lesions of sPLA2 transgenic mice are increased compared with control mice after 12 weeks on an atherogenic diet. Bars represent mean±SEM in µm2 of 27 nontransgenic (white bars; 13 male, 14 female) and 28 transgenic (black bars; 13 male, 15 female) animals.

Spontaneous Aortic Fatty Streak Lesions in sPLA₂ Transgenic Mice Maintained on a Low-Fat Chow Diet
Because of the very significant increase in lesions in transgenic sPLA₂ mice compared with nontransgenic littermates, we examined aortic lesions in 6-month-old mice maintained on a low-fat chow diet. We observed significant lesions in all 7 transgenic females examined, whereas 7 control female C57BL/6J mice exhibited no lesions or very small lesions (Figure 4). The average lesion size of the transgenic mice was 790±294 µm²/section and that of the nontransgenic mice was 18±18 µm²/section (P<0.03). The finding of such spontaneous lesions was surprising, given the relatively low levels of LDL/VLDL in these mice (see below).

View larger version (14K): [in this window] [in a new window]   Figure 4. Six-month-old, sPLA2 transgenic mice (dark circles) maintained on a low-fat chow diet develop significant atherosclerotic lesions of the aortic sinus compared with controls.

sPLA₂ Transgenic Mice Exhibit Decreased Levels of HDL-Cholesterol and Increased Levels of LDL/VLDL-Cholesterol
To examine possible changes in the lipoprotein profile of the sPLA₂ transgenic mice on the high-fat diet, we quantified plasma lipids and lipoproteins in transgenic and nontransgenic littermates. Tables 1 and 2 show the results in males and females, respectively. In addition, we examined elution profiles after fractionation by gel filtration (Figure 5). Although total plasma cholesterol and cholesteryl ester concentrations did not differ significantly, plasma levels of free cholesterol were approximately doubled in the transgenic mice compared with controls (Tables 1 and 2). This difference was highly significant and was not related to the type of diet. HDL cholesterol levels were significantly reduced in the transgenic mice compared with nontransgenic littermates on both the chow diet and after 12 weeks on high-fat diet (Tables 1 and 2, Figure 5). In addition, there was a small but significant increase in the levels of LDL/VLDL cholesterol in the transgenic mice (Tables 1 and 2, Figure 5). On the high-fat diet, total plasma cholesterol levels tripled and the pro-atherogenic lipoprotein profile become more accentuated in the transgenic mice compared with nontransgenic littermates (Tables 1 and 2, Figure 5). Total triglyceride concentrations did not differ between transgenic and control mice on the chow diet.

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid Concentrations in Male sPLA2 Transgenic Mice (sPLA2 TG) and Nontransgenic Littermates (Controls) on a Chow Diet and After 12 Weeks on an Atherogenic Diet (Fat, 15.75%; Cholesterol, 1.25%; and Cholate, 0.5%)

View this table: [in this window] [in a new window]   Table 2. Plasma Lipid Concentrations in Female sPLA2 Transgenic Mice (sPLA2 TG) and Non-transgenic Littermates (Controls) on a Chow Diet and After 12 Weeks on an Atherogenic Diet (Fat, 15.75%; Cholesterol, 1.25%; and Cholate, 0.5%)

View larger version (33K): [in this window] [in a new window]   Figure 5. Plasma lipoprotein profiles of sPLA2 transgenic mice () and nontransgenic littermates () on chow or atherogenic diets. Equal volumes of plasma pooled from 5 mice (400 µL) were injected onto 2 Superose 6 columns connected in series and lipoproteins were separated by gel filtration as described.13 Fractions (0.5 mL) were collected and assayed for cholesterol and triglyceride concentrations.

Paraoxonase Activity Is Reduced in Transgenic Mice
The changes in lipoprotein profiles in the sPLA₂ in transgenic mice are unlikely to entirely explain the differences in atherogenesis (see Discussion). Because the enzyme paraoxonase appears to be responsible, in part, for the protective effects of HDL,^{19 20 21 22} we measured levels of the enzyme in transgenic and nontransgenic mice. As shown in Figure 6A, paraoxonase activity was 3-fold lower in pooled plasma from transgenic mice (34.5±4.1 U/mL) than in controls (86.7±3.9 U/mL). FPLC fractions containing HDL of transgenic mice also showed a marked decrease in activity compared with controls, and paraoxonase in the transgenic mice was associated with smaller HDL particles (Figure 6B).

View larger version (27K): [in this window] [in a new window]   Figure 6. Decreased paraoxonase activity in plasma and in HDL. (A) Plasma paraoxonase activity. Pooled plasma samples (5 mice each) from the sPLA2 transgenic and from control littermates were assayed for paraoxonase activity (paraoxon hydrolyzing units/mL plasma) as described in Methods. The values are mean±SEM of triplicate determinations. (B) Equal volumes of pooled plasma (5 mice each) from groups of sPLA2 transgenic (solid line) and nontransgenic (broken line) mice were fractionated by gel filtration. Paraoxonase activity (paraoxon hydrolyzing units/mL) was determined in the fractions as described in Methods. It should be noted that the fraction numbers in these experiments differ from those in Figure 5 because different fraction volumes were collected.

	Discussion

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Previous studies have suggested that sPLA₂ may contribute to the development of atherosclerotic lesions,^{9 10 14} and we have examined this hypothesis directly using transgenic mice expressing human sPLA₂. Consistent with previous studies,^{9 10} we observed abundant immunohistochemically localized sPLA₂ in atherosclerotic lesions. The transgenic mice exhibited significantly increased lesions on a high-fat atherogenic diet as well as on a low-fat chow diet. The increase in lesion development appeared to result, in part, from decreased HDL and elevated LDL/VLDL levels. The levels of paraoxonase, an enzyme associated with HDL that protects against LDL oxidation and atherogenesis, were also substantially reduced in the sPLA₂ transgenic mice compared with nontransgenic littermates. These points are discussed below.

The sPLA₂ transgenic line used in these studies has previously been characterized with respect to sPLA₂ expression in plasma and various tissue(s). The transgenic mice displayed severe hyperkeratosis and nearly complete loss of fur, but showed little or no inflammation in various tissues examined.⁸ To analyze lesion development, the transgene was transferred onto the genetic background of the C57BL/6J inbred strain. This strain has been studied extensively as a model for atherosclerosis because it is susceptible to the development of aortic fatty streak lesions when fed an atherogenic diet rich in saturated fat, cholesterol, and cholic acid.^{23 24} Studying the effects of the sPLA₂ transgene on an inbred genetic background is also advantageous for reducing variability caused by genetic factors other than sPLA₂. Moreover, the C57BL/6J strain exhibits an insertional point mutation causing a frameshift in exon 3 of the mouse sPLA₂ gene, resulting in multiple transcripts that do not encode functional products.¹¹ The genetic background of C57BL/6J, therefore, provided a very good contrast for the effects of the human transgene.

Immunohistochemical staining with a polyclonal antibody to human sPLA₂ revealed the presence of the enzyme in atherosclerotic lesions of transgenic mice but not nontransgenic littermates. Previous studies have revealed the presence of sPLA₂ in human atherosclerotic lesions.^{9 10} Liver sections of transgenic mice also displayed positive immunohistochemical sPLA₂ staining and perivascular inflammatory infiltration, whereas sections from nontransgenic littermates were negative (data not shown). Human sPLA₂ transgene expression was previously found to be highest in liver and to correlate well with serum activity.⁸

Our results demonstrate that the transgenic mice had significantly increased atherosclerotic lesions on an atherogenic diet as well as a low-fat diet. The latter results were surprising, given the relatively low levels of LDL and VLDL in the transgenic mice maintained on a chow diet. The lesions in sPLA₂ transgenics fed a chow diet were similar in size and characteristic to those observed in apoA2 transgenic mice maintained on a chow diet.^{13 25} HDLs from apoA2 transgenic mice and from sPLA₂ transgenic mice were similar in that both had reduced levels of paraoxonase and both failed to protect against LDL oxidation in cocultures of aortic endothelial cells and smooth muscle cell (discussed in the accompanying article, Reference 18¹⁸ ). The lesions in female mice, in both the transgenic and nontransgenic groups, were larger than lesions in male mice. This is a characteristic of both diet-induced lesions (Reference 23²³ and references therein) and lesions in genetically engineered hypercholesterolemic mice (Reference 24²⁴ and references therein).

Because previous studies have shown that plasma lipoproteins are substrates of sPLA₂,¹⁴ we examined lipoprotein profiles of sPLA₂ transgenic mice and nontransgenic littermates. Significant differences were observed between the groups on both the chow and the atherogenic diets. On both diets, sPLA₂ transgenic mice exhibited significant elevations of LDL/VLDL-cholesterol and significant reductions of HDL-cholesterol, consistent with a pro-atherogenic change, and these correlated with the lesion sizes of the mice. Plasma-free fatty acids were not elevated in the transgenic mice, but this was not unexpected because free fatty acids are rapidly cleared from plasma. An unexpected result was the significantly increased levels of nonesterified cholesterol in the sPLA₂ transgenic mice, which were nearly doubled compared with control mice both on chow and on atherogenic diets. The explanation for this is unclear but it may involve altered function of lecithin cholesterol acyl transferase (LCAT), an enzyme found in plasma that catalyzes the transfer of a fatty acid residue from the sn-2 position of lecithin to cholesterol to form cholesteryl ester, utilizing apoA1 as a cofactor. Conceivably, sPLA₂ could alter LCAT function indirectly by changing HDL structure with respect to phospholipid composition or by inhibiting the interaction of apoA1 with LCAT on early discoidal HDL particles.²⁶ Another interesting possibility is that LCAT may be inactivated by various reactive oxygen species. Bielicke et al²⁷ have shown that oxidized LDL is a potent inhibitor of LCAT, and as shown in the accompanying studies,¹⁸ sPLA₂ transgenic mice exhibit evidence of increased LDL oxidation. It is noteworthy that purified acute-phase serum amyloid A (SAA) can exchange for apoA1 on HDL particles and that the SAA can stimulate sPLA₂ activity in vitro.²⁸ SAA could contribute to our findings because SAA gene expression is markedly increased by the atherogenic diet in C57BL/6J mice.^{29 30}

It seems unlikely that the observed changes in HDL and LDL/VLDL levels can entirely explain the dramatic effects of the sPLA₂ transgene on atherosclerosis. Based on our previous studies with apoA2 transgenic mice^{13 25} and on in vitro studies of HDL,^{22 29} we hypothesized that there may be functional alterations of HDL as well. In particular, the capacity of HDL to inhibit LDL oxidation is attributed, in part, to the activity of the enzyme paraoxonase, which can act as an sn-2 ester hydrolyzing phospholipase, destroying biologically active phospholipid peroxides.²⁰ Paraoxonase markedly inhibits cell-mediated LDL oxidation, and the inhibition of paraoxonase reduces HDL protection against LDL oxidation in the coculture model.^{20 22} We found that paraoxonase activity was significantly decreased in plasmas from the transgenic mice compared with controls. Serum paraoxonase is produced only in liver, and in serum it is tightly associated with apoA1. Displacement of apoA1 from HDL by SAA, as discussed above, also leads to a loss of paraoxonase activity.³¹ It is possible that the reduction in paraoxonase activity is because of reduced HDL levels. We have further examined the functional characteristics of plasma lipoproteins from the transgenic mice in the accompanying manuscript.¹⁸ It is also noteworthy that the uptake of LDL by macrophages is stimulated by PLA₂ treatment.³²

The relevance of these results for atherosclerosis in human populations is as yet unknown. However, it is interesting to speculate that sPLA₂ could contribute to the increased atherosclerosis observed in various inflammatory diseases such as rheumatoid arthritis.³³ Levels of sPLA₂ in the circulation can rise many-fold during the acute phase response and in chronic inflammatory conditions such as arthritis and inflammatory bowel disease.⁶ Also, a variety of cells relevant to atherosclerosis, including neutrophils and macrophages, can secrete sPLA₂ in response to inflammatory cytokines such as tumor necrosis factor and interleukin 1.^{34 35} The hypothesis that sPLA₂ contributes to the inflammatory aspects of human atherogenesis warrants further investigation.

	Acknowledgments

 
This work was supported by National Institute of Health grants HL30568 (A.J.L., A.M.F.), AG10886 (F.C.d.B.), the Council for Tobacco Research (F.C.d.B.), and Veterans Administration Research Funds (F.C.d.B.). We thank Jay Lee for discussions. F. de Beer and A. Lusis contributed equally to this study.

Received January 12, 1998; accepted October 5, 1998.

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