Secretory Phospholipase A 2 Group V

【摘要】  Objective- To study the distribution of group V secretory phospholipase A 2 (sPLA 2 ) in human and mouse lesions and compare its expression by human vascular cells, its activity toward lipoproteins, and the interaction with arterial proteoglycans (proteoglycans) with those of sPLA 2 -IIA. In addition, we also investigated the effect of a Western diet and lipopolysaccharide challenge on the aortic expression of these enzymes in mouse models.

Methods and Results- Immunohistochemistry showed sPLA 2 -V in human and mouse lesions to be associated with smooth muscle cells and also surrounding foam cells in lipid core areas. mRNA of the enzyme was expressed in human lesions and human vascular cells, supporting the immunohistochemistry data. sPLA 2 -V but not sPLA 2 -IIA was active on lipoproteins in human serum. The association with proteoglycans enhanced 2- to 3-fold sPLA 2 -V activity toward low-density lipoproteins but not that of the group IIA enzyme. Experiments in mouse models showed that treatment with a Western diet induced expression of sPLA 2 -V but not that of sPLA 2 -IIA in aorta. On the other hand, lipopolysaccharide-induced acute inflammation augmented the expression of sPLA 2 -IIA but not that of sPLA 2 -V.

Conclusions- These results indicate that these phospholipases could have different roles in atherosclerosis.

SPLA 2 -V was observed in human and mouse lesions associated with smooth muscle cells and surrounding foam cells in lipid cores. Proteoglycans increased the activity of sPLA 2 -V toward low-density lipoproteins. Western diet induced sPLA 2 -V expression in mouse aorta but not that of sPLA 2 -IIA. These enzymes may contribute to atherosclerosis by different pathways.

【关键词】  phospholipase atherogenesis inflammation lipoproteinretention proteoglycans

Introduction

During atherosclerosis development, there is a progressive decrease of the lesion phospholipid content and enrichment in cholesterol. 1 Furthermore, apolipoprotein B (apoB) lipoproteins isolated from human and rabbit lesions contain less phosphatidylcholine (PC) and more sphingomyelin than circulating lipoproteins. 2,3 Therefore, lipoproteins trapped in the intima appear to be hydrolyzed by secretory phospholipases. 4 These enzymes may contribute to atherosclerosis by hydrolysis of low-density lipoprotein (LDL) phospholipids that induce fusion and increase binding of cholesterol-rich particles to intima proteoglycans, triggering further modifications. 5-8 In addition, phospholipase(s) A 2 contribute to local release of lyso-phospholipids and nonesterified fatty acids, which have proinflammatory properties in arterial cells. 9-12 See page 1421

Secretory phospholipase A 2 (sPLA 2 ) group IIA is present in human atherosclerotic lesions, and experimental and clinical evidence suggest its involvement in atherosclerosis and cardiovascular disease. 13-17 sPLA 2 -IIA and the more recently cloned sPLA 2 -V are members of a family of enzymes that hydrolyze the fatty acids at the sn-2 position of glycerophospholipids. Both enzymes have low molecular weight (14 kDa), are histidine and calcium dependent, rich in disulfide bonds, are basic, and share structure similarities. 18 Several of these properties stabilize and enhance their activity in the extracellular milieu. The genes of sPLA 2 -IIA and sPLA 2 -V enzymes are located at close positions in homologous regions in mouse chromosome 4 and human chromosome 1 and share the same promoter. 19 This region was identified as an atherosclerosis susceptibility locus in the LDL receptor-deficient mouse and is considered a human candidate locus. 20 The C57BL/6 mouse strain is a natural knockout of sPLA 2 -II because a frame shift mutation in exon 3 blocks gene translation. 21 Therefore, either sPLA 2 -IIA does not contribute to atherogenesis in this mouse strain, or other(s) sPLA 2 compensates its absence. On the other hand, the human sPLA 2 -IIA transgenic mouse is more susceptible to atherosclerosis than its littermates that only express the group V. 13,16 This suggests that the expression of both enzymes may contribute to lesion formation. The group V sPLA 2 is also present in human atherosclerotic lesions, but the cell source and regulation of sPLA 2 -V activity in lesions are unknown. 22 Here, we report on the immunohistochemical localization and cell association of sPLA 2 -V in human and mouse lesions. We compared the mRNA expression in human lesions and vascular cells, the activities on lipoproteins, and modulation by extracellular arterial proteoglycans of sPLA 2 -V and sPLA 2 -IIA. In addition, we investigated in mouse models the effect of a Western diet and acute inflammation on the aortic expression of the enzymes.

Materials and Methods

The Materials and Methods section is available online at http://atvb.ahajournals.org.

Results

Immunohistochemical Staining of sPLA 2 -V in Human Lesions

The specificity of all used antibodies was evaluated by Western blotting, and no cross-reactivity of the antibodies against the 2 enzymes was detected (supplemental Figure I, available online at http://atvb.ahajournals.org). The antibodies against sPLA 2 -IIA that we used in our previous studies 5,23 were also specific and did not cross-react with sPLA 2 -V. Figure 1 shows serial sections of 3 human atherosclerotic lesions. Figure 1A, 1D, 1G, 1J, 1N, and 1 O are from an advanced lesion characterized by a thick intima and a well-formed necrotic lipid core. The second column ( Figure 1B, 1E, 1H, 1K, 1L, 1P, and 1 Q) shows sections from an intermediate type of aortic lesion characterized by a thick neointima with foam cells and a defined media, still with no tissue damage. The third column ( Figure 1C, 1F, 1I, 1M, and 1 R) shows sections from an advanced type of lesion in coronary artery characterized by a confluent mass of extracellular lipids (lipid core) and a structural altered intima. Actin-positive smooth muscle cells (SMCs) were present in the media and intima in all 3 lesion types ( Figure 1A through 1 C, arrows). CD68-positive macrophages were present only in the intima and lipid core of the lesions ( Figure 1D through 1 F, arrows). Immunostained sPLA 2 -V ( Figure 1G through 1 I, double arrows; 1N and 1O) was associated with SMCs colocalizing with actin in the media in intermediate lesions and the neointima of more advanced lesions. sPLA 2 -V was detected only extracellularly surrounding macrophages (M ) foam cell-rich areas and cholesterol crystals ( Figure 1G through 1J and 1L and 1 M, arrows). sPLA 2 -V was also detected in the endothelium of advanced lesions ( Figure 1S and 1 T, arrow) colocalizing with the marker for endothelial cells, von Willebrand factor (vWF), in serial consecutive sections ( Figure 1 U, arrow). vWF-positive endothelial cells in the vasa vasorum also showed positive immunostaining with sPLA 2 -V ( Figure 1V and 1 Z, respectively, arrows). sPLA 2 -V was not present in the adventitia, where we have previously shown sPLA 2 -IIA to be prominent 4 ( Figure 1B and 1 H; supplemental Figure II). All the respective negative controls were devoid of staining ( Figure 1P through 1R and 1W and 1 Y).

Figure 1. Immunohistochemistry of human atherosclerotic lesions. A, D, G, J, N, and O are from an advanced aortic lesion. B, E, H, K, L, P, and Q are from an intermediate type of lesion with foam cells and a defined media. C, F, I, and M and are from an advanced lesion from a coronary artery with a lipid core and a structural altered intima. Actin-positive SMCs are shown in sections A through C (arrows). CD68-positive macrophages are shown in sections D through F (arrows). Immunostained sPLA 2 -V is shown in G through I (double arrows); K, N, and O (arrows) are associated with SMCs colocalizing with actin in the media in intermediate lesions and neointima of more advanced lesions. SPLA 2 -V detected extracellularly surrounding MØ foam cell-rich areas and cholesterol crystals but not intracellularly in these cells shown in G, H, I, J, L, and M (arrows). Frames in G, H, and I are shown at larger magnification in H, K, L, and M. Serial consecutive sections showing positive immunostaining of sPLA 2 -V detected in the endothelium of advanced lesions in S and T (arrows) colocalizing with vWF (U, arrow) and in vasa vasorum (V and Z, arrows). Controls devoid of staining are shown in P, Q, R, W, and Y. Bars shown at the bottom left indicate the true scale for the microscope objective magnification used when taking the pictures. A through I, x 4 objective; bar=250 µm/L. J through S and V, W, and Z, x 40 objective; bar=50 µm/L. T, U, X, and Y, x 60 objective; bar=20 µm/L. A through R are oriented with the arterial lumen to the right. The blue color corresponds to hematoxilin staining.

Immunohistochemical Staining of sPLA 2 -V in Mouse Lesions

Figure 2 shows serial sections from lesions of 2 individual apolipoprotein E and low-density lipoprotein receptor (apoE x LDLr) double deficient mice ( Figure 2A through 2E and 2 H). There were similarities in the immunohistochemical distribution of sPLA 2 -V and that described above for human lesions, with the exception that the enzyme was not detected in mice endothelium (supplemental Figure III). This could have been caused by endothelium disruption during perfusion. Immunostaining showed shared areas positive for sPLA 2 -V ( Figure 2A and 2 E, arrows) and apoB ( Figure 2B and 2 F, arrows) using serial consecutive sections. More extensive colocalization was observed between macrophage-rich areas ( Figure 2D and 2 H) and sPLA 2 -V ( Figure 2A and 2 E) indicated with stars. Colocalization of sPLA 2 -V ( Figure 2 E) with actin-positive cells was also observed ( Figure 2 G, arrowheads).

Figure 2. Immunohistochemistry of mouse atherosclerotic lesions. Serial consecutive sections from the brachiocephalicus artery of lesion from 2 apoExLDLr double-deficient mice (A through D) and fed Western diet (E through H). Sections are oriented with the lumen facing down. Sections show positive staining for sPLA 2 -V (A and E); apoB (B and F); actin (C and G), and macrophages (D and H). A through D, x 20 objective; bar=100 µm/L. E through H, x 40 objective; bar=50 µm/L. Black arrows show shared areas positive for sPLA 2 -V immunostaining (A) and apoB (B); arrowheads show colocalization of sPLA 2 -V (E) with actin (G), and star colocalization of sPLA 2 -V (A) with macrophage immunostaining (D). The blue color corresponds to hematoxilin stain.

mRNA Expression of sPLA 2 -V and sPLA 2 -IIA in Human Vascular Cells, Human Lesions, and CD14-Positive Macrophages From Carotid Artery Lesions

The levels of sPLA 2 -IIA mRNA were 1000-fold higher than those of sPLA 2 -V in all 3 different sources of SMCs in culture, but the relative order of expression for different cells was similar ( Figure 3 ). However, no expression of sPLA 2 -IIA was detected in M, M -loaded with lipids by incubation with acetylated LDL, or in arterial endothelial cells, whereas low levels of sPLA 2 -V mRNA were consistently present in these cells. The expression levels for both enzymes in human fibrotic lesion samples and CD14-positive lesion macrophages were different between samples (donors) but always 2-fold higher for sPLA 2 -IIA than sPLA 2 -V (supplemental Figure IV). Expression levels of the housekeeping gene, 36B4, were similar between the different tissue samples and cultured cells (data not shown).

Figure 3. RT-PCR measurements of sPLA 2 -V and sPLA 2 -IIA mRNA in vascular cells. Each bar corresponds to the cell type used in the experiment: human uterine SMC (uSMC), aortic SMC (aSMC), coronary SMC (cSMC), arterial endothelial cells (aECs), THP-1-derived macrophages (M ), and M preloaded 48 hours with acetylated LDL. Values in the y axis show relative expression of sPLA 2 -V (A) and sPLA 2 -IIA (B) normalized to the housekeeping gene 36B4. Bars are average ±SD values of triplicate determinations from 3 individual cultures for each cell type.

Activity of Recombinant sPLA 2 -IIA and sPLA 2 -V on Human Serum and Lipoproteins

Both enzymes were equally active hydrolyzing phosphoethanolamine micelles and showed substrate specificity (supplemental Figure V). At equal enzyme molar concentrations (14 nmol/L or 200 ng/mL), sPLA 2 -V but not sPLA 2 -IIA hydrolyzed lipoprotein phospholipids in human sera and phospholipids of isolated very low-density lipoprotein (VLDL), LDL, and high-density lipoprotein (HDL; supplemental Figures VI and VII). Kinetic analysis showed that the VLDL surface phospholipids was the preferential substrate for sPLA 2 -V with a K m =546 µmol/L (substrate concentration that gives one-half the maximum velocity), followed by HDL and LDL with a K m of 1320 µmol/L and 3000 µmol/L, respectively ( Figure 4 ). High-performance liquid chromatography analysis of all lipid classes in VLDL, LDL, and HDL incubated 24 hours with recombinant sPLA 2 -V showed a decrease in PC content of all lipoproteins. The phosphatidylethanolamine content was also decreased significantly in VLDL and LDL but not in HDL. This was accompanied by a significant increase in the corresponding lyso-phospholipids ( Table ). No changes were observed in other lipid classes (data not shown), thus indicating that sPLA 2 -V is specific for lipoprotein phospholipids. Complex formation of LDL with proteoglycans can contribute to retention of the LDL in the intima. 24 We found that treatment with sPLA 2 -V increased the LDL-proteoglycans complex formation 20- to 25-fold. Using similar conditions, treatment of LDL with sPLA 2 -II resulted in no increase of the LDL-proteoglycans complex (supplemental Figure VIII).

Figure 4. Binding of sPLA 2 -IIA and sPLA 2 -V to arterial proteoglycans. A, Increasing amounts of sPLA 2 -IIA and sPLA 2 -V were added to 35 S-labeled proteoglycans (2.000 cpm, 0.030 µg) purified from human arterial SMCs. Apparent affinity dissociation constants were determined by 1-site hyperbola binding curves fitted to the data. Added sPLA 2 -IIA and sPLA 2 -V are indicated in the x axis. B, LDL (20 µg apoB, 1.38 µmol/L of phospholipids) was added to free or proteoglycans-bound sPLA 2 -IIA and sPLA 2 -V samples (shown in Figure 4 A). After 2-hour incubation at 37°C, enzymatic activity was monitored by measuring the free fatty acids content. sPLA 2 -IIA proteoglycans-bound ( ) and -free ( ); sPLA 2 -V proteoglycans-bound () and -free ( ). Values are means of duplicate values and are representative of 3 separate experiments.

Lipid Analysis of VLDL and LDL After Incubation With or Without sPLA 2 -V

Interaction of sPLA 2 With SMC Proteoglycans

sPLA 2 -IIA resides in the extracellular intima associated to proteoglycans, a situation that facilitates its action on lipoproteins also bound to the sulfated polysaccharides. The immunohistochemical results discussed above suggest that this could also occur with the group V enzyme ( Figure 1 ). Gel mobility shift assay with metabolically labeled chondroitin-6-sulfate proteoglycans synthesized by human aortic SMCs showed that SPLA 2 -IIA was bound to proteoglycans with an apparent affinity constant ( k d ) of 19 nmol/L, whereas sPLA 2 -V bound with a lower-affinity k d of 951 nmol/L ( Figure 5 A). To study the effect of binding to proteoglycans on the enzymatic activity, LDL was incubated with increasing concentrations of free or proteoglycans-bound sPLA 2 -IIA and sPLA 2 -V. These experiments indicated that LDL is a better substrate for sPLA 2 -V than for sPLA 2 -IIA, corroborating results shown in supplemental Figure VI. More important, the data in printed Figure 5 B demonstrate that when sPLA 2 -V was bound to proteoglycans, the hydrolysis of LDL phospholipids increased significantly. This upregulation of enzymatic activity on LDL phospholipids by binding to proteoglycans was not observed for sPLA 2 -IIA under similar conditions ( Figure 4 B).

Figure 5. Induction of sPLA 2 -V and sPLA 2 -IIA expression in mouse aorta after Western diet and lipopolysaccharide (LPS) challenge, respectively. A, RT-PCR analysis of sPLA 2 -V mRNA from aorta of C57BL/6 mice (n=5) and human sPLA 2 -IIA transgenic mice (n=5). B, Immunoblotting of sPLA 2 -V (100 µg protein per well) and sPLA 2 -IIA (35 µg protein per well) protein extracted from aorta of C57BL/6, apoExLDLr double knockout mice and human sPLA 2 -IIA transgenic mice. C indicates controls on chow diet; LPS, tissue collected 48 hours after an intraperitoneal injection with lipopolysaccharide; WD, tissue from animals on Western diet for 4 weeks. * P <0.01; *** P <0.001.

Induction of sPLA 2 -V and sPLA 2 -IIA Expression in Mouse Aorta

After 4 weeks on a Western diet (0.15% cholesterol, 21% cacao fat), sPLA 2 -V mRNA and protein were induced significantly in the aortas of C57BL/6 mice compared with animals on chow diet ( Figure 5 A). Furthermore, the apoE x LDL receptor double-deficient mice, which develop hyperlipidemia and spontaneous atherosclerosis without a Western diet, showed elevated sPLA 2 -V protein expression in aorta similar to that of C57BL/6 on the Western diet ( Figure 5 B). There was no effect of the Western diet treatment on the spontaneous high levels of enzyme expression in the double knockout mice. On the other hand, acute inflammation triggered by an intraperitoneal injection with lipopolysaccharide (5 mg/kg) lipopolysaccharide did not change sPLA 2 -V expression in C57BL/6 mice but induced significantly the expression of sPLA 2 -IIA in the aorta of the mice transgenic for this human enzyme. Treatment with the Western diet did not affect the aortic expression of sPLA 2 -IIA in the transgenic mice, but similar to the C57BL/6 mice, it increased the expression of sPLA 2 -V (data not shown).

Discussion

Expression of different phospholipase A 2 enzymes in arteries may represent redundant activities because of similar properties. However, our results indicate that group IIA and V sPLA 2 have dissimilar characteristics that could differentially modulate their postulated contribution to atherosclerosis. We found strong immunostaining of sPLA 2 -V extracellularly surrounding macrophage-like foam cells and in lipid-rich core areas. SPLA 2 -V was also present in SMCs in the media and neointima of intermediate and advanced lesions. However, only endothelial cells of advanced lesions showed positive immunostaining for sPLA 2 -V colocalizing with vWF. SPLA 2 -V was not detected in adventitia ( Figure 1; supplemental Figure II). This tissue distribution of sPLA 2 -V in atherosclerotic lesions differs in some aspects from that described previously for group IIA sPLA 2 by our laboratory and other laboratories. 4,23,25-28 These apparent differences should be confirmed using common serial sections of the same tissue samples immunostained with antibodies specific against each enzyme. Levels of sPLA 2 -IIA and sPLA 2 -V mRNA in human lesions and in vitro human cell cultures indicate that cells present in lesions can produce the enzymes (supplemental Figure IV; Figure 3 ). We have previously shown that SMCs are the main source of sPLA 2 -IIA in vivo in arteries and in cell cultures. 4,29 The present data indicate that nonproliferating human coronary SMCs and aortic SMCs have a higher mRNA expression for sPLA 2 -IIA and sPLA 2 -V than those of uterine SMCs, aortic endothelial cells, M, and lipid-loaded M ( Figure 3 ). There are large differences in expression levels of the 2 enzymes in vascular cells and human lesions ( Figure 3; supplemental Figure IV) that could be caused by dissimilar transcription levels or differences in mRNA stability. Interestingly and in accord with the immunohistochemistry data, sPLA 2 -V mRNA but not sPLA 2 -IIA mRNA was detected in human endothelial cells and M and lipid-loaded M ( Figure 3 ). sPLA 2 -IIA mRNA was detected in CD14-positive M from human lesions; however, no mRNA could be detected in THP-1-derived M in vitro, even when loaded with lipids. This discrepancy between ex vivo and in vitro RT-PCR results suggests that sPLA 2 -IIA gene transcription may require both differentiation and local exposure of macrophages to specific stimuli. 30

In the C57BL/6 mouse, no group IIA enzyme is expressed. 21 However, we found sPLA 2 -V expression in the plaques of apoE x LDL receptor double knockout of the same strain ( Figure 2; supplemental Figure III). The distribution pattern of sPLA 2 -V in mouse lesions was similar to that observed in human lesions. In addition, mouse showed shared areas positive for sPLA 2 -V and apoB. This could be caused by sPLA 2 -V entering the subendothelial space associated with apoB lipoproteins. However, we could not detect any association of the enzyme with lipoproteins after fractionation of serum in deuterium oxide gradients at physiological salt concentrations 31 (supplemental Figure IX).

How sPLA 2 -IIA and sPLA 2 -V could modify circulating lipoproteins is not clear. We found that sPLA 2 -V but not sPLA 2 -IIA hydrolyzed lipoprotein phospholipids in the presence of complete serum. When purified plasma lipoproteins were used as substrates, the sPLA 2 -V preferentially hydrolyzed the phospholipidsin VLDL ( K m =546 µmol/L), followed by those of HDL ( K m =1.3 mmol/L) and LDL ( K m =3 mmol/L; supplemental Figures VI and VII). Differences in sphingomyelin content on the lipoproteins surface monolayer may be responsible in part for the dissimilar activities observed. 32 The group IIA sPLA 2 did not hydrolyze phospholipids of any of the native lipoproteins (supplemental Figure VI). SPLA 2 -V has tryptophan residues in the interfacial binding region, which are absent in sPLA 2 -IIA. 33 This probably enables sPLA 2 -V but not type IIA to penetrate and hydrolyze phospholipid monolayers from and lipoproteins in extracellular fluid.

Treatment with sPLA 2 -V increased the association of LDL with arterial proteoglycans (supplemental Figure VIII), thus suggesting that it is better suited than group II sPLA 2 for acting on lipoproteins in the extracellular arterial intima. We speculate that modification of apoB-containing lipoproteins by extracellular sPLA 2 -V may contribute to 2 atherogenic mechanisms: the increased entrapment of partially modified LDL and the generation of proinflammatory lipid products. 7,34 This hypothesis is supported by recent data showing that sPLA 2 -V-modified LDL induces foam cell formation by a process that involves proteoglycans. 35 However, in spite of VLDL being a better substrate for the type V enzyme, we found no consistent increase of its binding to proteoglycans, probably because of the much lower affinity of proteoglycans for triglyceride-rich particles. 36 The consequences of HDL hydrolysis by sPLA 2 -V are unknown and deserve further investigation because a PC-deficient lipoprotein in the intima could be a poor acceptor of excess cell cholesterol. 37

Proteoglycans in the intima may not only contribute to the extracellular accumulation of the 2 enzymes but also to enhancing its activity. sPLA 2 -IIA associated to proteoglycans with higher affinity than sPLA 2 -V ( Figure 5 A). These differences are probably caused by the higher content of basic amino acids in sPLA 2 -IIA. However, the binding of sPLA 2 -V to proteoglycans resulted in a more significant increase in its capacity to hydrolyze LDL phospholipids than for sPLA 2 -IIA ( Figure 5 B). Mutational studies indicate that in human sPLA 2 -V, the interfacial-binding surface is separated from its glycosaminoglycan-binding surface, but in sPLA 2 -IIA, these areas partially overlap, and the glycosaminoglycan-binding surface is more diffuse. 33 Such differences suggest that in sPLA 2 -V, binding to proteoglycans may increase the exposure of the interfacial catalytic domain, thus facilitating the interaction with the substrate. In contrast, the binding of sPLA 2 -IIA to proteoglycans may block the interfacial domain and, as a consequence, impair association with the substrate.

Despite group IIA and V sPLA 2 enzymes being structurally similar, we found that a hyperlipidemic high-fat diet upregulates the expression in aorta of group V but not that of the group IIA. On the other hand, an acute inflammatory stimulus increased group IIA but not group V aortic expression ( Figure 5 ). The response of sPLA 2 -IIA to inflammatory stimuli is well known. 38,39 However, we believe that our finding of the effect of dyslipidemia on the type V enzyme is novel. We speculate that this phenomenon may be related to the described low-level inflammation associated with hyperlipidemia. 40

In conclusion, our results showed clear differences between the 2 enzymes in the expression levels in vascular cells and in their ability to use human lipoproteins in serum as substrates. They also differ in the functional response to associations with arterial proteoglycans. Interestingly, in the mice models used, the enzymes differ in their response to high-fat diet and inflammatory challenge. The described properties profiles of these enzymes suggest that they can affect atherosclerosis by different pathways.

Acknowledgments

This research was conducted with the financial support of the Swedish Heart-Lung Foundation and AstraZeneca, R&D, Mölndal, Sweden. The authors thank Germán Camejo for critical review of this manuscript.

【参考文献】
  Small DM. Progression and regression of atherosclerotic lesions. Insights from lipid physical biochemistry. Arteriosclerosis. 1988; 8: 103-129.

Camejo G, Hurt E, Romano M. Properties of lipoprotein complexes isolated by affinity chromatography from human aorta. Biomed Biochim Acta. 1985; 44: 389-401.

Daugherty A, Zweifel BS, Sobel BE, Schonfeld G. Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits. Arteriosclerosis. 1988; 8: 768-777.

Hurt-Camejo E, Anderssen S, Standal R, Rosengren B, Sartipy P, Stadberg E, Johansen B. Localization of non pancreatic secretory phospholipase A 2 in normal and atherosclerotic arteries: activity of the isolated enzyme on low density lipoprotein. Arterioscler Thromb Vasc Biol. 1997; 17: 300-309.

Sartipy P, Johansen B, Camejo G, Rosengren B, Bondjers G, Hurt-Camejo E. Binding of human phospholipase A 2 type II to proteoglycans: differential effect of glycosaminoglycans on enzyme activity. J Biol Chem. 1996; 271: 26307-26314.

Schissel SL, Jiang X-C, Tweedie-Hardman J, Jeong T-S, Hurt-Camejo E, Najib J, Rapp JH, Williams KJ, Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. J Biol Chem. 1998; 273: 2738-2746.

Camejo G, Hurt-Camejo E, Wiklund O, Bondjers G. Association of apoB lipoproteins with arterial proteoglycans: pathological significance and molecular basis. Atherosclerosis. 1998; 139: 205-222.

Hakala JK, Öörni K, Pentikäinen MO, Hurt-Camejo E, Kovanen PT. Lipolysis of LDL by human secretory phospholipase A 2 induces particle fusion and enhances the retention of LDL to human aortic proteoglycans. Arterioscler Thromb Vasc Biol. 2001; 21: 1053-1058.

Hurt-Camejo E, Camejo G, Peilot H, Öörni K, Kovanen P. Phospholipase A 2 in vascular disease. Circ Res. 2001; 89: 298-304.

Chang MY, Tsoi C, Wight Tn, Chait A. Lysophophatidylcholine regulates synthesis of biglycan and the proteoglycan form of macrophage colony stimulating factor. Arterioscler Thromb Vasc Biol. 2003; 23: 809-815.

Rinker KD, Kirkpatrick AP, Ting-Beall HP, Shephered RD, Levin JD, Irick J, Thomas JL, Truskey GA. Linoleic acid increases monocyte deformation and adhesion to endothelium. Atherosclerosis. 2004; 177: 275-285.

Benitez S, Camacho M, Arcelus R, Vila L, Bancells C, Ordonez-Llanos J, Sanchez-Quesada JL. Increased lysophosphaidylcholine and non-esterified fatty acid content in LDL induces chemokine release in endothelial cells. Relationship with electronegative LDL. Atherosclerosis. 2004; 177: 299-305.

Ivandic B, Castellini LW, Wang X-P, Qiao J-H, Mehrabian M, Navab M, Fogelman AM, Grass DS, Swanson ME, Beer MCD, Beer FD, Lusis AJ. Role of group II secretory phospholipase A 2 in atherosclerosis. 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A 2. Arterioscler Thromb Vasc Biol. 1999; 19: 1284-1290.

Hurt-Camejo E, Paredes S, Masana L, Camejo G, Sartipy P, Rosengren B, Pedreno J, Vallve JC, Benito P, Wiklund O. Rheumatoid arthritis patients have elevated levels of small low density lipoprotein with high affinity for arterial matrix components: possible contribution of phoshoplipase A2 to this atherogenic profile. Arthritis Rheum. 2001; 44: 2761-2767.

Leinonen E, Hurt-Camejo E, Wiklund O, Mattson-Hultén L, Hiukka A, Taskinen M-R. Insulin resistance and adipocity correlate with acute-phase reaction and soluble cell adhesion molecules in type 2 diabetes. Atherosclerosis. 2003; 166: 387-394.

Tietge UJF, Pratico D, Ding T, Funk C, Hildebrand R, Berkel TV, Eck MV. Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress. J Lipid Res. 2005; 46: 1604-1614.

Boekholdt SM, Keller TT, Wareham NJ, Luben R, Bingham SA, Day NE, Sandhu MS, Jukema JW, Kastelein JJP, Hack CE, Khaw K-T. Serum levels of type II secretory phospholipase A 2 and the risk of future coronary artery disease in apparently healthy men and women. The EPIC-Norfolk prospective population study. Arterioscler Thromb Vasc Biol. 2005; 25: 839-846.

Six DA, Dennis EA. The expanding superfamily of phospholipase A 2 enzymes: classification and characterization. Biochim Biophys Acta. 2000; 1488: 1-19.

Tischfield J, Xia Y, Shih D, Klisak I, Chen J, Engle S, Siakotos A, Winstead M, Seilhamer J, Allamand V, Gyapay G, Lusis A. Low-molecular-weight, calcium-dependent phospholipase A 2 genes are linked and map to homologous chromosome regions in mouse and human. Genetics. 1996; 32: 328-333.

Welch CL, Bretschger S, Latib N, Bezouevski M, Guo Y, Pleskac N, Liang C-P, Barlow C, Dansky H, Breslow JL, Tall AR. Localization of atherosclerosis susceptibility loci to chromosome 4 and 6 using the LDLr knockout mouse model. Proc Natl Acad Sci U S A. 2001; 98: 7946-7951.

Kennedy BP, Payette P, Mudgett J, Vadas P, Pruzanski W, Kwan M, Tang C, Rancourt DE, Cromlish WAA. Natural disruption of the secretory group II phospholipase A 2 gene in inbred mouse strains. J Biol Chem. 1995; 270: 22378-22385.

Wooton-Kee CR, Boyanovsky BB, Nasser MS, Villiers WJd, Webb NR. Group V sPLA2 hydrolysis of low-density lipoprotein results in spontaneous particle aggregation and promotes macrophages foam cell formation. Arterioscler Thromb Vasc Biol. 2004; 24: 762-767.

Romano M, Romano E, Björkerud S, Hurt-Camejo E. Ultrastructural localization of secretory type II phospholipase A 2 in atherosclerotic and nonatherosclerotic regions of human arteries. Arterioscler Thromb Vasc Biol. 1998; 18: 519-525.

Camejo G. The interaction of lipids and lipoproteins with the intercellular matrix of arterial tissue: its possible role in atherogenesis. Adv Lipid Res. 1982; 19: 1-53.

Menschikowski M, Kasper M, Lattke P, Schiering A, Schiefer S, Stockinger H, Jaross W. Secretory group II phospholipase A 2 in human atherosclerotic plaques. Atherosclerosis. 1995; 118: 173-181.

Elinder LS, Dumitrescu A, Larsson P, Hedin U, Fostergård J, Claesson H-E. Expression of phospholipase A2 isoforms in human normal and atherosclerotic arterial wall. Arterioscler Thromb Vasc Biol. 1997; 17: 2257-2263.

Sartipy P, Johansen B, Gåsnik K, Hurt-Camejo E. Molecular basis for the association of group IIA phospholipase A 2 and decorin in human atherosclerotic lesions. Circ Res. 2000; 86: 707-714.

Menschikowski M, Eckey R, Pietsch J, Auffenanger J, Kumpf R, Nelz P, Jaross W. Expression of human secretory group IIA phospholipase A 2 is associated with reduced concentrations of plasma cholesterol in transgenic mice. Inflammation. 2000; 24: 227-237.

Peilot H, Rosengren B, Bondjers G, Hurt-Camejo E. IFN-gamma induces secretory group IIA phospholipase A 2 in human arterial smooth muscle cells. Involvement of cell differentiation, STAT-3 activation and modulation byother cytokines. J Biol Chem. 2000; 275: 22895-22904.

Anthonsen M, Stengel D, Hourton D, Ninio E, Johansen B. Mildly oxidized LDL induces expression of group IIa secretory phospholipase A 2 in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol. 2000; 20: 1276-1282.

Davidsson P, Hulthe J, Fagerberg B, Olsson B-M, Hallberg C, Dahllöf B, Camejo G. A proteomic study of the apolipoproteins in LDL aubclasses in patients with the metabolic syndrome and type 2 diabetes. J Lipid Res. 2005; 46: 1999-2006.

Gesquiere L, Cho W, Subbaiah P. Role of group IIa and group V secretory phospholipase A 2 in the metabolism of lipoproteins. Substrate specificities of the enzymes and the regulation of their activities by sphingomyelin. Biochemistry. 2002; 41: 4911-4920.

Cho W. Structure, function, and regulation of group V phospholipase A 2. Biochim Biophys Acta. 2000; 1488: 48-58.

Williams KJ, Tabas I. Lipoprotein retention and clues for atheroma regression. Arterioscler Thromb Vasc Biol. 2005; 25: 1536-1540.

Boyanovsky BB, Westhuyzen DRVD, Webb NR. Group V secretory phsopholipase A2-modified low density lipoprotein promotes foam cell formation by a SR-A- and CD36-independnet process that involves cellular proteoglycans. J Biol Chem. 2005; 280: 32746-32752.

Olsson U, Camejo G, Bondjers G. Binding of a synthetic apolipoprotein B-100 peptide and peptide analogues to chondroitin-6-sulfate: effect of the lipid environment. Biochemistry. 1993; 32: 1858-1865.

Chung B-H, Franklin F, Liang P, Doran S, Cho BHS, Curcio CA. Phosphatidylcholine-rich acceptors, but not native HDL or its apolipoproteins, mobilize cholesterol from cholesterol-rich insoluble components of human atherosclerotic plaques. Biochim Biophys Acta. 2005; 1733: 76-89.

Laine VJO, Grass DS, Nevalainen TJ. Protection by group II phospholipase A 2 against Staphylococcus aureu s. J Immunol. 1999; 162: 7402-7408.

Tietge UJ, Maugeais C, Cain W, Rader DJ. Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2. Acta Physiol Scand. 2003; 285: E403-E411.

Lewis KE, Kirk EA, McDonald TO, Wang S, Wight TN, Wallick S, Andersson M, Aikawa K, Kahn SE. Increased in serum amyloid A evoked by dietary cholesterol is associated with increased atherosclerosis in mice. Circulation. 2004; 110: 540-545.

作者单位：Birgitta Rosengren; Helena Peilot; Mia Umaerus; Ann-Cathrine Jönsson-Rylander; Lillemor Mattsson-Hultén; Carina Hallberg; Philippe Cronet; Mariam Rodriguez-Lee; Eva Hurt-CamejoFrom AstraZeneca (B.R., M.U., A.-C.J.-R., C.H., P.C., E.H.-C.), R&D, Molecular Pharmacology, Mölndal, S