Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.     

Group V secretory phospholipase A2-modified low density lipoprotein promotes foam cell formation by a SR-A- and CD36-independent process that involves cellular proteoglycans

Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR-/-), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR-/- compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors.     

Macrophage foam cell formation with native low density lipoprotein

This investigation has elucidated a mechanism for development of macrophage foam cells when macrophages are incubated with native low density lipoprotein (LDL). LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. We have found that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate (PMA) stimulates LDL uptake and degradation and acyl-CoA:cholesterol acyltransferase-mediated esterification of LDL-derived cholesterol, resulting in massive macrophage cholesterol accumulation that could exceed 400 nmol/mg of cell protein. Cholesterol accumulation showed a biphasic linear LDL concentration dependence with LDL levels as high as 4 mg/ml, similar to LDL levels in artery intima. Protein kinase C mediated the PMA-stimulated macrophage uptake of LDL because the protein kinase C inhibitors, G?6983 and GF109203X, inhibited cholesterol accumulation. LDL receptors did not mediate macrophage cholesterol accumulation because accumulation occurred with reductively methylated LDL and in the presence of an anti-LDL receptor-blocking monoclonal antibody. LDL-induced cholesterol accumulation was not inhibited by antioxidants, was not accompanied by increased LDL binding to macrophages, did not depend on the apoB component of LDL, and was not down-regulated by prior cholesterol enrichment of macrophages. We have shown that the mechanism of LDL uptake by macrophages was PMA-stimulated endocytosis of LDL taken up as part of the bulk phase fluid (i.e. fluid phase endocytosis). The amount of LDL taken up with the bulk phase fluid was measured with [(3)H]sucrose and accounted for a minimum of 83% of the LDL cholesterol delivery and accumulation in PMA-activated macrophages. This novel mechanism of macrophage cholesterol accumulation shows that modification of LDL is not necessary for foam cell formation to occur. In addition, the findings direct attention to macrophage fluid phase endocytosis as a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification

Low density lipoprotein (LDL) can be oxidatively modified by cultured endothelial cells or by cupric ions, resulting in increased macrophage uptake of the lipoprotein. This process could be relevant to the formation of macrophage-derived foam cells in the early atherosclerotic lesion. The mechanism of endothelial cell modification of LDL is unknown. In the present work we show that incubation of LDL with purified soybean lipoxygenase, in the presence of pure phospholipase A2, can mimic endothelial cell-induced oxidative modification. Typically, incubation with lipoxygenase plus phospholipase A2 caused: 1) generation of about 15 nmol of thiobarbituric acid-reactive substances per mg of LDL protein; 2) a 4- to 7-fold increase in the rate of subsequent macrophage degradation of the LDL; 3) a 10-fold decrease in recognition by fibroblasts; 4) a marked increase in electrophoretic mobility in agarose gels; and, 5) disappearance of intact apoprotein B on SDS polyacrylamide gels. Degradation of the enzymatically modified LDL by macrophages was competitively inhibited by endothelial cell-modified LDL and by polyinosinic acid, but only partially suppressed by acetylated LDL. The lipoxygenase plus phospholipase A2-induced modification of LDL is not necessarily identical to endothelial cell modification, but it is a useful model for studying the mechanism of oxidative modification of LDL. This work also represents the first example of oxidative modification of LDL by specific enzymes leading to enhanced recognition by macrophages.     

Group V and X secretory phospholipase A(2)s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions

The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A(2) (sPLA(2)-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA(2)-X as well as group V secretory PLA(2) (sPLA(2)-V), another group of sPLA(2) that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA(2)s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA(2)-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA(2)-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA(2)-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA(2)-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA(2)-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.     

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.     

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice

The role of macrophage lipoprotein lipase (LPL) expression in atherosclerotic lesion formation was examined in low density lipoprotein receptor (LDLR(-/-)) mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions. First, LDLR(-/-) mice chimeric for macrophage LPL expression were created by transplantation of lethally irradiated female LDLR(-/-) mice with LPL(-/-) (n = 12) or LPL(+/+) (n = 14) fetal liver cells as a source of hematopoietic cells. To induce fatty streak lesions, these mice were fed a Western diet for 8 weeks, resulting in severe hypercholesterolemia. There were no differences in plasma post-heparin LPL activity, serum lipid levels, or lipoprotein distribution between these two groups. The mean lesion area in the proximal aorta in LPL(-/-) --> LDLR(-/-) mice was significantly reduced by 33% compared with LPL(+/+) --> LDLR(-/-) mice, and a similar reduction (38%) in lesion area was found by en face analysis of the aortae. To induce complex atherosclerotic lesions, female LDLR(-/-) mice were lethally irradiated, transplanted with LPL(-/-) (n = 14), LPL(+/-) (n = 13), or LPL(+/+) (n = 14) fetal liver cells, and fed the Western diet for 19 weeks. Serum cholesterol and triglyceride levels did not differ between the three groups. After 19 weeks of diet, the lesions in the proximal aorta were complex with relatively few macrophages expressing LPL protein and mRNA in LPL(+/+) --> LDLR(-/-) mice. Analysis of cross-sections of the proximal aorta demonstrated no differences in the extent of lesion area between the groups, whereas en face analysis of the aortae revealed a dose-dependent effect of macrophage LPL on mean aortic lesion area in LPL(-/-) --> LDLR(-/-), LPL(-/+) --> LDLR(-/-), and LPL(+/+) --> LDLR(-/-) mice (1.8 +/- 0. 2%, 3.5 +/- 0.5% and 5.9 +/- 0.8%, respectively). Taken together, these data indicate that macrophage LPL expression in the artery wall promotes atherogenesis during foam cell lesion formation, but this impact may be limited to macrophage-rich lesions.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.     

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A₂ (sPLA₂-IIa and sPLA₂-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA₂-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA₂-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA₂-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.     

A modification of apolipoprotein B accounts for most of the induction of macrophage growth by oxidized low density lipoprotein

It has recently been shown that macrophage proliferation occurs during the progression of atherosclerotic lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this include the interaction of oxidized LDL with integral plasma membrane proteins coupled to signaling pathways, the release of growth factors and autocrine activation of growth factor receptors, or the potentiation of mitogenic signal transduction by a component of oxidized LDL after internalization. The present study was undertaken to further elucidate the mechanisms involved in the growth-stimulating effect of oxidized LDL in macrophages. Only extensively oxidized LDL caused significant growth stimulation, whereas mildly oxidized LDL, native LDL, and acetyl LDL were ineffective. LDL that had been methylated before oxidation (to block lysine derivatization by oxidation products and thereby prevent the formation of a scavenger receptor ligand) did not promote growth, even though extensive lipid peroxidation had occurred. The growth stimulation could not be attributed to lysophosphatidylcholine (lyso-PC) because incubation of oxidized LDL with fatty acid-free bovine serum albumin resulted in a 97% decrease in lyso-PC content but only a 20% decrease in mitogenic activity. Similarly, treatment of acetyl LDL with phospholipase A2 converted more than 90% of the initial content of phosphatidylcholine (PC) to lyso-PC, but the phospholipase A2-treated acetyl LDL was nearly 10-fold less potent than oxidized LDL at stimulating growth. Platelet-activating factor receptor antagonists partly inhibited growth stimulation by oxidized LDL, but platelet-activating factor itself did not induce growth. Digestion of oxidized LDL with phospholipase A2 resulted in the hydrolysis of PC and oxidized PC but did not attenuate growth induction. Native LDL, treated with autoxidized arachidonic acid under conditions that caused extensive modification of lysine residues by lipid peroxidation products but did not result in oxidation of LDL lipids, was equal to oxidized LDL in potency at stimulating macrophage growth. Albumin modified by arachidonic acid peroxidation products also stimulated growth, demonstrating that LDL lipids are not essential for this effect. These findings suggest that oxidatively modified apolipoprotein B is the main growth-stimulating component of oxidized LDL, but that oxidized phospholipids may play a secondary role.     

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation

Accumulation of macrophage foam cells in atherosclerotic blood vessel intima is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by coimmunoprecipitation and pull-down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2. The MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify potential targets for antiatherosclerosis therapy.     

A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation.

To evaluate the contribution of the macrophage low density lipoprotein receptor (LDLR) to atherosclerotic lesion formation, we performed bone marrow transplantation studies in different mouse strains. First, LDLR(-/-) mice were transplanted with either LDLR(+/+) marrow or LDLR(-/-) marrow and were challenged with an atherogenic Western type diet. The diet caused severe hypercholesterolemia of a similar degree in the two groups, and no differences in the aortic lesion area were detected. Thus, macrophage LDLR expression does not influence foam cell lesion formation in the setting of extreme LDL accumulation. To determine whether macrophage LDLR expression affects foam cell formation under conditions of moderate, non-LDL hyperlipidemia, we transplanted C57BL/6 mice with either LDLR(-/-) marrow (experimental group) or LDLR(+/+) marrow (controls). Cholesterol levels were not significantly different between the two groups at baseline or after 6 weeks on a butterfat diet, but were 40% higher in the experimental mice after 13 weeks, mostly due to accumulation of beta-very low density lipoprotein (beta-VLDL). Despite the increase in cholesterol levels, mice receiving LDLR(-/-) marrow developed 63% smaller lesions than controls, demonstrating that macrophage LDLR affects the rate of foam cell formation when the atherogenic stimulus is beta-VLDL. We conclude that the macrophage LDLR is responsible for a significant portion of lipid accumulation in foam cells under conditions of dietary stress.     

Potential role of group X secretory phospholipase A(2) in cyclooxygenase-2-dependent PGE(2) formation during colon tumorigenesis

Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.     

Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis

Plasma levels of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA₂ is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA₂ and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA₂ protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA₂ mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA₂ were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA₂ were negatively correlated (r = − 0.578). The ratio of Lp-PLA₂ to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA₂ existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA₂ epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA₂ and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.     

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.