Acid sphingomyelinase plays important roles in ceramide homeostasis, which has been proposed to be linked to insulin resistance. To test this association in vivo, acid sphingomyelinase deletion (asm ؊/؊ ) was transferred to mice lacking the low density lipoprotein receptor (ldlr ؊/؊ ), and then offsprings were placed on control or modified (enriched in saturated fat and cholesterol) diets for 10 weeks. The modified diet caused hypercholesterolemia in all genotypes; however, in contrast to asm ؉/؉ /ldlr ؊/؊ , the acid sphingomyelinase-deficient littermates did not display hepatic triacylglyceride accumulation, although sphingomyelin and other sphingolipids were substantially elevated, and the liver was enlarged. asm ؊/؊ /ldlr ؊/؊ mice on a modified diet did not accumulate body fat and were protected against diet-induced hyperglycemia and insulin resistance. Experiments with hepatocytes revealed that acid sphingomyelinase regulates the partitioning of the major fatty acid in the modified diet, palmitate, into two competitive and inversely related pools, triacylglycerides and sphingolipids, apparently via modulation of serine palmitoyltransferase, a rate-limiting enzyme in de novo sphingolipid synthesis. These studies provide evidence that acid sphingomyelinase activity plays an essential role in the regulation of glucose metabolism by regulating the hepatic accumulation of triacylglycerides and sphingolipids during consumption of a diet rich in saturated fats.

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This review merely scratches the surface of some of the actions of sPLA(2)s in innate immunity, inflammation, and atherosclerosis. The goal is to provide an overview of recent findings involving sPLA(2)s and to point to potential pathophysiologic mechanisms that may become targets for therapy.

Objectives-Secretory phospholipase A 2 (sPLA 2 ) enzymes hydrolyze the sn-2 fatty acyl ester bond of phospholipids to produce a free fatty acid and a lysophospholid. Group V sPLA 2 is expressed in cultured macrophage cells and has high affinity for phosphatidyl choline-containing substrates. The present study assesses the presence of group V sPLA 2 in human and mouse atherosclerotic lesions and its activity toward low-density lipoprotein (LDL) particles. Methods and Results-Group V sPLA 2 was detected in human and mouse atherosclerotic lesions by immunohistochemical staining. Electron microscopic analysis showed that mouse group V sPLA 2 -modified LDL is significantly smaller (mean diameterϮSEMϭ25.3Ϯ0.25 nm) than native LDL (mean diameterϮSEMϭ27.7Ϯ0.29 nm). Hydrolysis by group V sPLA 2 induced spontaneous particle aggregation; the extent of aggregation was directly proportional to the degree of LDL hydrolysis. Group V sPLA 2 modification of LDL led to enhanced lipid accumulation in cultured mouse peritoneal macrophage cells. Conclusions-Group V sPLA 2 may play an important role in promoting atherosclerotic lesion development by modifying LDL particles in the arterial wall, thereby enhancing particle aggregation, retention, and macrophage uptake. Key Words: atherosclerosis Ⅲ group V secretory phospholipase A 2 Ⅲ LDL aggregation Ⅲ macrophages A critical event in early atherogenesis is the retention of low-density lipoprotein (LDL) particles in the subendothelium. Accumulating evidence points to LDL aggregation and LDL fusion as key elements of atherogenic lipid accumulation in the artery wall. 1 Aggregated lipoproteins that appear to be derived from LDL are prominent in early atherosclerotic lesions. 2,3 Aggregated LDL is taken-up by macrophages in vitro at an enhanced rate compared with non-aggregated LDL, leading to macrophage cholesterol accumulation and foam cell formation. 4,5 Because native LDL particles do not form aggregates, LDL modification appears to be a prerequisite for aggregation/fusion. Studies in vitro indicate that hydrolysis of LDL by secretory phospholipases A 2 (sPLA 2 ) may be linked to LDL aggregation and/or fusion and enhanced retention in the subendothelium. 3,6 The sPLA 2 family comprises a group of enzymes that hydrolyze the fatty acid esterified at the sn-2 position of glycerophospholipids. 7 The secreted enzymes are of low molecular weight (14 kDa), highly enriched in disulfide bonds, and require 1 to 10 mmol/L calcium for activity. The major secreted form present in synovial fluid, termed group IIa, has been proposed to act as a mediator of inflammatory responses. During acute or chronic inflammation, the concentration of group IIa sPLA 2 can increase by Ͼ100-fold in inflammatory fluids and plasma. 8,9 Immunohistochemistry studies have established that group IIa sPLA 2 is present in normal arterial tissue and increased in atherosclerotic lesions. 10 -12 We recently reported that macrophage expression of human group IIa sPLA 2 significantly enhances atherosclerotic lesion formation in...

Accumulating evidence indicates that secretory phospholipase A 2 (sPLA 2 ) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA 2 (GV sPLA 2 ) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA 2 -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. 125 I-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 NaClO 3 , 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.A critical event in early atherogenesis is the formation of lipid-laden macrophages ("foam cells") (1-4). According to the "response-to-retention" hypothesis (5), conditions leading to enhanced LDL 2 entrapment in the subendothelium trigger this process. Once retained in the vessel wall, LDL undergoes various modifications in both protein and phospholipid (PL) moieties that promote retention and lead to enhanced macrophage uptake. Several types of modifications of LDL, such as oxidation (6 -8), depletion of sphingomyelin by secretory sphingomyelinase (9), hydrolysis of glycero-PLs by sPLA 2 enzymes (10 -12), and aggregation (13-18) have been implicated in lipid accumulation in the vessel wall.The sPLA 2 family comprises a group of enzymes that hydrolyze the acyl-ester bond at the 2 position (sn-2) of glycero-PLs. Of the 10 sPLA 2 isozymes that have been described in mammals, three members (Group IIA, Group V, and Group X) have been detected in human and/or mouse atherosclerotic lesions (10,11,19). Accumulating evidence indicates that sPLA 2 hydrolysis of LDL-PL results in structural alterations of the particles that promote lipid accumulation in the vessel wall and enhances macrophage uptake. Hydrolysis of LDL by sPLA 2 in vitro results in an increased affinity for proteoglycans, which would be expected to incre...

Objective-Group V secretory phospholipase A 2 (GV sPLA 2 ) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA 2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo. Methods and Results-We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA 2 in atherogenesis in LDL receptor-deficient mice. Compared with control mice, animals overexpressing GV sPLA 2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow-derived GV sPLA 2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta. Conclusions-Our data in mouse models provide the first in vivo evidence that GV sPLA 2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease. Key Words: Group V secretory phospholipase A 2 Ⅲ atherosclerosis Ⅲ retrovirus-mediated gene transfer Ⅲ bone marrow transplantation T he secretory phospholipase A 2 (sPLA 2 ) family of enzymes hydrolyze the fatty acid esterified at the sn-2 position of glycerophospholipids. 1 Of the 10 sPLA 2 s described in mammals, Group IIA (GIIA), Group V (GV), and Group X (GX) sPLA 2 have been detected in human and/or mouse atherosclerotic lesions. [2][3][4] These enzymes have been proposed to exert multiple proatherogenic effects in the arterial wall. Phospholipid hydrolysis by sPLA 2 generates potentially bioactive lipids, namely free fatty acids and lysophospholipids, which may promote various proinflammatory processes. Hydrolysis by either GV or GX sPLA 2 markedly reduces the capacity of HDL to promote cellular cholesterol efflux from lipid-loaded macrophages. 5 Hydrolysis of LDL by sPLA 2 in vitro results in an increased affinity for extracellular matrix proteoglycans and promotes LDL aggregation. 3,6 When incubated with mouse peritoneal macrophages, LDL hydrolyzed by either GV or GX sPLA 2 induces foam cell formation. 2,3 Thus, in vitro studies suggest that sPLA 2 s could promote atherogenesis by increasing the retention of LDL particles in the subendothelium and by generating potent inducers of macrophage foam cells. See page 445In this study, we directly tested the hypothesis that GV sPLA 2 promotes atherosclerosis in vivo. Using both gain-offunction and loss-of-function approaches, we demonstrate for the first time that bone marrow-derived GV sPLA 2 contributes to atherogenesis in LDL receptor-deficient mice. Methods Generation of Retroviral VectorsR...

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation. Macrophage binding and uptake of oxidized low density lipoprotein (OxLDL) has been proposed to play a key role in the initiation of atherosclerotic lesion development, the formation of lipid-laden foam cells. CD36 is one of several OxLDL receptors contributing to this process (1, 2). CD36 is an 88 kDa plasma membrane glycoprotein that binds a diverse array of ligands in addition to OxLDL, including thrombospondin-1 (3), the native lipoproteins LDL, HDL, and VLDL (4), long-chain fatty acids (5), anionic phospholipids (6), and apoptotic cells (7). As a result of its broad specificity, CD36 has been reported to contribute to various normal and pathologic processes, such as apoptotic cell clearance, fatty acid transport, angiogenesis, atherosclerosis, inflammation, and lipid metabolism (reviewed in Ref. 8). CD36 is expressed in a range of cells and tissues that includes monocytes/macrophages, platelets, mammary epithelial cells, vascular endothelial cells, and adipose tissues (9). Studies performed ex vivo indicate that 60-70% of macrophage foam cell formation induced by OxLDL may be CD36-dependent (10-12). CD36-mediated OxLDL degradation has been reported in cell lines in addition to macrophages, including adipocytes (13). Studies in Chinese hamster ovary (CHO) cells transfected with CD36 cDNA and C32 cells with endogenous expression of CD36 have investigated CD36 trafficking after OxLDL binding. The results indicate that binding of OxLDL to CD36 leads to the internalization of CD36 and OxLDL into endosomal compartments that do not contain caveolin-1 or transferrin but do contain a glycosylphosphatidylinositol-a...