Abstract
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.
Highlights
A critical event in early atherogenesis is the formation of lipid-laden macrophages (“foam cells”) [1,2,3,4]
2 The abbreviations used are: LDL, low density lipoprotein; PL, phospholipid; Cholesterol Ester (CE), cholesterol ester; GV sPLA2, group V secretory phospholipase A2; GV sPLA2-modified LDL (GV-LDL), LDL hydrolyzed by Group V sPLA2; mock-LDL, LDL incubated in hydrolysis buffer in the absence of enzyme; ox-LDL, Cu2ϩ-oxidized LDL; vx-LDL, LDL aggregated by vortexing; heparan sulfate proteoglycans (HSPG), heparan sulfate proteoglycan; BSA, bovine serum albumin; WT, wild type, DKO, double knock-out; LDL receptorrelated protein (LRP), LDL receptor-related protein
We have focused on modification of LDL by Group V sPLA2, a member of the secretory phospholipase A2 family of enzymes shown by others to have potent activity toward phosphatidylcholine-containing substrates [40, 41]
Summary
A critical event in early atherogenesis is the formation of lipid-laden macrophages (“foam cells”) [1,2,3,4]. Several types of modifications of LDL, such as oxidation (6 – 8), depletion of sphingomyelin by secretory sphingomyelinase [9], hydrolysis of glycero-PLs by sPLA2 enzymes (10 –12), and aggregation [13,14,15,16,17,18] have been implicated in lipid accumulation in the vessel wall. Accumulating evidence indicates that sPLA2 hydrolysis of LDL-PL results in structural alterations of the particles that promote lipid accumulation in the vessel wall and enhances macrophage uptake. Our results indicate that Group V sPLA2-hdyrolyzed LDL promotes macrophage foam cell formation through a pathway that is distinct from other modified forms of LDL and involves cell-surface proteoglycans
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