Fusion and Atherogenic Properties of Enzymatically Modified Low Density Lipoprotein Particles

A is an inflammatory disease characterized by the appearance of small extracellular lipid droplets, formation of foam cells, and secretion of pro-inflammatory cytokines by vascular cells in the arterial intima. There is substantial evidence that the extracellular lipid droplets are derived directly from aggregated and fused low density lipoprotein (LDL) particles. However, native LDL particles do not aggregate or fuse unless they have been extensively modified. In fact, a variety of proteolytic, lipolytic, and oxidative enzymes and agents that are capable of degrading LDL in vitro, have also been found in the arterial wall. Human Type IIA secretory phospholipase A2 (PLA2) is an enzyme whose plasma level may increase dramatically during inflammatory diseases. It has been found in all the stages of atherosclerotic lesions, and has been shown to be able to degrade LDL in vitro. In addition, there is indirect evidence that once apolipoprotein B-100 containing lipoproteins enter the intima, hydrolysis of phosphatidylcholine, the major phospholipid of the LDL surface, by a PLA2-like activity takes place. In this study, Type IIA secretory PLA2 was shown to induce fusion of the LDL particles in the presence of proteoglycans (PGs), an important structural component of the arterial intima. In fact, the presence of PGs appeared to be a prerequisite for the lipolytic particle fusion. In addition, the binding strength of the fused LDL particles to PGs appeared to be increased, which promoted accumulation of the lipolyzed LDL to the PG-matrix. Enrichment of the PLA2-treated LDL with free fatty acids and lysophosphatidylcholines abolished fusion of the lipolyzed particles and detached a fraction of the PG-bound particles, suggesting that removal of the reaction product may have an important regulatory role in the formation of lipid droplets and their retention by PGs. Extracellular discharge of the lysosomal enzymes has been shown to be a common physiologic response to a variety of inflammatory stimuli. Cathepsin D and lysosomal acid lipase are lysosomal hydrolases that play major roles in the degradation of LDL in lysosomal compartments. In this study, we could show that both enzymes exist extracellularly in the human atherosclerotic intima and that, upon activation, human monocyte-derived macrophages can secrete these enzymes into the cell culture media. Incubation of LDL with the macrophage-conditioned media containing cathepsin D and lysosomal acid lipase (among other secreted hydrolases) induced fusion of LDL particles in vitro. Analysis of this hydrolase-modified LDL revealed that cathepsin D and lysosomal acid lipase had major roles in the degradation of LDL by macrophage-conditioned media. These particles were taken up avidly by macrophages and human coronary artery smooth muscle cells, which were transformed into foam cells. Hydrolase-modified LDL also induced expression and secretion of pro-inflammatory chemokines and cytokines, such as interleukin 8, monocyte chemoattractant protein 1, and interleukin 6. It was found that secretion of interleukin 8 from hydrolase-modified LDL-treated macrophages involved activation of p38 mitogen-activated protein kinase and nuclear translocation of nuclear-factor kappa B. Taken together, our in vitro data are compatible with the idea that PLA2 and lysosomal acid hydrolases, which have both been found in human atherosclerotic lesions, are able to transform LDL into fused particles, which resemble extracellular lipid droplets found in the arterial intima during atherogenesis.