Objective-Human atherosclerotic lesions have been shown to contain lipid droplets and vesicles resembling those of in vitro enzymatically modified LDL. However, little is known about the hydrolytic enzymes in the arterial intima that induce fusion of LDL particles and so produce lipid droplets or that induce foam cell formation. Methods and Results-Human coronary atherosclerotic lesions obtained at surgery and at autopsy were stained for lysosomal acid lipase and cathepsin D. The extracellular areas of macrophage-rich intimal regions of the atherosclerotic lesions stained positively for both cathepsin D and lysosomal acid lipase, whereas normal arteries were negative. When monocyte-derived macrophages were incubated with opsonized zymosan to stimulate the release of lysosomal enzymes from the cells and LDL was incubated with the macrophage-conditioned media, the apolipoprotein B-100, cholesteryl esters, and triacylglycerols of LDL were hydrolyzed. These hydrolytic modifications rendered the LDL particles unstable and induced their fusion. Cultured macrophages and smooth muscle cells took up the hydrolase-modified LDL particles avidly and were transformed into foam cells. Key Words: macrophage Ⅲ lysosomal enzymes Ⅲ LDL Ⅲ atherosclerosis A therogenesis is characterized by accumulation of LDLderived lipids in the extracellular matrix of the arterial intima, recruitment of monocyte-derived macrophages, and their transformation into lipid-laden foam cells. 1 The lipid accumulates extracellularly in atherosclerotic lesions in the form of small lipid droplets and vesicles. 2 Modification of LDL in vitro by proteolysis, lipolysis, and oxidation has been shown to produce particles resembling those observed in the arterial intima. [3][4][5][6][7][8][9][10] Lipid particles enriched in unesterified cholesterol have been isolated from the human arterial intima, 11 and evidence for the presence of the particles resembling in vitro enzymatically modified LDL 3 has been provided by the immunohistochemistry. 12 However, the enzymes responsible for such LDL modification in the arterial intima have remained uncharacterized. Conclusions-Our See page 1312Two enzymes that participate in the hydrolysis of endocytosed LDL in the lysosomal compartment of cells are cathepsin D and lysosomal acid lipase (LAL). The former initiates the lysosomal degradation of apolipoprotein B-100 (apoB-100) 13 and is found in atherosclerotic intima. 14,15 The latter is normally responsible for lysosomal hydrolysis of cholesteryl esters and triacylglycerols of the endocytosed LDL particles. 16 Extracellular discharge of lysosomal enzymes is a common physiological response to a variety of inflammatory stimuli. 17,18 Under pathologic conditions, cathepsin D has been found extracellularly in many tissues, eg, in the invading edge of the rheumatoid synovium, 19 MethodsAn online Methods section is available at http://atvb.ahajournals.org. ResultsThe presence of the lysosomal enzymes cathepsin D and LAL was studied by immunohistochemistry in normal and in ather...

Abstract-The first morphological sign of atherogenesis is the accumulation of extracellular lipid droplets in the proteoglycan-rich subendothelial layer of the arterial intima. Secretory nonpancreatic phospholipase A 2 (snpPLA 2 ), an enzyme capable of lipolyzing LDL particles, is found in the arterial extracellular matrix and in contact with the extracellular lipid droplets. We have recently shown that in the presence of heparin, lipolysis of LDL with bee venom PLA 2 induces aggregation and fusion of the particles. Here, we studied the effect of human snpPLA 2 on the integrity of LDL particles and on their interaction with human aortic proteoglycans. In addition, the capacity of the proteoglycans to retain PLA 2 -lipolyzed LDL particles was tested in a microtiter well assay. We found that lipolysis of LDL induced fusion of proteoglycan-bound LDL particles, which increased their binding strength to the proteoglycans. Moreover, lipolysis of LDL with snpPLA 2 under physiological salt and albumin concentrations induced a 3-fold increase in the amount of LDL bound to proteoglycans. The results imply a role for PLA 2 in the retention and accumulation of LDL to the proteoglycan matrix in atherosclerosis. Key Words: phospholipases Ⅲ LDL Ⅲ fusion Ⅲ retention Ⅲ proteoglycans I nitiation of atherosclerosis in both humans 1 and experimental animals 2-4 is characterized by the appearance of extracellular lipid droplets in the proteoglycan (PG)-rich subendothelial layer. Experimental models have shown that similar droplets can be formed directly from LDL particles both in situ 2,5 and in vivo. 2,6 Moreover, both chemical analyses 7,8 and measurements of the size 9,10 of the extracellular lipid droplets in human arterial lesions suggest that the majority of the droplets originate from LDL particles. Because native LDL particles do not fuse into such lipid droplets, the LDL particles must undergo modification in the arterial intima. Indeed, the lipid droplets isolated from the arterial intima have features suggesting that they may have been derived from plasma LDL by extensive modification (reviewed by Öörni et al 11 ). Modification of LDL in vitro by proteolytic enzymes, 12-14 by oxidative compounds, 15 or by lipolytic enzymes such as sphingomyelinase, 13,15-17 phospholipase C, 18,19 or phospholipase A 2 (PLA 2 ) 17,20 has been shown to induce aggregation, fusion, or both aggregation and fusion of the particles. See p 884Phospholipase A 2 s are enzymes that catalyze the hydrolysis of the sn-2 fatty acyl ester bond in phospholipids, yielding a free fatty acid and a lysophospholipid. Recently, type II secretory nonpancreatic phospholipase A 2 (snpPLA 2 ), which is capable of lipolyzing LDL, 21 has been shown to be located in both atherosclerotic and nonatherosclerotic arterial intima [22][23][24][25][26] and to be associated with extracellular matrix structures and lipid droplets. 24 Interestingly, lipolysis of LDL by bee venom PLA 2 reduces the size of the LDL particles in the absence of glycosaminoglycans (GAGs) 17,20,27 but ...