Electronegative LDL induces priming and inflammasome activation leading to IL-1ß release in human monocytes and macrophages.

BACKGROUND: Electronegative LDL (LDL(−)), a modified LDL fraction found in blood, induces the release of inflammatory mediators in endothelial cells and leukocytes. However, the inflammatory pathways activated by LDL(−) have not been fully defined. We aim to study whether LDL(−) induced release of the first-wave proinflammatory IL-1ß in monocytes and monocyte-derived macrophages (MDM) and the mechanisms involved. METHODS: LDL(−) was isolated from total LDL by anion exchange chromatography. Monocytes and MDM were isolated from healthy donors and stimulated with LDL(+) and LDL(−) (100 mg apoB/L). RESULTS: In monocytes, LDL(−) promoted IL-1ß release in a time-dependent manner, obtaining at 20 h-incubation the double of IL-1ß release induced by LDL(−) than by native LDL. LDL(−)-induced IL-1ß release involved activation of the CD14-TLR4 receptor complex. LDL(−) induced priming, the first step of IL-1ß release, since it increased the transcription of pro-IL-1ß (8-fold) and NLRP3 (3-fold) compared to native LDL. Several findings show that LDL(−) induced inflammasome activation, the second step necessary for IL-1ß release. Preincubation of monocytes with K+ channel inhibitors decreased LDL(−)-induced IL-1ß release. LDL(−) induced formation of the NLRP3-ASC complex. LDL(−) triggered 2-fold caspase-1 activation compared to native LDL and IL-1ß release was strongly diminished in the presence of the caspase-1 inhibitor Z-YVAD. In MDM, LDL(−) promoted IL-1ß release, which was also associated with caspase-1 activation. CONCLUSIONS: LDL(−) promotes release of biologically active IL-1ß in monocytes and MDM by induction of the two steps involved: priming and NLRP3 inflammasome activation. SIGNIFICANCE: By IL-1ß release, LDL(−) could regulate inflammation in atherosclerosis.

Myeloperoxidase and hypochlorite, but not copper ions, oxidize heparin-bound LDL particles and release them from heparin.

A key factor in atherosclerosis is the retention of low density lipoprotein (LDL) in the extracellular matrix of the arterial intima, where it binds to the negatively charged glycosaminoglycan chains of proteoglycans. Oxidation may lead to modification of the lysine residues of apolipoprotein B-100 of LDL, which normally mediate the binding of LDL to glycosaminoglycans. Here, we studied whether various modes of oxidation can release LDL from heparin, a glycosaminoglycan with a strong negative charge, in vitro. We found that copper ions were unable to oxidize heparin-bound LDL particles because of their redox inactivation by the glycosaminoglycans. In contrast, myeloperoxidase and hypochlorite, a product of myeloperoxidase, were able to oxidize heparin-bound LDL, and this oxidation led to the release of the oxidized particles from heparin. When the released LDL particles were compared with the residual heparin-bound LDL particles, the released particles were more electronegative and contained more modified lysine residues than did the particles that remained bound. Because human atherosclerotic lesions contain catalytically active myeloperoxidase and (lipo)proteins modified by hypochlorite, the results suggest that myeloperoxidase-secreting monocytes/macrophages in the arterial intima can oxidize and extract LDL from the extracellular matrix with ensuing uptake by the macrophages of the oxidized and released LDL, with eventual formation of foam cells.

Phospholipase A(2) in vascular disease.

Secretory phospholipase A(2) (PLA(2)) can be proatherogenic both in the circulation and in the arterial wall. In blood plasma, PLA(2) can modify the circulating lipoproteins and so induce formation of small dense LDL particles, which are associated with increased risk for cardiovascular disease. In the arterial wall, PLA(2) can hydrolyze lipoproteins. The PLA(2)-modified lipoproteins bind tightly to extracellular proteoglycans, which may lead to their enhanced retention in the arterial wall. The modified lipoproteins may also aggregate and fuse, which can lead to accumulation of their lipids within the extracellular matrix. The PLA(2)-modified particles are more susceptible to further modifications by other enzymes and agents and can be taken up by macrophages, leading to accumulation of intracellular lipids. In addition, lysophospholipids and free fatty acids, the hydrolysis products of PLA(2), promote atherogenesis. Thus, these lipid mediators can be carried, either by the PLA(2)-modified lipoproteins themselves or by albumin, into the arterial cells, which then undergo functional alterations. This may, in turn, lead to specific changes in the extracellular matrix, which increase the retention and accumulation of lipoproteins within the matrix. In the present article, we discuss the possible actions of PLA(2) enzymes, especially PLA(2)-IIA, in the arterial wall during atherogenesis.

Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.

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.

Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles.

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH(3))(3) resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.

Changes in a phospholipid bilayer induced by the hydrolysis of a phospholipase A2 enzyme: a molecular dynamics simulation study.

Phospholipase A2 (PLA2) enzymes are important in numerous physiological processes. Their function at lipid-water interfaces is also used as a biophysical model for protein-membrane interactions. These enzymes catalyze the hydrolysis of the sn-2 bonds of various phospholipids and the hydrolysis products are known to increase the activity of the enzymes. Here, we have applied molecular dynamics (MD) simulations to study the membrane properties in three compositionally different systems that relate to PLA2 enzyme action. One-nanosecond simulations were performed for a 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and for two of its PLA2-hydrolyzed versions, i.e., bilayers consisting of lysophospholipids and of either free charged linoleate or free uncharged linoleic acid molecules. The results revealed loosening of the structure in the hydrolyzed bilayer due to increased mobility of the molecules in the direction normal to the bilayer. This loss of integrity due to the hydrolysis products is in accord with observations that not only the presence of hydrolysis products, but also a variety of other perturbations of the membrane may activate PLA2. Additionally, changes were observed in other structural parameters and in the electrostatic potential across the membrane-water interface. These changes are discussed in relation to the simulation methodology and the experimental observations of PLA2-hydrolyzed membranes.

Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.

Modified LDL - trigger of atherosclerosis and inflammation in the arterial intima.

Atherosclerosis is characterized by chronic inflammation of an injured intima. The pathological processes are initiated by accumulation of morphologically distinct, modified forms of LDL, and followed by cellular infiltration and foam cell formation. Activated intimal cells secrete enzymes and agents capable of modifying LDL, and the modified lipids of LDL, in turn, are able to activate intimal cells and to trigger various inflammatory signals. These processes can initiate and maintain a vicious circle in the intima and lead to lesion progression. In this review, we focus on the LDL modifications relevant to the initial lipid accumulation and discuss their pro-inflammatory effects.

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL.

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.

Lipolytic modification of LDL by phospholipase A2 induces particle aggregation in the absence and fusion in the presence of heparin.

One of the first events in atherogenesis is modification of low density lipoprotein (LDL) particles in the arterial wall with ensuing formation of aggregated and fused lipid droplets. The accumulating particles are relatively depleted in phosphatidylcholine (PC). Recently, secretory phospholipase A2 (PLA2), an enzyme capable of hydrolyzing LDL PC into fatty acid and lysoPC molecules, has been found in atherosclerotic arteries. There is also evidence that both LDL and PLA2 bind to the glycosaminoglycan (GAG) chains of extracellular proteoglycans in the arterial wall. Here we studied the effect of heparin GAG on the lipolytic modification of LDL by PLA2. Untreated LDL, heparin-treated LDL, and heparin-bound LDL were lipolyzed with bee venom PLA2. In the presence of albumin, lipolysis resulted in aggregation in all 3 preparations of the LDL particles. Lipolysis of untreated LDL did not result in aggregation if albumin was absent from the reaction medium, and the lipolytic products accumulated in the particles rendering them negatively charged. However, heparin-treated and heparin-bound lipolyzed LDL particles aggregated even in the absence of albumin. Importantly, in the presence of albumin, some of the heparin-treated and heparin-bound lipolyzed LDL particles fused, the proportion of fused particles being substantially greater when LDL was bound to heparin during lipolysis. In summary, lipolysis of LDL PC by PLA2 under physiological conditions, which allow transfer of the lipolytic degradation products to albumin, leads to fusion of LDL particles in the presence, but not in the absence, of heparin. Thus, it is possible that within the GAG meshwork of the arterial intima, PLA2-induced modification of LDL is one source of the lipid droplets during atherogenesis.

Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles. Two distinct mechanisms leading to increased binding strength of LDL to human aortic proteoglycans.

During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles. Sphingomyelinase (SMase) and phospholipase A2 (PLA2) have been found in the arterial wall, and, moreover, lesional LDL shows signs of hydrolysis of both sphingomyelin and phosphatidylcholine. We have now studied the effects of these two lipolytic modifications on the aggregation and fusion of LDL particles by hydrolyzing the particles with Bacillus cereus SMase or bee venom PLA2. In addition, the binding strengths of the modified LDL to human aortic proteoglycans (PG) were analyzed on an affinity column. We found that SMase induced aggregation and fusion of LDL, but PLA2 induced only aggregation of the particles. In addition, the SMase-induced aggregation and fusion of LDL was promoted by pretreatment of LDL with PLA2. Determination of the binding strengths of the hydrolyzed LDL revealed that mere lipolysis of LDL without aggregation or fusion, either by SMase or PLA2, did not affect the binding of the particles to PG. Aggregation and fusion of lipolyzed LDL particles, however, increased their strength of binding to PG. Active lysine residues in apolipoprotein B-100 (apoB-100) appear to be involved in the binding of LDL to PG, and, in fact, quantitative 13C NMR analysis revealed that, in the fused LDL particles, the number of active lysine residues per apoB-100 moiety was increased. Moreover, aggregation and fusion of LDL increased the number of apoB-100 copies and, consequently, the number of active lysine residues per aggregate or fused particle. Our present findings therefore (i) show that treatment of LDL with SMase and PLA2 generates modified LDL particles, which then bind to human aortic PG with increased strength, and (ii) suggest that SMase- and PLA2-induced aggregation and fusion of LDL are potential mechanisms leading to focal retention of extracellular lipid in the arterial wall.

Human arterial proteoglycans increase the rate of proteolytic fusion of low density lipoprotein particles.

Low density lipoprotein (LDL) particles can undergo fusion in the arterial intima, where they are bound to proteoglycans. Here we studied the effect of human arterial proteoglycans on proteolytic fusion of LDL in vitro. For this purpose, an assay was devised based on fluorescence resonance energy transfer that allowed continuous monitoring of fusion of proteoglycan-bound LDL particles. We found that addition of human arterial proteoglycans markedly increased the rate of proteolytic fusion of LDL. The glycosaminoglycans isolated from the proteoglycans also increased the rate of fusion, demonstrating that this effect was produced by the negatively charged sulfated polysaccharides in the proteoglycans. Furthermore, heparin, chondroitin 6-sulfate, and dextran sulfate, three commercially available sulfated polysaccharides, also increased the rate of LDL fusion, with heparin and chondroitin 6-sulfate being as effective as and dextran sulfate more effective than human proteoglycans. The ability of the sulfated polysaccharides to increase the rate of proteolytic fusion of LDL depended critically on their ability to form insoluble complexes with LDL, which, in turn, resulted in an increased rate of LDL proteolysis and, in consequence, in an increased rate of LDL fusion. The results reveal a novel mechanism regulating LDL fusion and point to the potentially important role of arterial proteoglycans in the generation of LDL-derived lipid droplets in the arterial intima during atherogenesis.

Oxidation of low density lipoprotein particles decreases their ability to bind to human aortic proteoglycans. Dependence on oxidative modification of the lysine residues.

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

The proteoglycan decorin links low density lipoproteins with collagen type I.

Decorin is a small dermatan sulfate-rich proteoglycan which binds to collagen type I in vitro and in vivo. In atherosclerotic lesions the contents of low density lipoprotein (LDL), decorin, and collagen type I are increased, and ultrastructural studies have suggested an association between LDL and collagen in the lesions. To study interactions between LDL, decorin, and collagen type I, we used solid phase systems in which LDL was coupled to a Sepharose column, or in which LDL, decorin, or collagen type I was attached to microtiter wells. The interaction between LDL and decorin in the fluid phase was evaluated using a gel mobility shift assay. We found that LDL binds to decorin by ionic interactions. After treatment with chondroitinase ABC, decorin did not bind to LDL, showing that the glycosaminoglycan side chain of decorin is essential for LDL binding. Acetylated and cyclohexanedione-treated LDL did not bind to decorin, demonstrating that both lysine and arginine residues of apoB-100 are necessary for the interaction. When collagen type I was attached to the microtiter plates, only insignificant amounts of LDL bound to the collagen. However, if decorin was first allowed to bind to the collagen, binding of LDL to the decorin-collagen complexes was over 10-fold higher than to collagen alone. Thus, decorin can link LDL with collagen type I in vitro, which suggests a novel mechanism for retention of LDL in collagen-rich areas of atherosclerotic lesions.