Plasmin and kallikrein reduce HDL-induced cholesterol efflux from foam cells

Arterial intima contains metabolically active factors such as proteases, which may act on high-density lipoprotein (HDL) and impair its ability to accept cholesterol. In this study we treated human HDL(3) with human plasmin and human plasma kallikrein, two proteases also found in the human arterial intima, in order to study their effect on the ability of HDL(3) to promote cholesterol efflux from human macrophage foam cells. After exposure to plasmin or plasma kallikrein for 15 min, HDL(3) showed a decrease of about 60% in its ability to promote cholesterol efflux from the macrophage foam cells. SDS-PAGE analysis of the degraded HDL(3) particles showed that plasmin had generated cleavage products less than 15 kDa in size and plasma kallikrein had generated a major product of about 19 kDa. However, there was only a slight loss of intact apolipoproteins, suggesting degradation of a small subpopulation of HDL(3) particles. Agarose gel electrophoresis showed that a decrease in cholesterol efflux was accompanied by total loss of the HDL(3) with prebeta-mobility, but no apparent change in those with alpha mobility. These results suggest that the presence of active plasmin or plasma kallikrein in the atherosclerotic arterial intima promotes atherogenesis by blocking cholesterol efflux from macrophage foam cells.     

Paraoxonases 1, 2, and 3, oxidative stress, and macrophage foam cell formation during atherosclerosis development

Paraoxonases PON1 and PON3, which are both associated in serum with HDL, protect the serum lipids from oxidation, probably as a result of their ability to hydrolyze specific oxidized lipids. The activity of HDL-associated PON1 seems to involve an activity (phospholipase A2-like activity, peroxidase-like activity, lactonase activity) which produces LPC. To study the possible role of PON1 in macrophage foam cell formation and atherogenesis we used macrophages from control mice, from PON1 knockout mice, and from PON1 transgenic mice. Furthermore, we analyzed PON1-treated macrophages and PON1-transfected cells to demonstrate the contribution of PON1 to the attenuation of macrophage cholesterol and oxidized lipid accumulation and foam cell formation. PON1 was shown to inhibit cholesterol influx [by reducing the formation of oxidized LDL (Ox-LDL), increasing the breakdown of specific oxidized lipids in Ox-LDL, and decreasing macrophage uptake of Ox-LDL]. PON1 also inhibits cholesterol biosynthesis and stimulates HDL-mediated cholesterol efflux from macrophages. PON2 and PON3 protect against oxidative stress, with PON2 acting mainly at the cellular level. Whereas serum PON1 and PON3 were inactivated under oxidative stress, macrophage PON2 expression and activity were increased under oxidative stress, probably as a compensatory mechanism against oxidative stress. Intervention to increase the paraoxonases (cellular and humoral) by dietary or pharmacological means can reduce macrophage foam cell formation and attenuate atherosclerosis development.     

Mild oxidation promotes and advanced oxidation impairs remodeling of human high-density lipoprotein in vitro

High-density lipoproteins (HDLs) prevent atherosclerosis by removing cholesterol from macrophages and by exerting antioxidant and anti-inflammatory effects. Oxidation is thought to impair HDL functions, yet certain oxidative modifications may be advantageous; thus, mild oxidation reportedly enhances cell cholesterol uptake by HDL whereas extensive oxidation impairs it. To elucidate the underlying energetic and structural basis, we analyzed the effects of copper and hypochlorite (which preferentially oxidize lipids and proteins, respectively) on thermal stability of plasma spherical HDL. Circular dichroism, light scattering, calorimetry, gel electrophoresis, and electron microscopy showed that mild oxidation destabilizes HDL and accelerates protein dissociation and lipoprotein fusion, while extensive oxidation inhibits these reactions; this inhibition correlates with massive protein cross-linking and with lipolysis. We propose that mild oxidation lowers kinetic barriers for HDL remodeling due to diminished apolipoprotein affinity for lipids resulting from oxidation of methionine and aromatic residues in apolipoproteins A-I and A-II followed by protein cross-linking into dimers and/or trimers. In contrast, advanced oxidation inhibits protein dissociation and HDL fusion due to lipid redistribution from core to surface upon lipolysis and to massive protein cross-linking. Our results help reconcile the apparent controversy in the studies of oxidized HDL and suggest that mild oxidation may benefit HDL functions.     

Cloning and expression of a cellular high density lipoprotein-binding protein that is up-regulated by cholesterol loading of cells.

Plasma membranes of cultured cells contain high affinity receptors for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol. Recent studies using ligand blot analysis have identified a 110-kDa membrane protein which has features predicted for an HDL receptor, in that it preferentially binds HDL apolipoproteins and undergoes up-regulation in response to cholesterol loading of cells. In this study, we isolated a cDNA clone from an expression library using an antibody raised against partially purified 110-kDa HDL-binding protein. This clone encodes a novel cell protein, designated HBP, comprised mostly of 14 imperfect tandem repeats of approximately 70 amino acids in length. Each repeat appears to contain two amphipathic helices. Expression of HBP in cultured cells was increased severalfold when cells were loaded with cholesterol, as evident by increases in both HBP mRNA and membrane-associated protein. Overexpression of HBP in mammalian cell transfectants was associated with higher HDL binding to isolated cell protein and with modest increases in HDL binding to the cell surface. Proteins identified by ligand blot analysis had lower apparent M(r) than the primary HBP gene product and varied in M(r) and in HDL binding activity between cell types, suggesting that HBP undergoes cell-specific processing. These results provide preliminary evidence that HBP is a component of a cellular pathway that facilitates removal of excess cholesterol from cells, perhaps through its interaction with HDL. However, the predicted structure of HBP does not conform to that of any known receptor, suggesting that it does not function as a classic plasma membrane receptor.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Influence of apolipoproteins AI, AII, and Cs on the metabolism of membrane and lysosomal cholesterol in macrophages.

We have demonstrated previously that HDL-mediated efflux of plasma membrane cholesterol is independent of specific binding of apolipoproteins to the high density lipoprotein (HDL) receptor in either control or cholesterol-enriched cells (Karlin, J. B., Johnson, W. J., Benedict, C. R., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1987) J. Biol. Chem. 262, 12557-12564 and Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1988) J. Biol. Chem. 263, 14099-14106). The present studies were conducted to determine if the process for removal of intracellular (lysosomal) cholesterol is similar to that of membrane cholesterol or if, in contrast, it is selectively regulated by specific apolipoproteins of HDL. For these reasons, we examined the influence of each of the major apolipoproteins of human HDL, apoAI, apoAII, and apoCs on the metabolism of membrane and lysosomal cholesterol in a macrophage foam cell model. We developed an experimental system which allows, for the first time, the simultaneous determination of lysosomal hydrolysis of cholesteryl ester and efflux and esterification of both lysosomal and membrane cholesterol. J774 and elicited mouse peritoneal macrophages were loaded with cholesteryl ester within lysosomes through phagocytosis of sonicated lipid droplets. Membrane and lysosomal pools of cholesterol were differentially radiolabeled. Discoidal complexes of egg phosphatidylcholine and purified apolipoproteins having a similar size and composition were used as cholesterol acceptors. Our results demonstrate that lysosomal hydrolysis of cholesteryl ester is independent of the presence of extracellular acceptors. Lysosomal production of cholesterol stimulates the esterification by acyl-CoA:cholesterol acyltransferase of membrane and lysosomal cholesterol. All the particles tested induce the efflux of both pools of cholesterol at a similar ratio. As efflux is stimulated, esterification by acyl-CoA:cholesterol acyltransferase is reduced. We conclude that none of these apolipoproteins selectively influences the efflux or the esterification of membrane of lysosomal cholesterol. In addition, we observe that particles containing apoAI are the most efficient acceptors, but this effect is not linked to specific binding to the HDL receptor.     

Oxysterol efflux from macrophage foam cells: the essential role of acceptor phospholipid.

Oxidized forms of cholesterol (oxysterols) are present in atherosclerotic lesions and may play an active role in lesion development. For example, 7-ketocholesterol (7KC) inhibits cholesterol efflux from macrophage foam cells induced by apolipoprotein A-I (apoA-I). Such oxysterols may promote foam cell formation in atherosclerotic lesions by preventing effective clearance of excess cholesterol. ApoA-I also induces phospholipid (PL) export from foam cells and it has been suggested that cholesterol efflux is dependent upon PL association with the apolipoprotein. In the current study, the effect of oxysterol enrichment of foam cells on phospholipid efflux was measured. Export of cellular PL to apoA-I from 7KC-enriched foam cells was inhibited to the same extent as cholesterol, indicating that the reduced cholesterol export may be a consequence of a decline in the capacity of the foam cells to generate PL/apoA-I particles capable of accepting cellular cholesterol. Incubation of foam cells with pre-formed PL/apoA-I discs increased cholesterol export from 7KC-enriched cells to levels seen in 7KC-free cells. Foam cells produced by uptake of oxidized LDL, which contain similar amounts of 7KC plus other oxidation products, expressed a more profound inhibition of PL export to apoA-I. Cholesterol efflux from these cells improved only partially by provision of PL-containing acceptors. Efflux of 7KC from both foam cell types occurred to PL/apoA-I discs but was only minimal to lipid-free apoA-I, indicating that export of this oxysterol is more dependent than cholesterol upon the presence of extracellular phospholipid.     

Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL < or =10(th) and HDL > or =90(th) Finnish age/sex-specific percentile. Cholesterol efflux from [(3)H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL(2), and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL(2), and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of prebeta-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

Scavenger receptor BI mediates the selective uptake of oxidized cholesterol esters by rat liver

High density lipoprotein (HDL) can protect low density lipoprotein (LDL) against oxidation. Oxidized cholesterol esters from LDL can be transferred to HDL and efficiently and selectively removed from the blood circulation by the liver and adrenal in vivo. In the present study, we investigated whether scavenger receptor BI (SR-BI) is responsible for this process. At 30 min after injection, the selective uptake of oxidized cholesterol esters from HDL for liver and adrenal was 2.3- and 2.6-fold higher, respectively, than for native cholesterol esters, whereas other tissues showed no significant difference. The selective uptake of oxidized cholesterol esters from HDL by isolated liver parenchymal cells could be blocked for 75% by oxidized LDL and for 50% by phosphatidylserine liposomes, both of which are known substrates of SR-BI. In vivo uptake of oxidized cholesterol esters from HDL by parenchymal cells decreased by 64 and 81% when rats were treated with estradiol and a high cholesterol diet, respectively, whereas Kupffer cells showed 660 and 475% increases, respectively. These contrasting changes in oxidized cholesterol ester uptake were accompanied by similar contrasting changes in SR-BI expression of parenchymal and Kupffer cells. The rates of SR-BI-mediated selective uptake of oxidized and native cholesterol esters were analyzed in SR-BI-transfected Chinese hamster ovary cells. SR-BI-mediated selective uptake was 3.4-fold higher for oxidized than for native cholesterol esters (30 min of incubation). It is concluded that in addition to the selective uptake of native cholesterol esters, SR-BI is responsible for the highly efficient selective uptake of oxidized cholesterol esters from HDL and thus forms an essential mediator in the HDL-associated protection system for atherogenic oxidized cholesterol esters.     

Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues

Cultured extrahepatic cells possess a specific high affinity binding site (receptor) for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. To characterize the binding recognition site(s) on HDL, the ability of HDL to interact with cultured human fibroblasts was assayed after chemical alteration of specific apoprotein amino acid residues. Reduction and alkylation, acetylation, and cyclohexanedione treatment of HDL3 had little or no effect on its cellular binding. Treatment of HDL3 with tetranitromethane (TNM), however, caused a large dose-dependent decrease in binding, with maximum inhibition at 3 mM. Amino acid analysis of the TNM-treated particles showed specific alteration of tyrosine residues, but sodium dodecyl sulfate-gel electrophoresis demonstrated apoprotein cross-linking coincident with decreased binding. These results suggest that modification of HDL tyrosine residues and/or cross-linking of HDL apoproteins alters the ligand site recognized by the HDL receptor. Gradient gel electrophoresis, molecular sieve chromatography, and electron microscopy showed only minor changes in size distribution and shape of HDL3 particles after treatment with 3 mM TNM, but at higher TNM concentrations, coalescence and aggregation of particles was evident. Treatment of HDL3 with 3 mM TNM affected neither its promotion of the low affinity (receptor-independent) cholesterol efflux from cells nor its ability to accept cholesterol from an albumin suspension, yet promotion of high affinity (receptor-dependent) cholesterol efflux from cells was abolished. The finding that TNM treatment of HDL3 decreases both its receptor binding and its promotion of cholesterol efflux from cells without substantial alteration of its physical properties supports the hypothesis that the HDL receptor functions to facilitate cholesterol transport from cells.     

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXRα/β expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking.     

Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-gamma

The Th1-derived cytokine gamma interferon, IFN-gamma, is present within the microenvironment of an atheromatous lesion and likely contributes to lesion progression through macrophage activation. While the inflammatory effects of IFN-gamma are well known, the role of this cytokine in cholesterol metabolism in macrophage derived foam cells is unclear. In the present study, the incubation of foam cells with IFN-gamma resulted in the reduction of HDL(3)-mediated cholesterol efflux. The decrease in cholesterol efflux was not observed with other macrophage-activating factors as colony-stimulating factors failed to demonstrate a similar effect. The reduction in cholesterol efflux was independent of apoE synthesis or SR-BI expression and was associated with a redistribution of intracellular cholesterol with an increase in cholesteryl ester accumulation. The increase in the esterified pool, primarily in cholesterol eicosapentadenoate, docosapentaenoate, arachidonate, and linoleate was associated with a 2-fold increase in acyl-CoA:cholesterol-O-acyltransferase, ACAT, activity and message without any change in neutral cholesteryl ester hydrolase activity. While CD36 message was reduced in IFN-gamma-treated foam cells, the ability to reverse the decrease in efflux by the ACAT inhibitor A58035 in a dose-dependent manner suggests that the IFN-gamma effect on efflux is primarily through the modulation of ACAT expression. Therefore, in addition to its inflammatory effects, IFN-gamma can contribute to the progression of an atherosclerotic lesion by altering the pathway of intracellular cholesterol trafficking in macrophage derived foam cells.     

Modifying Apolipoprotein A-I by Malondialdehyde,but Not by an Array of Other Reactive Carbonyls,Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.     

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [³H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [³H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.     

Neutrophil proteinase 3 — An LDL- and HDL-proteolyzing enzyme with a potential to contribute to cholesterol accumulation in human atherosclerotic lesions

Neutrophil proteinase 3 (PR3) is a multifunctional neutral serine protease involved in the regulation of pro-inflammatory processes, but its potential causal roles in the lipid-driven responses in atherosclerosis have remained unexplored. This study aimed to investigate the presence of PR3 in human atherosclerotic lesions and the ability of this protease to modify the structure and functions of LDL and HDL particles in vitro. Coronary artery segments were collected from autopsied subjects and immunostained for PR3. Atherosclerotic lesions but not normal intima contained PR3. Incubation of LDL particles with the PR3 led to extensive degradation of their apoB-100 component and strongly increased their binding strength to isolated human aortic proteoglycans in vitro. Moreover, cultured human monocyte-derived macrophages avidly ingested the PR3-modified LDL particles and were converted into foam cells. Incubation of HDL particles with PR3 led to proteolysis of their major apolipoproteins (apoA-I, apoA-II, and apoE) and impaired their ability to promote cholesterol efflux from the macrophage foam cells. We conclude that PR3 is present in human atherosclerotic lesions and that this neutral serine protease has proatherogenic properties. Thus, by proteolytically modifying LDL and HDL particles, PR3 may promote cholesterol accumulation both extra- and intracellularly in atherosclerotic lesions, and so contribute to the lipid-driven component of atherogenesis.