High density lipoprotein oxidation: in vitro susceptibility and potential in vivo consequences

Elevated levels of plasma high density lipoprotein (HDL) are strongly predictive of protection against atherosclerotic vascular disease. HDL particles likely have several beneficial actions in vivo, including the initiation of reverse cholesterol transport. The apparent importance of oxidative modification of low density lipoprotein in atherogenesis raises the question of how oxidative modification of HDL might affect its cardioprotective actions. HDL is readily oxidized using numerous models of lipoprotein oxidation. In vitro evidence suggests oxidation might impair some protective actions, but actually enhance other mechanisms induced by HDL that prevent the accumulation of cholesterol in the artery wall. This article reviews the current literature concerning the relative oxidizability of HDL, the structural changes induced in HDL by oxidation in vitro, and the potential consequences of oxidative modification on the protective actions of HDL in vivo.     

Oxidized cholesterol in the diet is a source of oxidized lipoproteins in human serum

The aim of this study was to determine in humans whether oxidized cholesterol in the diet is absorbed and contributes to the pool of oxidized lipids in circulating lipoproteins. When a meal containing 400 mg cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-epoxy cholesterol) was fed to six controls and three subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol in serum was found in chylomicron/chylomicron remnants (CM/RM) and endogenous (VLDL, LDL, and HDL) lipoproteins. In controls, alpha-epoxy cholesterol in CM/RM was decreased by 10 h, whereas in endogenous lipoproteins it remained in the circulation for 72 h. In subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol was mainly in CM/RM. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. In contrast, no transfer was observed when human serum was substituted with rat serum, suggesting that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into the CM/RM fraction and then transferred to LDL and HDL, contributing to lipoprotein oxidation. Moreover, LDL containing alpha-epoxy cholesterol displayed increased susceptibility to further copper oxidation in vitro. It is possible that oxidized cholesterol in the diet accelerates atherosclerosis by increasing oxidized cholesterol levels in circulating LDL and chylomicron remnants.     

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.     

Chondroitin-4-sulfate protects high-density lipoprotein against copper-dependent oxidation

We investigated the effect of chondroitinsulphate (CS), the major glycosaminoglycan of the arterial wall, on the oxidation of human high-density lipoprotein (HDL) by kinetic analysis. Chondroitin-4-sulfate (C4S) increased the lag time and reduced the maximum rate of HDL oxidation induced by Cu2+, as assessed by monitoring both conjugated diene formation and low-level chemiluminescence. On the contrary, chondroitin-6-sulfate (C6S) was ineffective. Dermatansulfate exhibited an inhibitory effect comparable to that of C4S. C4S protected also the protein moiety of HDL, as it reduced tryptophan destruction by lipid-oxidizing species and delayed the formation of fluorescent adducts between end products of lipid peroxidation and amino acid residues. Again, C6S was ineffective. C4S was able to bind Cu2+; this resulted in less Cu2+ available for HDL oxidation and likely represented the mechanism of the protective effect. Neither C4S nor C6S affected HDL oxidation by peroxyl radicals, indicating that free radical scavenging activity was not involved in the protective effect. These results suggest that C4S might prevent the oxidative modification of HDL in arterial wall, thus preserving its antiatherogenic potential for reverse cholesterol transport and, possibly, for clearance of oxidized lipids.     

Oxidation of cholesterol in low density and high density lipoproteins by cholesterol oxidase

The cholesterol oxidase-catalyzed oxidation of cholesterol in native low density (LDL) and high density lipoproteins (HDL3) as well as in monolayers prepared from surface lipids of these particles, has been examined. The objective of the study was to compare the oxidizability of cholesterol, and to examine the effects of lipid packing on oxidation rates. When [3H]cholesterol-labeled lipoproteins were exposed to cholesterol oxidase (Streptomyces sp.), it was observed that LDL [3H]cholesterol was oxidized much faster than HDL3 [3H]cholesterol. This was true both at equal cholesterol concentration per enzyme unit, and at equal amounts of lipoprotein particles per enzyme unit. About 95% of lipoprotein [3H]cholesterol was available for oxidation. The complete degradation of lipoprotein sphingomyelin by sphingomyelinase (Staphylococcus aureus) resulted in a 10-fold increase in the rate of LDL [3H]cholesterol oxidation, whereas the effects on rates of HDL3 [3H]cholesterol oxidation were less dramatic. A monolayer study with LDL surface lipids indicated that degradation of sphingomyelin loosened the lipid packing, because the ceramide formed occupied a smaller surface area than the parent sphingomyelin, and since the condensing effect of cholesterol on sphingomyelin packing was lost. The effects of sphingomyelin degradation on lipid packing in monolayers of HDL3-derived surface lipids were difficult to determine from monolayer experiments. Based on the finding that cholesterol oxidases are surface pressure-sensitive with regard to their catalytic activity, these were used to estimate the surface pressure of intact LDL and HDL3. The cut-off surface pressure of a Brevibacterium enzyme was 25 mN/m and 20 mN/m in monolayers of LDL and HDL3-derived surface lipids, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidatively modified high density lipoprotein promotes inflammatory response in human monocytes–macrophages by enhanced production of ROS,TNF-α, MMP-9, and MMP-2

It has been proposed that high-density lipoprotein (HDL) loses its cardioprotective ability through oxidative modifications by reactive oxygen species (ROS) and promote atherogenesis. However, the pro-atherogenic pathways undergone by oxidized HDL remain poorly understood. Since monocytes play a crucial role in atherogenesis, this study was aimed to investigate the influence of both native and oxidized HDL (oxHDL) on monocytes-macrophages functions relevant to atherogenesis. HDL particles were isolated from human blood samples by ultracentrifugation and subjected to in vitro oxidation with CuSO(4). The extent of oxidation was quantitated by measurement of lipid peroxides. Human peripheral blood mononuclear cells were isolated and cultured under standard conditions. Cells were treated with native and oxHDL at varying concentrations for different time intervals and used for several analyses. Intracellular ROS production was assessed based on ROS-mediated DCFH fluorescence of the cells. The release of TNF-α and matrix metalloproteinases (MMPs) was quantitated using ELISA kit and gelatine zymography, respectively. Treatment of cells with oxidized HDL enhanced the production of ROS in a concentration-dependent way, while native HDL had no such effect. Further, the release of TNF-α, MMP-9, and MMP-2 was found to be remarkably higher in cells incubated with oxHDL than that of native HDL. Results demonstrate that oxidative modification of HDL induces pro-inflammatory response and oxidative stress in human monocytes-macrophages.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

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.     

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu(2+)-mediated oxidation.

A recent study demonstrated that Cu(2+)-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457-6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.     

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.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Myeloperoxidase-mediated oxidation of high-density lipoproteins: fingerprints of newly recognized potential proatherogenic lipoproteins

Substantial evidence supports the notion that oxidative processes participate in the pathogenesis of atherosclerotic heart disease. Major evidence for myeloperoxidase (MPO) as enzymatic catalyst for oxidative modification of lipoproteins in the artery wall has been suggested in numerous studies performed with low-density lipoprotein. In contrast to low-density lipoprotein, plasma levels of high-density lipoprotein (HDL)-cholesterol and apoAI, the major apolipoprotein of HDL, inversely correlate with the risk of developing coronary artery disease. These antiatherosclerotic effects are attributed mainly to HDL's capacity to transport excess cholesterol from arterial wall cells to the liver during 'reverse cholesterol transport'. There is now strong evidence that HDL is a selective in vivo target for MPO-catalyzed oxidation impairing the cardioprotective and antiinflammatory capacity of this antiatherogenic lipoprotein. MPO is enzymatically active in human lesion material and was found to be associated with HDL extracted from human atheroma. MPO-catalyzed oxidation products are highly enriched in circulating HDL from individuals with cardiovascular disease where MPO concentrations are also increased. The oxidative potential of MPO involves an array of intermediate-generated reactive oxygen and reactive nitrogen species and the ability of MPO to generate chlorinating oxidants-in particular hypochlorous acid/hypochlorite-under physiological conditions is a unique and defining activity for this enzyme. All these MPO-generated reactive products may affect structure and function of HDL as well as the activity of HDL-associated enzymes involved in conversion and remodeling of the lipoprotein particle, and represent clinically useful markers for atherosclerosis.     

Formation of methionine sulfoxide-containing specific forms of oxidized high-density lipoproteins

Atherosclerosis is characterized by the accumulation of both lipoprotein-derived lipids and inflammatory cells in the affected vascular wall that results in a state of heightened oxidative stress and that is reflected by the accumulation of oxidized lipoproteins. Circulating oxidized low-density lipoprotein (oxLDL) is used as a surrogate marker for coronary artery disease, although the 'escape' of oxLDL from the vessel wall is hindered by the large size of this lipoprotein and its specific retention by the extracellular matrix. Also, the oxidation of lipoproteins in human atherosclerotic lesions is not limited to LDL. In fact, the lipids of all classes of lipoproteins are oxidized to a comparable extent. Examining the fate of lipid hydroperoxides, the primary lipid peroxidation products, in high-density lipoproteins (HDL) undergoing oxidation, revealed that they become reduced to the corresponding alcohols by specific Met residues of apolipoprotein A-I (apoA-I) and apoA-II. As a consequence, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. This review describes the characterization of specifically oxidized HDL with an emphasis on MetO formation, the structural and functional consequences of such oxidation, and the potential utility of specifically oxidized HDL as a surrogate marker of atherosclerosis.     

Human carotid atherosclerotic plaque increases oxidative state of macrophages and low-density lipoproteins,whereas paraoxonase 1 (PON1) decreases such atherogenic effects

Human atherosclerotic plaque contains a variety of oxidized lipids, which can facilitate further oxidation. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated esterase (lipolactonase), exhibiting antiatherogenic properties. The aims of the present study were to examine the oxidizing potency of the human carotid plaque lipid extract (LE), and the antiatherogenic role of PON1 on LE oxidation competence. Human carotid plaques were extracted by organic solvent, and the extract was incubated with lipoprotein particles, with macrophages, or with probes sensitive to oxidative stress, with or without preincubation with PON1, followed by oxidative-stress assessment. Our findings imply that the LE oxidized LDL, macrophages, and exogenous probes and decreases HDL-mediated cholesterol efflux from macrophages, in a dose-dependent manner. Incubation of PON1 with LE significantly affects LE composition, reduces LE atherogenic properties, and decreases the extract's total peroxide concentration by 44%, macrophage oxidation by 25%, and probe oxidation by up to 52%. We conclude that these results expand our understanding of how the plaque itself accelerates atherogenesis and provides an important mechanism for attenuation of atherosclerosis development by the antioxidant action of PON1 on the atherosclerotic plaque.     

Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation

The inhibition of low-density lipoprotein (LDL) oxidation by high-density lipoprotein (HDL) is a major antiatherogenic property of this lipoprotein. This activity is due, in part, to HDL associated proteins. However, whether these proteins interact in the antioxidant activity of HDL is unknown. LDL was incubated with apolipoprotein A1 (apo A1), lecithin:cholesterol acyltransferase (LCAT), and paraoxonase-1 (PON1) alone or in combination, in the presence or absence of HDL under oxidizing conditions. LDL lipid peroxide concentrations were determined. Apo A1, LCAT, and PON1 all inhibit LDL oxidation in the absence of HDL and enhance the ability of HDL to inhibit LDL oxidation. Their effect was additive rather than synergistic; the combination of these proteins significantly enhanced the length of time LDL was protected from oxidation. This seemed to be due to the ability of PON1 to prevent the oxidative inactivation of LCAT. Apo A1, LCAT, and PON1 can all contribute to the antioxidant activity of HDL in vitro. The combination of apo A1, LCAT, and PON1 prolongs the time that HDL can prevent LDL oxidation, due, at least in part, to the prevention LCAT inactivation.     

Is the oxidation of high-density lipoprotein lipids different than the oxidation of low-density lipoprotein lipids?

This article gives detailed insight into the kinetics of high-density lipoprotein (HDL) oxidation catalyzed by azobis(2-amidinopropane).dihydrochloride (ABAP) or by copper. ABAP initialized oxidation of human HDL 3-4 times faster than non-human primate HDL with a similar composition. The oxidizability of non-human primate HDL was 1000 times lower than the oxidizability calculated from rate constants derived from liposome oxidation, suggesting that there is a slow step in HDL oxidation not present in liposomes. Saturable binding of copper to HDL was a significant feature of copper-catalyzed oxidation. Binding constants (K(m)) for non-human primate HDL were 2-3-fold lower than those for human HDL. Copper-catalyzed oxidation of non-human primate HDL was slower than that of human HDL, but human HDL(2) and HDL(3) oxidized at about the same rate. Overall, the kinetics describing the oxidation of HDL were mechanistically similar to those reported for LDL, suggesting that HDL lipids were as easily oxidized as LDL lipids and that HDL will be easily oxidized in vivo when exposed to agents that oxidize LDL.     

Haptoglobin binding to apolipoprotein A-I prevents damage from hydroxyl radicals on its stimulatory activity of the enzyme lecithin-cholesterol acyl-transferase

Apolipoprotein A-I (ApoA-I), a major component of HDL, binds haptoglobin, a plasma protein transporting to liver or macrophages free Hb for preventing hydroxyl radical production. This work aimed to assess whether haptoglobin protects ApoA-I against this radical. Human ApoA-I structure, as analyzed by electrophoresis and MS, was found severely altered by hydroxyl radicals in vitro. Lower alteration of ApoA-I was found when HDL was oxidized in the presence of haptoglobin. ApoA-I oxidation was limited also when the complex of haptoglobin with both high-density lipoprotein and Hb, immobilized on resin beads, was exposed to hydroxyl radicals. ApoA-I function to stimulate cholesterol esterification was assayed in vitro by using ApoA-I-containing liposomes. Decreased stimulation was observed when liposomes oxidized without haptoglobin were used. Conversely, after oxidative stress in the presence of haptoglobin (0.5 microM monomer), the liposome activity did not change. Plasma of carrageenan-treated mice was analyzed by ELISA for the levels of haptoglobin and ApoA-I, and used to isolate HDL for MS analysis. Hydroxyproline-containing fragments of ApoA-I were found associated with low levels of haptoglobin (18 microM monomer), whereas they were not detected when the haptoglobin level increased (34-70 microM monomer). Therefore haptoglobin, when circulating at enhanced levels with free Hb during the acute phase of inflammation, might protect ApoA-I structure and function against hydroxyl radicals.     

Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages

Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (ΔA234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [³H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 36% at lipoprotein concentrations of 10 to 200 μg protein/ml. Cholesterol efflux correlated negatively with TBARS production (r= −0.97, P < 0.003) and ΔA234 (r = −0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.     

The inverse relation of HDL anti‐oxidative functionality with serum amyloid a is lost in metabolic syndrome subjects

Objective:

Anti‐oxidative properties of high density lipoproteins (HDL) are relevant for atheroprotection. HDL carry serum amyloid A (SAA), which may impair HDL functionality. We questioned whether HDL anti‐oxidative capacity is determined by SAA.

Design and Methods:

Relationships of HDL anti‐oxidative capacity (% inhibition of low density lipoprotein oxidation in vitro) with SAA were determined in 54 non‐diabetic subjects without metabolic syndrome (MetS) and 68 subjects with MetS (including 51 subjects with Type 2 diabetes mellitus).

Results:

SAA levels were higher in MetS subjects, coinciding higher high sensitive C‐reactive protein (hs‐CRP) and lower HDL cholesterol and apolipoprotein (apo) A‐I levels (P<0.001 for all). HDL anti‐oxidative capacity was not different between subjects with and without MetS (P=0.76), but the HDL anti‐oxidation index (HDL anti‐oxidative capacity multiplied by individual HDL cholesterol concentrations), as a measure of global anti‐oxidative functionality of HDL, was lower in Mets subjects (P<0.001). HDL anti‐oxidative capacity was correlated inversely with SAA levels in subjects without MetS (r=‐0.286, P=0.036). Notably, this relationship was independent of HDL cholesterol or apoA‐I (P<0.05 for both). In contrast, no relation of HDL anti‐oxidative capacity with SAA was observed in MetS subjects (r=0.032, P=0.80). The relationship of SAA with HDL anti‐oxidative capacity was different in subjects with MetS compared to subjects without MetS (P=0.039 for the interaction between the presence of MetS and SAA on HDL anti‐oxidative capacity) taking age and diabetes status into account.

Conclusion:

Higher SAA levels may impair HDL anti‐oxidative functionality. The relationship of this physiologically relevant HDL functionality measure with circulating SAA levels is apparently disturbed in metabolic syndrome.     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.