Oxidative Modification Of High-density Lipoprotein Research Articles

Biology of High-Density Lipoproteins: An Update49High density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-kB/STAT1-IRF1 signaling50Homocysteine accelerated the formation of THP-1 macrophages-derived foam cells and cholesterol disorder via regulating the expressions of LXRa, ABCA1 and ABCG151Protein components of HDL as markers of cardiovascular damage in patients with arterial hypertension

Biology of High-Density Lipoproteins: An Update49High density lipoproteins exert pro-inflammatory effects on macrophages via passive cholesterol depletion and PKC-NF-kB/STAT1-IRF1 signaling50Homocysteine accelerated the formation of THP-1 macrophages-derived foam cells and cholesterol disorder via regulating the expressions of LXRa, ABCA1 and ABCG151Protein components of HDL as markers of cardiovascular damage in patients with arterial hypertension

Abstract 450: Modification of HDL by Reactive Aldehydes Impairs HDL's Athero-protective Functions

High density lipoproteins (HDL) are athero-protective particles that promote the removal of excess cholesterol from lipid-loaded macrophages and stimulate their migration in order to protect against foam cell formation, a precursor to atherosclerotic plaque build-up. Recently, studies have shown that oxidative modification of HDL prevents HDL from protecting against atherosclerosis; however, the exact mechanisms by which this occurs are not well defined. We hypothesize that oxidative modification of HDL by reactive aldehydes such as acrolein (a major component of cigarette smoke) and 4-hydroxynonenal (HNE; a product of lipid peroxidation) impairs HDL’s athero-protective effects in macrophages. We tested our hypothesis using three different assays. First, we determined that modified forms of HDL upregulate mRNA levels of pro-atherogenic scavenger receptors such as cluster of differentiation 36 (CD36), a known oxidized LDL receptor. Incubation of macrophages with native HDL did not exert similar effects. Second, we tested the ability of oxidized HDL to prevent foam cell formation. Peritoneal macrophages isolated from WT C57Bl/J mice were cholesterol-loaded and incubated with native HDL, acrolein-modified HDL (acro-HDL), or HNE-modified HDL (HNE-HDL). Oil Red-O staining demonstrated that 24% of macrophages had foam cell formation upon incubation with native HDL, whereas 61% and 49% foam cell formation was observed for acro- and HNE-HDL, respectively. Preliminary data suggests this may be CD36-dependent. Finally, using a Boyden chamber assay, we demonstrated that both acro- and HNE-HDL, but not native HDL, had an impaired ability to promote macrophage migration (43% and 72% of HDL cell migration levels, respectively). We determined that the inability of acro- and/or HNE-HDL to stimulate macrophage migration may be due to an impaired ability of these modified lipoproteins to activate the PI3K pathway, as shown by decreased levels of phosphorylated protein kinase B (Akt). In conclusion, we have identified three independent mechanisms by which modification of HDL with acrolein or HNE impairs HDL’s cardio-protective effects and, instead, generates a particle that promotes pathways that lead to atherosclerosis.

15‐Lipoxygenase‐Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells

Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL3 on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL3 failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.

Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctional high-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorous acid (HOCl)

Oxidative modification of HDLs (high-density lipoproteins) by MPO (myeloperoxidase) compromises its anti-atherogenic properties, which may contribute to the development of atherosclerosis. Although it has been established that HOCl (hypochlorous acid) produced by MPO targets apoA-I (apolipoprotein A-I), the major apolipoprotein of HDLs, the role of the other major oxidant generated by MPO, HOSCN (hypothiocyanous acid), in the generation of dysfunctional HDLs has not been examined. In the present study, we characterize the structural and functional modifications of lipid-free apoA-I and rHDL (reconstituted discoidal HDL) containing apoA-I complexed with phospholipid, induced by HOSCN and its decomposition product, OCN- (cyanate). Treatment of apoA-I with HOSCN resulted in the oxidation of tryptophan residues, whereas OCN- induced carbamylation of lysine residues to yield homocitrulline. Tryptophan residues were more readily oxidized on apoA-I contained in rHDLs. Exposure of lipid-free apoA-I to HOSCN and OCN- significantly reduced the extent of cholesterol efflux from cholesterol-loaded macrophages when compared with unmodified apoA-I. In contrast, HOSCN did not affect the anti-inflammatory properties of rHDL. The ability of HOSCN to impair apoA-I-mediated cholesterol efflux may contribute to the development of atherosclerosis, particularly in smokers who have high plasma levels of SCN- (thiocyanate).

Specific binding of hypochlorite-oxidized HDL to platelet CD36 triggers proinflammatory and procoagulant effects

ObjectiveOxidative stress and systemic inflammation negatively affect several protective functions of high density lipoproteins (HDL) and oxidative modification of HDL by the inflammation-derived oxidant hypochlorite converts HDL into a potent platelet agonist. Therefore it was the aim of this work to clarify if these platelet-activating effects result from specific binding of hypochlorite-oxidized HDL (hyp-OxHDL) to the platelet surface and to identify responsible receptors. MethodsBinding and functional studies were performed with hyp-OxHDL in absence and presence of (potential) competitors in normal and CD36-deficient human platelets.Platelet aggregation was quantified by light transmission aggregometry. Surface expression of CD62P, phosphatidylserine and CD40L was quantified by flow cytometry. ResultsBinding studies reveal that hyp-OxHDL show specific and saturable high-affinity binding to the platelet surface. Hyp-OxHDL trigger platelet aggregation and in a dose dependent way provoke the release of significant amounts of CD40L as well as phosphatidylserine on the platelet surface. Blocking specific binding of hyp-OxHDL to the platelet surface interferes with the ability of hyp-OxHDL to stimulate human platelets.CD36-deficient human platelets show markedly reduced binding of hyp-OxHDL. Upon addition of hypochlorite-oxidized HDL, CD36-deficient platelets do not aggregate and completely fail to release CD40L or phosphatidylserine. ConclusionsFrom these results we conclude that specific binding of hyp-OxHDL to platelet CD36 is essential for the proinflammatory and procoagulant effects of hyp-OxHDL shown within this work. The contribution of other receptors besides CD36 to specific binding of hyp-OxHDL to the platelet membrane appears to be minimal, at best.

Divergent effects of alpha-tocopherol and vitamin C on the generation of dysfunctional HDL associated with diabetes and the Hp 2-2 genotype.

The haptoglobin (Hp) 2-2 genotype is associated with increased risk of cardiovascular disease (CVD) in diabetes (DM). We recently proposed this increased risk arises from the tethering of redox active hemoglobin (Hb) to high density lipoprotein (HDL), thereby resulting in oxidative modification of HDL. Clinical trials have demonstrated that vitamin E (alpha-tocopherol) decreases while vitamin C increases CVD in Hp 2-2 DM individuals. We sought to test the hypothesis that the interaction of alpha-tocopherol or vitamin C on CVD in Hp 2-2 DM was due to their divergent effects on HDL oxidation and function. Vitamin C significantly increased while alpha-tocopherol completely blocked oxidation mediated by glycosylated Hb-Hp 2-2. Vitamin C had no benefit while alpha-tocopherol completely restored HDL function in Hp 2-2 DM mice. Co-administration of vitamin C mitigated the protective effects of alpha-tocopherol on HDL. There exists a pharmacogenomic interaction between vitamin C and alpha-tocopherol and the Hp 2-2 genotype on HDL function and structure. Choosing the correct antioxidant in the correct subset of patients may be critical in order to demonstrate benefit from antioxidant therapy.

Effects of a high polyunsaturated fat diet and vitamin E supplementation on high-density lipoprotein oxidation in humans

Oxidative modification of high-density lipoproteins (HDL) impairs several biologic functions critical to its role in reverse cholesterol transport. We therefore investigated the effect of dietary polyunsaturated fat and vitamin E on the kinetics of HDL oxidation. Ten subjects were fed sequentially: a baseline diet in which the major fat source was olive oil; a high polyunsaturated fat diet in which the major fat source was safflower oil; and the safflower oil diet plus 800 I.U. vitamin E per day. Plasma lipoprotein levels, vitamin E content, fatty acid composition, and oxidation lag time and rate were determined after 3 weeks on each diet. The polyunsaturated fat diet increased the mean HDL 2 lag time from 45.8±12.5 to 83.3±11.6 min with no change in oxidation rate. Addition of vitamin E further increased the HDL 2 lag time to 115.6±4.4 min and decreased the HDL 2 oxidation rate 10-fold. Neither the polyunsaturated diet alone nor the diet with vitamin E supplementation had any effect on HDL 3 oxidation. We conclude that under conditions of controlled dietary fat intake, a high polyunsaturated fat intake does not increase the oxidation susceptibility of HDL subfractions, and that in this setting, vitamin E supplementation reduces the oxidation susceptibility of HDL 2. These data suggest that antioxidants could influence HDL function in vivo.

Oxidative modification of high-density lipoprotein 3 induced by human polymorphonuclear neutrophils. Protective effect of pentoxifylline.

The function of high-density lipoproteins (HDLs) in reverse cholesterol transport is impaired if HDLs are subjected to oxidative stress. Polymorphonuclear neutrophils (PMNs), which have been detected in the earliest stages of atherosclerotic lesions, are one of the most likely sources of the reactive oxygen species that cause such stress. In this study, we investigated the effect of a PMN oxidative burst on HDL3. We also studied the impact on these events of pentoxifylline, a drug that regulates granulocyte function. HDL3 (370 nmol.mL-1 cholesterol-HDL) was incubated with PMNs (2 x 106. mL-1) in NaCl/Pi in the presence or absence of an iron chelate complex (10 microm Fe-nitrilotriacetic acid) at 37 degreesC for 60 min or 24 h. Phorbol myristate acetate (PMA) or formyl-methionylleucyphenylalanine (fMetLeuPhe) was used to stimulate PMNs. In iron-free NaCl/Pi medium, PMA-stimulated PMNs had a 40% lower HDL3 alpha-tocopherol content, whatever the incubation time. In NaCl/Pi medium containing iron, there was 80% less HDL3 alpha-tocopherol at 60 min, and HDL3 alpha-tocopherol had almost disappeared after 24 h. In this latter condition, the amount of thiobarbituric acid-reactive substances was significantly higher than the respective control HDL3 (P < 0.05) and oxidation of HDL3 by PMA-stimulated PMNs was associated with cross-linking of apoprotein AI, which was detected by SDS/PAGE. Similar results were obtained with fMetLeuPhe-stimulated PMN except that HDL3 alpha-tocopherol was consumed much more slowly during the first 60 min. Pretreatment of PMNs with various concentrations of pentoxifylline (0.001-20 mm) led to the concentration-dependent inhibition of oxidative modification of HDL3 induced by stimulated PMNs. The addition of 20 mm pentoxifylline in the most extreme oxidative stress conditions resulted in 70% of HDL3 alpha-tocopherol being maintained, with no formation of thiobarbituric acid-reactive substances and a lower level of apoprotein AI cross-linking. Thus HDL3 is susceptible to oxidative modifications induced by stimulated PMNs, in the presence of an exogenous source of iron. Pentoxifylline inhibited the oxidative modification of HDL3 by PMNs.

The role of oxidized HDL in monocyte/macrophage functions in the pathogenesis of atherosclerosis in Rhesus monkeys

The effect of oxidative modification of high-density lipoprotein (HDL) was assessed by incubation of normal HDL (obtained from Rhesus monkeys fed a stock diet) with 5 microM CuSO4 at 37 degrees C for 12 h/24 h. The physicochemical properties of oxidized-HDL (Ox-HDL) were found to be affected in terms of lipid peroxidation, as observed by the increased level of thiobarbituric acid reactive substances (nmol MDA/mg HDL protein). The biological properties of HDL were altered, since a decrease in the efflux of free cholesterol into the medium was found in the presence of Ox-HDL24h compared with normal HDL (N-HDL). The binding, uptake and degradation of 125I-LDL by macrophages increased in the presence of Ox-HDL24h. The activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione-peroxidase) was reduced in monocytes in the presence of Ox-HDL. However, in the presence of N-HDL, the levels of antioxidant enzymes were maintained at a higher level than in the control (in the absence of HDL) monocytes. Furthermore, the number of monocytes adhered to aortic endothelium were found to be increased in the presence of Ox-HDL. These findings suggest that HDL is susceptible to oxidative modification. Since the parameters selected in the present study are involved in the pathogenesis of atherosclerosis, it can be postulated that the in vivo protection of HDL in atherosclerosis can be reversed in the circumstances in which HDL undergoes oxidative modification like low-density lipoprotein (LDL).

High density lipoprotein loses its effect to stimulate efflux of cholesterol from foam cells after oxidative modification.

In this study, we performed oxidative modification of high density lipoprotein (HDL) in vitro. The amount of lipid peroxide increased when either HDL2 or HDL3 was incubated with phosphate-buffered saline containing 5 microM CuSO4 for 24 h at 37 degrees C, indicating that both fractions of HDL were oxidatively modified. This modification resulted in denaturation of apolipoprotein AI on SDS/PAGE and increased the negative charge on agarose gel electrophoresis. When incubated with macrophage-derived foam cells, native HDL caused a marked efflux of cholesterol from them, leading to a decrease in the amount of cholesteryl ester in the cells. However, oxidized HDL showed a lessened effect on the decrease of cholesteryl ester in foam cells. These data suggest that oxidative modification of HDL may stimulate development of atherosclerosis by limiting efflux of cholesterol from foam cells.