HDL-mediated Macrophage Cholesterol Efflux Research Articles

The Capacity of APOB-Depleted Plasma in Inducing ATP-Binding Cassette A1/G1-Mediated Macrophage Cholesterol Efflux-But Not Gut Microbial-Derived Metabolites-Is Independently Associated with Mortality in Patients with ST-Segment Elevation Myocardial Infarction.

Impaired HDL-mediated macrophage cholesterol efflux and higher circulating concentrations of trimethylamine N-oxide (TMAO) levels are independent risk factors for cardiovascular mortality. The TMAO precursors, γ-butyrobetaine (γBB) and Trimethyllysine (TML), have also been recently associated with cardiovascular death, but their interactions with HDL-mediated cholesterol efflux remain unclear. We aimed to determine the associations between APOB depleted plasma-mediated macrophage cholesterol efflux and plasma TMAO, γBB, and TML concentrations and explore their association with two-year follow-up mortality in patients with acute ST-elevation myocardial infarction (STEMI) and unstable angina (UA). Baseline and ATP-binding cassette transporter ABCA1 and ABCG1 (ABCA1/G1)-mediated macrophage cholesterol efflux to APOB-depleted plasma was decreased in patients with STEMI, and the latter was further impaired in those who died during follow-up. Moreover, the circulating concentrations of TMAO, γBB, and TML were higher in the deceased STEMI patients when compared with the STEMI survivors or UA patients. However, after statistical adjustment, only ABCA1/G1-mediated macrophage cholesterol efflux remained significantly associated with mortality. Furthermore, neither the TMAO, γBB, nor TML levels altered the HDL-mediated macrophage cholesterol efflux in vitro. We conclude that impaired ABCA1/G1-mediated macrophage cholesterol efflux is independently associated with mortality at follow-up in STEMI patients.

Phenol-Enriched Virgin Olive Oil Promotes Macrophage-Specific Reverse Cholesterol Transport In Vivo.

The intake of olive oil (OO) enriched with phenolic compounds (PCs) promotes ex vivo HDL-mediated macrophage cholesterol efflux in humans. We aimed to determine the effects of PC-enriched virgin OO on reverse cholesterol transport (RevCT) from macrophages to feces in vivo. Female C57BL/6 mice were given intragastric doses of refined OO (ROO) and a functional unrefined virgin OO enriched with its own PC (FVOO) for 14 days. Our experiments included two independent groups of mice that received intragastric doses of the phenolic extract (PE) used to prepare the FVOO and the vehicle solution (saline), as control, for 14 days. FVOO intake led to a significant increase in serum HDL cholesterol and its ability to induce macrophage cholesterol efflux in vitro when compared with ROO group. This was concomitant with the enhanced macrophage-derived [3H]cholesterol transport to feces in vivo. PE intake per se also increased HDL cholesterol levels and significantly promoted in vivo macrophage-to-feces RevCT rate when compared with saline group. PE upregulated the expression of the main macrophage transporter involved in macrophage cholesterol efflux, the ATP binding cassette a1. Our data provide direct evidence of the crucial role of OO PCs in the induction of macrophage-specific RevCT in vivo.

Effects of Virgin Olive Oil and Phenol-Enriched Virgin Olive Oils on Lipoprotein Atherogenicity.

The atherogenicity of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins (TRLs) may be more significant than LDL cholesterol levels. Clinical trials which have led to increased high-density lipoprotein (HDL) cholesterol have not always seen reductions in cardiovascular disease (CVD). Furthermore, genetic variants predisposing individuals to high HDL cholesterol are not associated with a lower risk of suffering a coronary event, and therefore HDL functionality is considered to be the most relevant aspect. Virgin olive oil (VOO) is thought to play a protective role against CVD. This review describes the effects of VOO and phenol-enriched VOOs on lipoprotein atherogenicity and HDL atheroprotective properties. The studies have demonstrated a decrease in LDL atherogenicity and an increase in the HDL-mediated macrophage cholesterol efflux capacity, HDL antioxidant activity, and HDL anti-inflammatory characteristics after various VOO interventions. Moreover, the expression of cholesterol efflux-related genes was enhanced after exposure to phenol-enriched VOOs in both post-prandial and sustained trials. Improvements in HDL antioxidant properties were also observed after VOO and phenol-enriched VOO interventions. Furthermore, some studies have demonstrated improved characteristics of TRL atherogenicity under postprandial conditions after VOO intake. Large-scale, long-term randomized clinical trials, and Mendelian analyses which assess the lipoprotein state and properties, are required to confirm these results.

Impaired HDL (High-Density Lipoprotein)-Mediated Macrophage Cholesterol Efflux in Patients With Abdominal Aortic Aneurysm-Brief Report.

Objective- The ability of HDL (high-density lipoprotein) to promote macrophage cholesterol efflux is considered the main HDL cardioprotective function. Abdominal aortic aneurysm (AAA) is usually characterized by cholesterol accumulation and macrophage infiltration in the aortic wall. Here, we aim to evaluate the composition of circulating HDL particles and their potential for promoting macrophage cholesterol efflux in AAA subjects. Approach and Results- First, we randomly selected AAA and control subjects from Spain. The AAA patients in the Spanish cohort showed lower plasma apoA-I levels concomitantly associated with low levels of plasma HDL cholesterol and the amount of preβ-HDL particles. We determined macrophage cholesterol efflux to apoB-depleted plasma, which contains mature HDL, preβ-HDL particles and HDL regulatory proteins. ApoB-depleted plasma from AAA patients displayed an impaired ability to promote macrophage cholesterol efflux. Next, we replicated the experiments with AAA and control subjects derived from Danish cohort. Danish AAA patients also showed lower apoA-I levels and a defective HDL-mediated macrophage cholesterol efflux. Conclusions- AAA patients show impaired HDL-facilitated cholesterol removal from macrophages, which could be mechanistically linked to AAA.

Abstract 555: Investigation of 4-oxo-2-nonenal Mediated HDL Dysfunction and the Use of Lipid Dicarbonyl Scavengers in Preserving HDL Function

Background: Lipid peroxidation products such as hydroxy-2-nonenal (HNE) are elevated in atherosclerosis and cause HDL dysfunction. Generated in parallel to HNE is 4-oxo-2-nonenal (ONE), a less studied lipid dicarbonyl possibly due to its far greater reactivity. The consequences of ONE modification of HDL have not been studied. We have recently found that scavengers of lipid dicarbonyls such as salicylamine (SAM) and pentylpyridoxamine (PPM) prevent HDL dysfunction induced by malondialdehyde and isolevuglandin (IsoLG). In this study, we examine the impact of ONE adduct formation on HDL structure-function, and the scavenging abilities of various small molecules in preventing ONE modification. Methods and Results: By Western blot analysis, ONE crosslinked apoA-I on HDL at a concentration of 3 ONE molecules for every 10 apoA-I proteins (0.3 molar equivalence, eq.), which is 100 fold lower than HNE but comparable to IsoLG. ONE-mediated crosslinking of HDL proteins preferentially produced a 39 kDa band on SDS-PAGE, likely an apoA-I/apoA-II heterodimer. ONE-modified HDL partially inhibited the ability of HDL to protect against the inflammatory response of macrophages (as shown in TNFα and IL-1β mRNA expression), but did not render HDL pro-inflammatory. At 3 eq., ONE dramatically decreased the ability of apoA-I to exchange among HDLs, from ~46.5% to only ~18.4% (P<0.001). HDL-mediated macrophage cholesterol efflux was decreased by ~70.6% (P<0.005) and 56.1% (P<0.001) by HDL modified by 0.3 and 3 eq. ONE, respectively. Investigation of various scavenger analogues in protecting HDL from ONE modification showed that while SAM and its fluoro- and chloro- analogues partially prevented ONE-mediated HDL protein crosslinking, PPM nearly completely blocked crosslinking. PPM pretreatment of HDL prior to 3 eq. ONE modification was also able to restore HDL-mediated macrophage cholesterol efflux, from 56.1~ to ~83.0% (P<0.01) while the inactive analogue pentylpyridoxine had no effect. Conclusions: Our study is the first to show that ONE causes HDL dysfunction, and demonstrates that not all modified HDLs result in the same “dysfunction”. We also demonstrate the use of PPM in preferentially scavenging ONE in biological systems.

Abstract 576: Salicylamine, A γ-ketoaldehyde Scavenger, Improves HDL Function and Reduces Atherosclerosis in Apoe Deficient Mice

Background: Lipid peroxidation products impair the cholesterol efflux capacity of high-density lipoprotein (HDL) and promote the development of atherosclerosis. The impact of inhibition of malondialdehyde (MDA)-HDL adduct formation by scavengers on HDL function and whether small molecule aldehyde scavengers protect against the development of atherosclerosis was examined. Methods and Results: Western blot analysis of ApoAI revealed that the amount of ApoAI crosslinking increased with MDA concentration. In the presence of LPS, MDA-HDL (HDL modified by 1mM MDA) versus control HDL stimulated 2- and 1.8-fold more expression of TNF-α and IL-1β in Apoe-/- macrophages demonstrating that MDA-HDL has reduced anti-inflammatory function. HDL-mediated macrophage cholesterol efflux was decreased by ~ 42%, 55%, 70%, and 80%, respectively, for HDL modified with 0.125 mM, 0.25 mM, 0.5 mM, and 1mM MDA, demonstrating that MDA modification of HDL affects its cholesterol efflux capacity in a dose dependent manner. Analysis by Western blot demonstrated that 5mM of salicylamine (SAM) and 5mM of pentylpyridoxamine (PPM), γ-ketoaldehyde scavengers, attenuated MDA mediated crosslinking of apoA-I in HDL (molar ratio of MDA and HDL is 1:5) by 60% and 80 % (P<0.05), respectively. Both SAM and PPM maintained the cholesterol efflux capacity of MDA treated HDL in Apoe-/- macrophages. In addition, pretreatment of LDL with SAM prevented MDA-ApoB adduct formation, and compared to incubation with LDL containing MDA-ApoB adducts, SAM treatment resulted in 57% less cholesterol accumulation in J774 macrophages. Importantly, administration of the ketoaldehyde scavenger, SAM, versus the nonreactive analogue, 4-SAM, to Apoe-/- mice consuming a Western diet for 16 weeks reduced the extent of proximal aortic atherosclerosis by 28% (P<0.05). Conclusions: Treatment with salicylamine, a γ-ketoaldehyde scavenger: 1) inhibits MDA-ApoA1 adduct formation thereby preserving HDL cholesterol efflux capacity; 2) prevents MDA-apoB100 formation resulting in less macrophage cholesterol accumulation; 3) reduces atherosclerosis in Apoe -/- mice. These results support the therapeutic potential of salicylamine in the treatment of atherosclerotic cardiovascular disease.

Coenzyme Q10 consumption promotes ABCG1-mediated macrophage cholesterol efflux: a randomized, double-blind, placebo-controlled, cross-over study in healthy volunteers.

We have recently shown that coenzyme Q10 (CoQ10) enhances macrophage reverse cholesterol transport via activator protein-1/miRNA-378/ABCG1 signal pathway in vivo and in vitro. Whether CoQ10 exerts similar beneficial effects in human is currently unknown. The present study evaluated the effect of CoQ10 on ABCG1-mediated macrophage cholesterol efflux in 20 healthy volunteers. Participants were given 100 mg CoQ10 twice daily or placebo for 1 week with a 1-week washout period. Human monocyte-derived macrophages (MDMs) were differentiated under pooled sera obtained before (preCoQ10) or after CoQ10 (postCoQ10) consumption. The CoQ10-induced inhibition of MDMs foam cell formation was blocked by ABCG1 silencing in postCoQ10 MDMs incubated with postCoQ10 sera compared to preCoQ10 sera. The cholesterol efflux to HDL, and mRNA as well as protein expressions of ATP-binding cassette transporter G1 (ABCG1) were augmented in postCoQ10 MDMs incubated with postCoQ10 sera compared to preCoQ10 sera. The change in the serum CoQ10 concentration positively correlated with cholesterol efflux to HDL and ABCG1 mRNA level in the CoQ10 group. MDMs treated with purified CoQ10 had an enhanced cholesterol efflux to HDL. CoQ10 consumption may have an atheroprotective property by inducing ABCG1 expression and enhancing HDL-mediated macrophage cholesterol efflux in healthy individuals.

Reduced glutathione increases quercetin stimulatory effects on HDL- or apoA1-mediated cholesterol efflux from J774A.1 macrophages

In our in vitro study, we analyzed the effects of incubation of J774A.1 macrophages with reduced glutathione (GSH) and quercetin on the extent of cellular cholesterol efflux by high-density lipoprotein (HDL) or apolipoprotein A1 (apoA1). This combination was the most potent one among other exogenous and endogenous antioxidant combinations, since it significantly increased the extent of HDL-mediated cholesterol efflux from macrophages by 47% versus control cells, whereas quercetin (20 μM) or GSH (200 μM) alone increased it by only 37% or 17%, respectively. Similarly, apoA1-mediated cholesterol efflux was increased by 11% or 22% in quercetin or quercetin + GSH-treated cells, respectively, versus control cells. These stimulatory effects were noted only after 20 h of cell incubation. The combination of quercetin + GSH demonstrated high scavenging capacity of free radicals versus quercetin or GSH alone. In addition, quercetin + GSH significantly decreased macrophage oxidative stress as measured by the scavenging capacity of free radicals in the cells, the formation of reactive oxygen species, and the levels of cellular glutathione and lipid peroxides. There was no significant effect of quercetin + GSH on cellular HDL binding, on ATP-binding cassette A1 (ABCA1) activity, or on ABCG1 messenger RNA (mRNA) levels.In contrast, mRNA levels for ABCA1 and peroxisome proliferator-activated receptor alpha (PPARα) were both significantly increased by 89% and 93%, respectively, in quercetin + GSH-treated cells versus control cells. Quercetin alone increased the mRNA levels for ABCA1 or PPARα by 42% or 77%, respectively, whereas GSH alone increased it by 22% or 28%, respectively. Mass spectra analysis revealed that oxidized quercetin reacts with GSH to form a new adduct product. We thus conclude that the stimulatory effects of quercetin + GSH on apoA1- or HDL-mediated macrophage cholesterol efflux are related to the ability of GSH to preserve quercetin in its reduced form.

Cholesterol Efflux and Atheroprotection

High-density lipoprotein (HDL) has been proposed to have several antiatherosclerotic properties, including the ability to mediate macrophage cholesterol efflux, antioxidant capacity, antiinflammatory properties, nitric oxide–promoting activity, and ability to transport proteins with their own intrinsic biological activities.1 HDL particles are critical acceptors of cholesterol from lipid-laden macrophages and thereby participate in the maintenance of net cholesterol balance in the arterial wall and in the reduction of proinflammatory responses by arterial cholesterol-loaded macrophages. The pathways that regulate HDL-mediated macrophage cholesterol efflux and disposition of cholesterol involve cell membrane–bound transporters, plasma lipid acceptors, plasma proteins and enzymes, and hepatic cellular receptors (Figure 1). From the earliest proposed concept for HDL-mediated cholesterol efflux,2,3 the concentration of the cholesterol content in HDL particles has been considered a surrogate measurement for the efficiency of the “reverse cholesterol transport” (RCT) process; however, macrophage-derived cholesterol represents a minor component of the cholesterol transported by HDL particles.4–7 One important pathway for cholesterol-mediated efflux from macrophage foam cells involves interaction between the ATP-binding cassette transporter A1 (ABCA1) and cholesterol-deficient and phospholipid-depleted apolipoprotein (apo) A-I complexes (pre-β migrating HDL or very small HDL [HDL-VS]; Figure 2).1,8 Subsequently, the ATP-binding cassette transporter G1 (ABCG1) mediates macrophage cholesterol efflux through interactions (Figure 3) with spherical, cholesterol-containing α-HDL particles (small HDL [HDL-S], medium HDL [HDL-M], large HDL [HDL-L], and very large (HDL-VL).1 In contrast, the scavenger receptor class B type I (SR-BI) is a multifunctional receptor that mediates bidirectional lipid transport in the macrophage, which is dependent on the content of cholesterol in lipid-laden macrophages. A more established role for SR-BI in cholesterol trafficking involves selective uptake of cholesteryl esters from mature HDL by the liver. Recent studies suggest that polymorphisms in SR-BI contribute to the functional capacity of this cholesterol …

Human macrophage cholesterol efflux potential is enhanced by HDL-associated 17β-estradiol fatty acyl esters

High-density lipoprotein (HDL) and 17β-estradiol independently provide protection against atherosclerosis. Estradiol fatty acyl esters incorporate into HDL and whether this association enhances the atheroprotective properties of HDL is unclear. The study objective was to clarify the role that HDL-associated estradiol fatty acyl esters play in mediating the initial steps of reverse cholesterol transport. Cholesterol efflux potential from cholesterol loaded macrophage cells to HDL-associated estradiol ester or between HDL from premenopausal women and age-matched males and the cellular receptors involved were examined. Human THP-1 macrophages, loaded with [ 3H]cholesterol oleate, acetylated low-density lipoprotein, were pretreated with or without SR-BI inhibitors or an estrogen receptor antagonist and incubated with either HDL-associated estradiol oleate, HDL lacking estradiol oleate, or isolated HDL from females and males, and cholesterol efflux was measured. Cellular internalization and hydrolysis of HDL-associated [ 3H]estradiol ester were determined. HDL-associated estradiol oleate and premenopausal female HDL demonstrated significantly higher cholesterol efflux capacity to media than male HDL. SR-BI and estrogen receptor inhibition significantly reduced this effect. Cells internalized and subsequently hydrolyzed HDL-associated [ 3H]estradiol ester to [ 3H]estradiol and again SR-BI inhibition reduced this internalization. These results demonstrate that HDL-mediated macrophage cholesterol efflux potential is enhanced by HDL-associated estradiol esters.

Di-oleoyl phosphatidylcholine (PC-18:1) stimulates paraoxonase 1 (PON1) enzymatic and biological activities: In vitro and in vivo studies

Objective Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme which possess anti-atherogenic properties. Our aim was to analyze the effect of HDL phospholipids on HDL-associated paraoxonase (PON1) catalytic and biological activities. Methods and results In HDL isolated from di-oleoyl-phosphatidylcholine (PC-18:1)-enriched serum, HDL–PC-18:1 levels, as well as PON1 lactonase, arylesterase and paraoxonase activities were increased by 23%, 35%, 47% and 63%, respectively, as compared to control HDL ( p < 0.01). Furthermore, PON1 contribution to HDL-mediated cholesterol efflux from J774A.1 macrophages was higher in PC-18:1-enriched HDL in comparison to control HDL. In vivo olive oil consumption by Balb C mice increased HDL phospholipids/protein (30%), and HDL–PON1 arylesterase (150%) and lactonase (94%) activities ( p < 0.01). Furthermore, in the olive oil-treated mice PON1 contribution to HDL-mediated macrophage cholesterol efflux was higher by 100%, in comparison to placebo mouse HDL ( p < 0.01). Similarly, olive oil consumption by healthy subjects increased HDL–PC-18:1 levels, HDL–PON1 arylesterase (88%), lactonase (52%), paraoxonase (140%) activities and PON1 stimulatory effect on HDL-mediated cholesterol efflux (53%) as compared to HDL before treatment ( p < 0.01). PC-18:1 stimulatory effect on recombinant PON1 mutant (lacks 20 amino acids at the N-terminal region) paraoxonase and lactonase activities was lower by 56% and 57%, respectively, in comparison to its effect on wild type PON1 ( p < 0.01). Conclusion Intervention to increase PON1 activities by HDL enrichment with PC-18:1 could be proven as a beneficial anti-atherogenic therapy.

Antiatherogenicity of extra virgin olive oil and its enrichment with green tea polyphenols in the atherosclerotic apolipoprotein-E-deficient mice: enhanced macrophage cholesterol efflux

The antiatherogenic properties of extra virgin olive oil (EVOO) enriched with green tea polyphenols (GTPPs; hereafter called EVOO-GTPP), in comparison to EVOO, were studied in the atherosclerotic apolipoprotein-E-deficient (E 0) mice. E 0 mice (eight mice in each group) consumed EVOO or EVOO-GTPP (7 μl/mouse/day, for 2 months) by gavage feeding. The placebo group received only water. At the end of the study, blood samples, peritoneal macrophages and aortas were collected. Consumption of EVOO or EVOO-GTPP resulted in a minimal increase in serum total and high-density lipoprotein (HDL) cholesterol levels (by 12%) and in serum paraoxonase 1 activity (by 6% and 10%). EVOO-GTPP (but not EVOO) decreased the susceptibility of the mouse serum to AAPH-induced lipid peroxidation (by 18%), as compared to the placebo-treated mice. The major effect of both EVOO and EVOO-GTPP consumption was on HDL-mediated macrophage cholesterol efflux. Consumption of EVOO stimulated cholesterol efflux rate from mouse peritoneal macrophages (MPMs) by 42%, while EVOO-GTPP increased it by as much as 139%, as compared to MPMs from placebo-treated mice. Finally, the atherosclerotic lesion size of mice was significantly reduced by 11% or 20%, after consumption of EVOO or EVOO-GTPP, respectively. We thus conclude that EVOO possesses beneficial antiatherogenic effects, and its enrichment with GTPPs further improved these effects, leading to the attenuation of atherosclerosis development.

S-Glutathionylation regulates HDL-associated paraoxonase 1 (PON1) activity

HDL-associated paraoxonase 1 (PON1) undergoes inactivation under oxidative stress and is preserved by dietary antioxidants. PON1 cysteines can affect PON1 enzymatic activities. S-Glutathionylation, a redox regulatory mechanism characterized by the formation of a mixed disulfide between a protein thiol and oxidized glutathione (GSSG), was shown to preserve some enzymes from irreversible inactivation under pathological conditions. We questioned whether PON1 activity is regulated by S-glutathionylation. Incubation of PON1 or HDL with GSSG indeed resulted in a dose-dependent inactivation of PON1 activities, including its physiological activity to increase HDL-mediated macrophage cholesterol efflux. This PON1 inactivation was associated with the formation of a mixed disulfide bond between GSSG and PON1’s cysteine residue(s), as detected by immunoblotting with anti-glutathione IgG. PON1 activity was recovered following the addition of a reducing agent, DL-Dithiothreitol (DTT), to the PON1–SSG complex. We thus conclude that HDL-associated serum PON1 can undergo S-glutathionylation under oxidative stress with a consequent reversible inactivation.

Paraoxonase 1 (PON1) enhances HDL-mediated macrophage cholesterol efflux via the ABCA1 transporter in association with increased HDL binding to the cells: a possible role for lysophosphatidylcholine

We investigated the role of HDL-associated paraoxonase 1 (PON1) in HDL-mediated macrophage cholesterol efflux by using HDL derived from wild type mice (Control-HDL), from human PON1-transgenic mice (HDL-PON1Tg) or from PON1-knockout mice (HDL-PON1 0). Cholesterol efflux from mouse peritoneal macrophages (MPM) or from J774 A.1 macrophage cell line by HDL-PON1Tg, was significantly increased (by 60%) compared to HDL-PON1 0. We demonstrated that this PON1 effect was associated with an increased HDL binding to the cells, as the binding of HDL-PON1Tg (or HDL-PON1 0 that was enriched with PON1) was increased by 50% compared to that of HDL-PON1 0. Using either a cAMP analogue, to increase ABCA1 receptor expression, or rabbit anti-mouse SR-BI specific antibody to block the SR-BI receptor, PON1 stimulation of HDL binding and of HDL-mediated macrophage cholesterol efflux, were both found to involve the ABCA1 transporter. Studies with PON1 specific inhibitors revealed that PON1 activity was required for its stimulation of HDL-mediated macrophage cholesterol efflux. Upon incubation of macrophages with Control-HDL or with HDL-PON1Tg, macrophage lysophosphatidylcholine (LPC) content was increased by 3.7- and 7.5-fold, respectively. Such an LPC enrichment of macrophages resulted in up to 60% increased HDL binding to the cells, and a 41% increased HDL-mediated cholesterol efflux. Similarly, macrophage loading with LPC (by either adding LPC, or PON1 or phospholipase A 2) significantly increased apolipoprotein A-I (apoA-I) mediated cholesterol efflux by 104, 65 and 56%, respectively, in ABCA1 overexpressing macrophages. We conclude that HDL-associated PON1 may contribute to the attenuation of atherosclerosis development by its ability to act on macrophage phospholipids, to form LPC, in turn, stimulates HDL binding and HDL-mediated macrophage cholesterol efflux via the ABCA1 transporter.