Cholesterol Influx Research Articles

Mitochondrial heat shock protein participates in placental steroidogenesis

The human placenta, which does not express the StAR protein, synthesizes large amounts of progesterone. The rate-limiting step for steroidogenesis is the transport of cholesterol which is divided into two steps: 1) cholesterol flux from cytoplasm to outer membrane mitochondria, and 2) cholesterol transport from outer to inner mitochondrial membrane. The proteins mediating placental cholesterol influx have not been clearly identified. We investigated the proteins involved in the transport of cholesterol in syncytiotrophoblast mitochondria from human placenta. Two proteins, one of 30 kDa, and another of 60 kDa, were identified using anti-MLN64 antibodies. The 30 kDa protein corresponds to a fragment of MLN64, and the 60 kDa protein was identified as a heat shock protein. During steroidogenesis, mitochondria released MLN64 protein to supernatant. When this supernatant was added to fresh isolated mitochondria, progesterone synthesis increased; a similar result was obtained with the addition of the recombinant MLN64-START protein. In the presence of flurescein-5-maleimide or N-ethyl-maleimide, the mitochondrial synthesis of progesterone was inhibited in a dose-dependent fashion without changes in mitochondrial respiration. 2D-electrophoretic pattern showed that flurescein-5-maleimide- fluorescence was associated with HSP60. Both MLN64 and HSP60 were identified in mitochondrial contact sites. The results suggest that HSP60 is involved in the steroidogenic metabolism of human placenta. A tight association between MLN64 and HSP60 is suggested for cholesterol transport in the human placenta.

NF-E2–Related Factor 2 Promotes Atherosclerosis by Effects on Plasma Lipoproteins and Cholesterol Transport That Overshadow Antioxidant Protection

To test the hypothesis that NF-E2-related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties. Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E-null background by sequential breeding, resulting in Nrf2(-/-), apoE(-/-) (KO), Nrf2(-/+), apoE(-/-) (HET) and Nrf2(+/+), and apoE(-/-) wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36. Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.

MODULATION OF OXIDIZED LDL MEDIATED CELLULAR RESPONSES BY DIFFERENT FORMS OF HDL: PP.2.69

Objectives: Initial phase of atherosclerosis involves influx of low density lipoprotein cholesterol (LDL- C) accumulation, generation of reactive oxygen species and oxidative modulation of LDL-C. The effect of different form of high density lipoprotein (HDL) was studied on oxidized-LDL induced cellular responses in differentiated human monocytic leukemic cell line(THP-1 cells) and monocytes of patients with normal LDL and high LDL. Expression of pro-apoptotic and anti-apoptotic molecules were evaluated at mRNA and protein level. Designs and Methods: Different forms of lipoproteins were separated by ultracentrifugation from normal human plasma. Apolipoprotein A-1 (ApoA-1) was purified and used for preparation of normal high density lipoprotein (nHDL). Differentiated cells (THP-1 cells/cells from hypercholesteremic patients and controls differentiated with 100 nm PMA) were either cultured in presence of oxidized-LDL (Ox-LDL) or preincubated with normal HDL(nHDL) or reconstituted HDL (rHDL-prepared by method of Jonas) or oxidized HDL (Ox-HDL) followed by coincubation with Ox-LDL. Expression of pro-apoptotic (Bax and caspase-3) and anti-apoptotic molecules (Bcl-2 and Bcl-Xl) were assessed. Results: DNA fragmentation analysis revealed that Ox-LDL induced apoptosis in differentiated monocytes. The nHDL/rHDL inhibited Ox-LDL induced apoptosis in differentiated monocytes while Ox-LDL did not show this phenomenon. Studies from differentiated monocytes from hyperLDL patients revealed a higher percentage of apoptotic cells. OxLDL could result in detachment of differentiated THP-1 cells from surface of plate. This phenomenon was reduced by culturing with nHDL/rHDL. An elevated expression of Bax was found in differentiated cells cultured in presence of Ox-LDL while expression of Bcl-2 and Bcl-Xl was downregulated. Expression of Bcl-2 and Bcl-Xl was upregulated when preincubation with nHDL/rHDL was done. Expression of scavenger receptor LOX-1 was decreased in this group. Conclusions: Ox-LDL induced apoptosis in differentiated monocytes was downregulated in presence of nHDL/rHDL but not with OxHDL.Ox-LDL mediates apoptosis through scavenger receptor in differentiated THP- cells and monocytes from patients with high LDL.

Abstract 5096: Alterations in cholesterol regulation contribute to the production of intratumoral androgens in castration-resistant prostate cancer

Abstract BACKGROUND: Prostate cancer (CaP) is the most commonly diagnosed cancer and third leading cause of cancer-related death in North American males. Recent evidence suggests that androgens mediate disease progression as recurrent CaP tumors contain androgens in levels high enough to activate the androgen receptor (AR). We have previously shown that in the absence of exogenous androgens, CaP cells develop the ability to synthesize their own androgens. We hypothesize that a) androgens induce the in vitro expression and activity of cholesterol synthesis and esterification pathways and b) cholesterol synthesis, influx and efflux are altered in vivo to provide precursors for de novo androgen synthesis contributing to castration-resistant prostate cancer progression. METHODS: We have analyzed cholesterol metabolism in vitro (LNCaP, PC-3 and C4-2 cells in the presence of androgens) and in vivo (LNCaP xenograft model). The LNCaP xenograft model was used to mimic clinical progression of the disease. Tumors were excised from the same mouse at three stages: Androgen-Dependent (AD, prior to castration), Nadir (N, 8-days post castration) and Castration-Resistance (CRPC, 35-days post castration) and stored for further analysis. Protein expressions of AR, PSA and proteins involved in cholesterol synthesis and metabolism, influx and efflux as well as actin were determined by immunoblotting. Total cholesterol concentrations were determined within each excised tumor using a fluorescent assay and LC-MS. Tumoral steroid levels were assessed by LC-MS. RESULTS: Microsomal PC-3 and LNCaP cell HMG CoA reductase (cholesterol biosynthesis) activity was increased upon androgen treatment. ACAT1 activity (cholesterol esterification) was induced by androgens in PC-3 cells and this induction was AR- dependent as well as associated with increased ACAT1 protein expression. Tumor LDLr, SR-BI, HMG CoA reductase, ACAT1 and 2 as well as ABCA1 were altered during disease progression to CRPC likely to increase influx and synthesis of cholesterol. Total cholesterol levels increased in N and CRPC compared to AD. HMG CoA reductase expression was upregulated in N compared to AD (300%) and was maintained at this level at CRPC. Tumoral cholesterol biosynthesis ex vivo was increased from N to CRPC. Testosterone and dihydrosterone increased at CRPC to physiologically relevant levels and this may account for the increase in AR activation as determined by PSA expression at CRPC. CONCLUSIONS: Cellular cholesterol processes are altered by androgens and during progression to CRPC. Cholesterol from increased biosynthesis or uptake is likely a precursor for tumoral de novo androgen synthesis. ACKNOWLEDGEMENTS: Funding for this project was provided by the Canadian Institutes of Health Research (CIHR) and an NCIC-Terry Fox Group grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5096.

PPARγ regulates the expression of cholesterol metabolism genes in alveolar macrophages

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear transcription factor involved in lipid metabolism that is constitutively expressed in the alveolar macrophages of healthy individuals. PPARγ has recently been implicated in the catabolism of surfactant by alveolar macrophages, specifically the cholesterol component of surfactant while the mechanism remains unclear. Studies from other tissue macrophages have shown that PPARγ regulates cholesterol influx, efflux, and metabolism. PPARγ promotes cholesterol efflux through the liver X receptor-alpha (LXRα) and ATP-binding cassette G1 (ABCG1). We have recently shown that macrophage-specific PPARγ knockout (PPARγ KO) mice accumulate cholesterol-laden alveolar macrophages that exhibit decreased expression of LXRα and ABCG1 and reduced cholesterol efflux. We hypothesized that in addition to the dysregulation of these cholesterol efflux genes, the expression of genes involved in cholesterol synthesis and influx was also dysregulated and that replacement of PPARγ would restore regulation of these genes. To investigate this hypothesis, we have utilized a Lentivirus expression system (Lenti-PPARγ) to restore PPARγ expression in the alveolar macrophages of PPARγ KO mice. Our results show that the alveolar macrophages of PPARγ KO mice have decreased expression of key cholesterol synthesis genes and increased expression of cholesterol receptors CD36 and scavenger receptor A-I (SRA-I). The replacement of PPARγ (1) induced transcription of LXRα and ABCG1; (2) corrected suppressed expression of cholesterol synthesis genes; and (3) enhanced the expression of scavenger receptors CD36. These results suggest that PPARγ regulates cholesterol metabolism in alveolar macrophages.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Dietary Compound Quercitrin Dampens VEGF Induction and PPARγ Activation in Oxidized LDL-Exposed Murine Macrophages: Association with Scavenger Receptor CD36

Oxidized LDL (oxLDL) has been implicated in the pathogenesis of atherosclerosis accompanying lipid-laden cell appearance, inflammatory responses, and vascular dysfunction. This study examined the potentials of polyphenol quercitrin to inhibit oxLDL induction of scavenger receptor A (SR-A) and CD36 involving activation of peroxisome proliferator-activated receptor gamma (PPARgamma). J774A1 murine macrophages were cultured with 10 microg/mL Cu(2+)-oxLDL for various times in the presence of 1-10 micromol/L quercitrin. Cu(2+)-oxLDL at the given concentration facilitated macrophage proliferation and enhanced oxLDL uptake. Quercitrin dampened oxLDL uptake and lipid accumulation elevated in macrophages exposed to oxLDL. Western blot analysis revealed that 10 microg/mL oxLDL upregulated expression of SR-A and CD36, which was rapidly abolished at the transcriptional levels by 10 micromol/L quercitrin within 4 h. Quercitrin diminished production of proinflammatory and proatherogenic vascular endothelial growth factor that augmented through the oxLDL binding to CD36. Similarly, quercitrin repressed expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 involved in monocyte trafficking and macropahage migration. In addition, quercitrin attenuated oxLDL-induced transcriptional activation of PPARgamma leading to CD36 induction. Furthermore, quercitrin alleviated macrophage uptake of oxLDL through interfering with PKC-PPAR signaling cascades. These results demonstrate that quercitrin blocked oxLDL uptake, cholesterol influx and lipid-laden foam cell formation through inhibiting induction of SR and VEGF linked to PKCalpha-PPARgamma-responsive pathways. Therefore, quercitrin may be an antiatherogenic agent blocking foam cell formation pertaining to induction of SR and VEGF.

High glucose promotes intracellular lipid accumulation in vascular smooth muscle cells by impairing cholesterol influx and efflux balance

High glucose promotes macrophage-derived foam cell formation involved in increased influx or reduced efflux of lipids. The aim of this study is to investigate the influence of hyperglycaemia on foam cell transformation of vascular smooth muscle cells (VSMCs) and possible mechanisms contributing to these effects. The results showed that high glucose increased the expression of CD36, a regulator of lipid influx, and suppressed the expression and activity of the adenosine triphosphate-binding cassette (ABC) transporter ABCG1, a regulator of cholesterol efflux to high-density lipoprotein, in a dose- and time-dependent manner. However, cholesterol efflux to lipid-free apoAI was not impaired. VSMCs exposed to high glucose readily developed into lipid-loaded cells, as demonstrated by Oil Red O staining and cholesterol content analysis. In addition, high glucose-induced down-regulation of ABCG1 was reversed by nuclear factor-kappaB (NF-kappaB) inhibitors BAY 11-7085 and tosyl-phenylalanine chloromethyl ketone and by the antioxidant N-acetyl-L-cysteine (NAC). This reversal was accompanied by reduced cellular lipid content. Also, NAC and NF-kappaB inhibitors can effectively block the high glucose-induced activity of NF-kappaB binding to DNA and/or peroxide production. These results suggested that hyperglycaemia-induced foam cell formation in VSMCs was related to the imbalanced lipid flux by increasing CD36-mediated modified low-density lipoprotein uptake and reducing ABCG1-regulated cellular cholesterol efflux. Moreover, this effect was associated with increased oxidative stress and activated NF-kappaB pathway signalling.

High glucose stimulates macrophage SR-BI expression and induces a switch in its activity from cholesterol efflux to cholesterol influx

AimsDiabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions. Methods and resultsABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30mM) vs. low (5mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI. ConclusionsDiabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.

Alterations in cholesterol regulation contribute to the production of intratumoral androgens during progression to castration‐resistant prostate cancer in a mouse xenograft model

Emerging evidence suggests that androgens and the androgen receptor (AR) are important mediators of castration-resistant prostate cancer (CRPC) progression. Increased expression of several enzymes responsible for cholesterol synthesis and conversion into downstream androgens has been documented in human CRPC tumors in comparison to primary tumors. Based on these observations it is hypothesized that cholesterol and its overall regulation within the cell are altered, thus modifying precursor levels for de novo androgen synthesis within the castrate tumoral environment. Tumoral steroid levels were assessed by LC-MS. Free and esterified cholesterol was quantified by LC-MS and a fluorescent assay. Gene and protein expression were assessed by RT-PCR and immunoblotting. Herein, using a prostate cancer xenograft mouse model it is demonstrated by Western blot analysis that proteins responsible for cholesterol regulation (LDL-r, SR-B1, HMG-CoA reductase, ACAT1,2, ABCA1) are altered during disease progression to increase influx and synthesis of cholesterol as well as free cholesterol formation from cholesteryl ester stores. In turn this can provide increased amounts of precursor for intratumoral steroidogenesis after castration. Androgens- testosterone and dihydrotestosterone- coincidently increase at CRPC to physiologically relevant levels leading to the induction of AR expression and PSA production. Furthermore, cellular cholesterol homeostasis is maintained by increased cholesterol efflux at CRPC so that excess free cholesterol does not cause toxicity to the tumor cells. Cellular cholesterol regulation processes are altered during progression to CRPC. Free cholesterol from increased biosynthesis or uptake is likely a precursor for intratumoral de novo androgen synthesis.

An update on the lipid nephrotoxicity hypothesis

When the 'lipid nephrotoxicity hypothesis' was proposed in 1982, it brought together several disparate experimental findings in hyperlipidemia and renal disease to suggest that concomitant hyperlipidemia and proteinuria would cause self-perpetuating renal disease once the initial glomerular insult was no longer present. This process would be analogous to atherosclerosis. Since 1982, increasing evidence has supported the hypothesis that lipid abnormalities contribute to both atherosclerosis and glomerulosclerosis. In this Review, we discuss research developments that are relevant to the lipid nephrotoxicity hypothesis. We describe how inflammatory stress accompanying chronic kidney disease modifies lipid homeostasis by increasing cholesterol uptake mediated by lipoprotein receptors, inhibiting cholesterol efflux mediated by the ATP-binding cassette transporter 1 and impairing cholesterol synthesis in peripheral cells. As a result of these events, cholesterol relocates to and accumulates in renal, vascular, hepatic and possibly other tissues. The combination of increased cellular cholesterol influx and reduced efflux causes injury in some tissues and lowers the plasma cholesterol level. In addition, inflammatory stress causes a degree of statin resistance via unknown mechanisms. These phenomena alter traditional understanding of the pathogenesis of lipid-mediated renal and vascular injury and could influence the clinical evaluation of renal and cardiovascular risk and the role of lipid-lowering treatment in affected patients.

Molecular mechanisms underlying the cholesterol-lowering effect of Ginkgo biloba extract in hepatocytes: a comparative study with lovastatin

To explore the molecular mechanisms underlying the cholesterol-lowering effect of a Ginkgo biloba extract (GBE). Enzyme activity, cholesterol flux and changes in gene expression levels were assessed in cultured hepatocytes treated with GBE or lovastatin. GBE decreased the total cholesterol content in cultured hepatocytes and inhibited the activity of HMG-CoA reductase, as determined by an in vitro enzyme activity assay. In addition, GBE decreased cholesterol influx, whereas lovastatin increased cholesterol influx. GBE treatment induced significant increases in the expression of cholesterogenic genes and genes involved in cholesterol metabolism, such as SREBF2, as determined by cDNA microarray and real-time RT-PCR. Furthermore, INSIG2, LDLR, LRP1, and LRP10 were differentially regulated by GBE and lovastatin. The data imply that the two compounds modulate cholesterol metabolism through distinct mechanisms. By using a gene expression profiling approach, we were able to broaden the understanding of the molecular mechanisms by which GBE lowers cellular cholesterol levels. Specifically, we demonstrated that GBE exhibited dual effects on the cellular cholesterol pool by modulating both HMG-CoA reductase activity and inhibiting cholesterol influx.

PCSK9 and heart disease: quieting an outdated metabolic moderator

vesicles [4]. Within early endosomes, acidification causes dissociation of the receptor–ligand complex, after which the LDLR recycles to the cell surface. The internalized LDL particles are degraded in lysosomes and liberated cholesterol is then used for the synthesis of membranes, steroid hormones, lipoproteins and bile acids. This process is subject to a negative-feedback mechanism that maintains tight control of cellular cholesterol levels [5]. Cholesterol influx results in transcriptional suppression of genes encoding cholesterol biosynthetic enzymes and LDLR, thus preventing potentially toxic cholesterol overaccumulation. Conversely, when cholesterol levels are depleted the expression of these same genes is stimulated. Cholesterol-lowering statin drugs modulate this regulatory circuit by directly inhibiting HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. I-LDL turnover studies in humans demonstrated an increased clearance rate of LDL from circulation following statin therapy, indicating increased liver LDLR function as a secondary beneficial effect of statins [6,7].

Abstract: 75 HDL – ASSOCIATED PARAOXONASE 1 (PON1) ATTENUATES MACROPHAGE FOAM CELL FORMATION AND ATHEROGENESIS

Data about glucocorticoids role in the development of atherosclerosis are controversial showing different effects in human than in experimental animal models. Atherosclerosis is the result of a chronic inflammatory response to an injured endothelium where an uncontrolled uptake of OxLDL by macrophages triggers the development of foam cells, the main component of fatty streaks in atherosclerotic plaque. There are few data about the direct effect of glucocorticoids in macrophages of atherosclerotic plaque. The aim of the study was to elucidate the role of glucocorticoids in the development of foam cells in atherosclerosis initiation.For this purpose we used THP1 cells differentiated to macrophages with phorbol esters and incubated with OxLDL alone or with cortisol or cortisone. THP1 cells were also incubated with cortisone plus an inhibitor of 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) activity to determine the role of this enzyme on glucocorticoid action in this process.Ours results showed that cortisol and cortisone decreased significantly the inflammation promoted by OxLDL, and also diminished the expression of genes involved in influx and efflux of cholesterol resulting in a reduced lipid accumulation. Likewise cortisol and cortisone decreased 11βHSD1 expression in THP1 cells. The presence of the inhibitor of 11βHSD1 abolished all the effects elicited by cortisone.Our results indicate a direct effect of glucocorticoids on macrophages braking atherosclerosis initiation, reducing pro-inflammatory markers and OxLDL uptake and cholesterol re-esterification, but also inhibiting cholesterol output. These effects appear to be mediated, at least in part, by 11βHSD1 activity.

A new insight into resveratrol as an atheroprotective compound: Inhibition of lipid peroxidation and enhancement of cholesterol efflux

Resveratrol, a polyphenolic constituent of red wine, is known for its anti-atherogenic properties and is thought to be beneficial in reducing the incidence of cardiovascular diseases (CVD). However, the mechanism of action by which it exerts its anti-atherogenic effect remains unclear. In this study, we investigated the relationship between the antioxidant effects of resveratrol and its ability to promote cholesterol efflux. We measured the formation of conjugated dienes and the rate of lipid peroxidation, and observed that resveratrol inhibited copper- and irradiation-induced LDL and HDL oxidation as observed by a reduction in oxidation rate and an increase in the lag phase ( p < 0.05). We used DPPH screening to measure free radical scavenging activity and observed that resveratrol (0–50 μM) significantly reduced the content of free radicals ( p < 0.001). Respect to its effect on cholesterol homeostasis, resveratrol also enhanced apoA-1-mediated cholesterol efflux ( r 2 = 0.907, p < 0.05, linear regression) by up-regulating ABCA-1 receptors, and reduced cholesterol influx or uptake in J774 macrophages ( r 2 = 0.89, p < 0.05, linear regression). Incubation of macrophages (J774, THP-1 and MPM) with Fe/ascorbate ion, attenuated apoA-1 and HDL 3-mediated cholesterol efflux whereas resveratrol (0–25 μM) significantly redressed this attenuation in a dose-dependent manner ( p < 0.001). Resveratrol thus appears to be a natural antioxidant that enhances cholesterol efflux. These properties make it a potential natural antioxidant that could be used to prevent and treat CVD.

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL2 = HDL3. Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL3. ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r2 = 0.7), apolipoprotein A-I (apoA-I) (r2 = 0.5), HDL-C (r2 = 0.4), HDL-PL (r2 = 0.4), α-2 HDL (r2 = 0.4), and preβ HDL (r2 = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL3. ABCG1 expression did not increase the association of HDL3 with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

Thieno[2,3-c]Isoquinolin-5-one, a Potent Poly(ADP-Ribose) Polymerase Inhibitor, Promotes Atherosclerotic Plaque Regression in High-Fat Diet-Fed Apolipoprotein E-Deficient Mice: Effects on Inflammatory Markers and Lipid Content

We recently showed that poly(ADP-ribose) polymerase (PARP) is activated within atherosclerotic plaques in an animal model of atherosclerosis. Pharmacological inhibition of PARP or reduced expression in heterozygous animals interferes with atherogenesis and may promote factors of plaque stability, possibly reflecting changes in inflammatory and cellular factors consistent with plaque stability. The current study addresses the hypothesis that pharmacological inhibition of PARP promotes atherosclerotic plaque regression. Using a high-fat diet-induced atherosclerosis apolipoprotein E(-/-) mouse model, we demonstrate that administration of the potent PARP inhibitor, thieno[2,3-c]isoquinolin-5-one (TIQ-A), when combined with a regular diet regimen during treatment, induced regression of established plaques. Plaque regression was associated with a reduction in total cholesterol and low-density lipoproteins. Furthermore, plaques of TIQ-A-treated mice were highly enriched with collagen and smooth muscle cells, displayed thick fibrous caps, and exhibited a marked reduction in CD68-positive macrophage recruitment and associated foam cell presence. These changes correlated with a significant decrease in expression of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1, potentially as a result of a robust reduction in tumor necrosis factor expression. The PARP inhibitor appeared to affect cholesterol metabolism by affecting acyl-coenzymeA/cholesterol acyltransferase-1 expression but exerted no effect on cholesterol influx or efflux as assessed by an examination of the ATP-binding cassette transporter-1 and the scavenger receptor-A expression levels in the different experimental groups. In accordance, PARP inhibition may prove beneficial not only in preventing atherogenesis but also in promoting regression of preexisting plaques.

Origins of intestinal ABCA1-mediated HDL-cholesterol

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.

Antiinflammatory Actions of HDL

Atherosclerosis is an inflammatory disorder characterized by the accumulation of macrophages and T-lymphocytes in the arterial intima. The macrophages are derived from blood monocytes that adhere to, and transmigrate across, an activated or injured endothelial surface. The firm adhesion of monocytes to the endothelium requires expression of integrins such as CD11/CD18 on the monocyte surface and endothelial expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. Monocytes that are firmly tethered to the endothelium transmigrate into the subendothelial space in a process that is dependent on endothelial expression of chemokines such as monocyte chemotactic protein-1 (MCP-1).1 See accompanying article on page 2071 After entering the artery wall, monocytes differentiate into macrophages in processes that are mediated by macrophage colony stimulating factor (CSF) and granulocyte-macrophage CSF.2 Macrophages express a range of scavenger receptors that bind and internalize modified LDL in an unregulated manner. This leads to a progressive accumulation of lipids in the macrophages and the formation of foam cells: the hallmark cells of atherosclerotic lesions. The results of numerous epidemiological studies have established that …

Genetic Variation in ABC G5/G8 and NPC1L1 Impact Cholesterol Response to Plant Sterols in Hypercholesterolemic Men

ATP-binding cassette hetero-dimeric transporters G5 and G8 (ABCG5/G8) have been postulated to mediate intestinal cholesterol efflux, whereas Niemann-Pick C1 Like 1 (NPC1L1) protein is believed to be essential for intestinal cholesterol influx. The individual or combined genetic markers, such as single nuclear polymorphisms (SNPs), of these two transporter genes may explain inter-individual variations in plasma cholesterol response following plant sterol (PS) intervention. The present study was aimed at investigating the association between ABCG5/G8 and NPC1L1 genotype SNPs with sterol absorption and corresponding plasma concentrations. The study used a 4-week crossover design with 82 hypercholesterolemic men characterized by high vs. low basal plasma PS concentrations consuming spreads with or without 2 g/day of PS. For the ABCG8 1289 C > A (T400 K) polymorphism, the A allele carriers with high basal plasma PS concentrations demonstrated a 3.9-fold greater reduction (p < 0.05) in serum low density lipoprotein cholesterol (LDL-C) than their low basal plasma PS counterparts. For the NPC1L1 haplotype of 872 C > G (L272L) and 3929 G > A (Y1291Y), individuals carrying mutant alleles showed a 2.4-fold greater (p < 0.05) reduction in LDL-C levels, compared to wild type counterparts. Results suggest that genetic and metabolic biomarkers together may predict inter-individual lipid level responsiveness to PS-intervention, and thus could be useful in devising individualized cholesterol lowering strategies.