Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity

ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 mediate the efflux of cholesterol and other sterols. Both transporters are expressed on the fetal capillaries of the placenta and are involved in maternal-to-fetal cholesterol delivery. In this study, we report that ABCA1 and ABCG1 are also present on the syncytiotrophoblast, the maternal facing placental membrane. Syncytial ABCA1 expression is apical, suggesting a role in cholesterol efflux to the mother, while ABCG1 is expressed basolaterally indicating transport to the fetus. Silencing of ABCA1 expression in primary trophoblasts in culture, or pharmacological antagonism by glyburide, decreased cholesterol efflux to apolipoprotein A-I (apoA-I) compared to controls, while ABCG1-silencing decreased cholesterol efflux to high density lipoproteins (HDL). In contrast, treatment with endogenous or synthetic LXR α/β ligands such as T0901317 increased ABCA1 and ABCG1 expression and enhanced cholesterol efflux to apoA-I and HDL, respectively, while treatment with pharmacological PPAR-α or -γ ligands was without effect. Trophoblasts transfected with ABCA1 or ABCG1 siRNA were more sensitive to toxic oxysterols substrates (25-hydroxycholesterol and 7-ketocholesterol) compared to mock-transfected cells, while prior treatment with T0901317 reduced oxysterol-mediated toxicity. These results identify syncytial ABCA1 and ABCG1 as important, inducible cholesterol transporters which also prevent placental accumulation of cytotoxic oxysterols.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.     

Enhanced ABCG1 expression increases atherosclerosis in LDLr-KO mice on a western diet

ABCG1 promotes cholesterol efflux from cells, but ABCG1(-/-) bone marrow transplant into ApoE(-/-) and LDLr(-/-) mice reduces atherosclerosis. To further investigate the role of ABCG1 in atherosclerosis, ABCG1 transgenic mice were crossed with LDLr-KO mice and placed on a high-fat western diet. Increased expression of ABCG1 mRNA was detected in liver (1.8-fold) and macrophages (2.7-fold), and cholesterol efflux from macrophages to HDL was also increased (1.4-fold) in ABCG1xLDLr-KO vs. LDLr-KO mice. No major differences were observed in total plasma lipids. However, cholesterol in the IDL-LDL size range was increased by approximately 50% in ABCG1xLDLr-KO mice compared to LDLr-KO mice. Atherosclerosis increased by 39% (10.1+/-0.8 vs 6.1+/-0.9% lesion area, p=0.02), as measured by en face analysis, and by 53% (221+/-98 vs 104+/-58x10(3)microm(2), p =0.01), as measured by cross-sectional analysis in ABCG1xLDLr-KO mice. Plasma levels for MCP-1 (1.5-fold) and TNF-alpha (1.2-fold) were also increased in ABCG1xLDLr-KO mice. In summary, these findings suggest that enhanced expression of ABCG1 increases atherosclerosis in LDLr-KO mice, despite its role in promoting cholesterol efflux from cells.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [³H]-cholesterol efflux (by 50%) but did not reduce [³H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.     

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>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). 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) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) 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.     

ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation

Here we demonstrate that the ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. Targeted disruption of Abcg1 in mice has no effect on plasma lipids but results in massive accumulation of both neutral lipids and phospholipids in hepatocytes and in macrophages within multiple tissues following administration of a high-fat and -cholesterol diet. In contrast, overexpression of human ABCG1 protects murine tissues from dietary fat-induced lipid accumulation. Finally, we show that cholesterol efflux to HDL specifically requires ABCG1, whereas efflux to apoA1 requires ABCA1. These studies identify Abcg1 as a key gene involved in both cholesterol efflux to HDL and in tissue lipid homeostasis.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.     

The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.     

Stimulation of Akt poly-ubiquitination and proteasomal degradation in P388D1 cells by 7-ketocholesterol and 25-hydroxycholesterol

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H₂DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography–mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [³H]-cholesterol labeled HPEC and [¹⁴C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7β-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL₃ (78?±?17%, 40?±?9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol

Among oxysterols oxidized at C7 (7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), 7β-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1β secretion. TNF-α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α- or β-hydroxyl radical position plays a key role in apoptosis and IL-1β secretion.     

Restoration of PPARγ reverses lipid accumulation in alveolar macrophages of GM-CSF knockout mice

Pulmonary alveolar proteinosis (PAP) is a lung disease characterized by a deficiency of functional granulocyte macrophage colony-stimulating factor (GM-CSF) resulting in surfactant accumulation and lipid-engorged alveolar macrophages. GM-CSF is a positive regulator of PPARγ that is constitutively expressed in healthy alveolar macrophages. We previously reported decreased PPARγ and ATP-binding cassette transporter G1 (ABCG1) levels in alveolar macrophages from PAP patients and GM-CSF knockout (KO) mice, suggesting PPARγ and ABCG1 involvement in surfactant catabolism. Because ABCG1 represents a PPARγ target, we hypothesized that PPARγ restoration would increase ABCG1 and reduce macrophage lipid accumulation. Upregulation of PPARγ was achieved using a lentivirus expression system in vivo. GM-CSF KO mice received intratracheal instillation of lentivirus (lenti)-PPARγ or control lenti-eGFP. Ten days postinstillation, 79% of harvested alveolar macrophages expressed eGFP, demonstrating transduction. Alveolar macrophages showed increased PPARγ and ABCG1 expression after lenti-PPARγ instillation, whereas PPARγ and ABCG1 levels remained unchanged in lenti-eGFP controls. Alveolar macrophages from lenti-PPARγ-treated mice also exhibited reduced intracellular phospholipids and increased cholesterol efflux to HDL, an ABCG1-mediated pathway. In vivo instillation of lenti-PPARγ results in: 1) upregulating ABCG1 and PPARγ expression of GM-CSF KO alveolar macrophages, 2) reducing intracellular lipid accumulation, and 3) increasing cholesterol efflux activity.     

Relevance and mechanism of oxysterol stereospecifity in coronary artery disease

Cholesterol oxidation products (oxysterols) are markers for in vitro LDL oxidation. They are potent inducers of programmed cell death and are also found in high concentrations inside atherosclerotic lesions. Among physiologically occurring oxysterols, 7beta-OH-cholesterol suggests an increase of lipid peroxidation in vivo. In the underlying study, we quantified free plasma oxysterols by means of gas chromatography in patients with stable coronary artery disease (CAD). Total free plasma oxysterols were elevated more than 2-fold in patients with stable CAD (233 +/- 49 vs 108 +/- 19 ng/ml, n = 22, P < 0.05) compared to a control group (n = 20) with similar atherogenic risk profile and angiographically normal coronary arteries. We found that 7-ketocholesterol, as well as the beta-isomers of epoxide (25.7 +/- 10.0 vs 7.3 +/- 1.4 ng/ml, P = 0.07) and 7beta-OH-cholesterol (65.1 +/- 15.7 vs 19.4 +/- 8.9 ng/ml, P < 0.01), was mainly responsible for this increase. To elucidate a potential relevance of oxysterol stereospecificity in regard to endothelial damage, we further conducted in vitro experiments using human arterial endothelial cells (HAECs). Surprisingly, beta-isomers exerted an up to 10-fold higher amount of cell death in equivalent doses when compared to alpha-isomers. The greater cytotoxic potential of beta-isomers was due to increased apoptosis, preceded by mitochondrial release of cytochrome c with subsequent caspase-3 activation. Stereospecific release of cytochrome c depended on the presence of an intact cytoplasmic membrane, hinting at the existence of a putative oxysterol receptor or a direct stereospecific effect on membrane biology. Finally, both isoforms of oxysterols directly released cytochrome c only in conjunction with protein containing cytosol and endoplasmatic reticulum. Free plasma oxysterol levels, particularly 7-ketocholesterol, beta-epoxide and 7beta-OH-cholesterol, are elevated in patients with stable CAD, independent of their LDL cholesterol levels. Due to the highly increased cytotoxicity of oxysterol beta-isomers in vitro, they may represent important atherogenic risk factors.     

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20-25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/-0.9 mg/dl vs. WT: 1.2+/-0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.