John A. Stonik

Lipopolysaccharide Down Regulates Both Scavenger Receptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells

ABSTRACT Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor. The mechanism of LPS induced cholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPSs proatherogenic effects may at least in part reflect altered cholesterol metabolism. Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific 125I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression. Inhibitors of NF-κB activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-κB. These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.

5A Apolipoprotein Mimetic Peptide Promotes Cholesterol Efflux and Reduces Atherosclerosis in Mice

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.

Asymmetry in the Lipid Affinity of Bihelical Amphipathic Peptides A STRUCTURAL DETERMINANT FOR THE SPECIFICITY OF ABCA1-DEPENDENT CHOLESTEROL EFFLUX BY PEPTIDES

ApoA-I contains a tandem array of amphipathic helices with varying lipid affinity, which are critical in its ability to bind and remove lipids from cells by the ABCA1 transporter. In this study, the effect of asymmetry in the lipid affinity of amphipathic helices in a bihelical apoA-I mimetic peptide, 37pA, on lipid efflux by the ABCA1 transporter was examined. Seven peptide variants of 37pA were produced by substituting a varying number of hydrophobic amino acids for alanine on either one or both helices. The 5A peptide with five alanine substitutions in the second helix had decreased helical content compared with 37pA (5A, 12 ± 1% helicity; 37pA, 28 ± 2% helicity) and showed less self-association but, similar to the parent peptide, was able to readily solubilize phospholipid vesicles. Furthermore, 5A, unlike the parent peptide 37pA, was not hemolytic (37pA, 27 ± 2% RBC lysis, 2 h, 18 μm). Finally, the 5A peptide stimulated cholesterol and phospholipid efflux by the ABCA1 transporter with higher specificity (ABCA1-transfected versus untransfected cells) than 37pA (5A, 9.7 ± 0.77%, 18 h, 18 μm versus 1.5 ± 0.27%, 18 h, 18 μm (p < 0.0001); 37pA, 7.4 ± 0.85%, 18 h, 18μm versus 5.8 ± 0.20%, 18 h, 18μm (p = 0.03)). In summary, we describe a novel bihelical peptide with asymmetry in the lipid affinity of its helices and properties similar to apoA-I in terms of specificity for cholesterol efflux by the ABCA1 transporter and low cytotoxicity.

Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein

Rationale: Apolipoprotein (apoA)-I mimetic peptides are a promising type of antiatherosclerosis therapy, but how the structural features of these peptides relate to the multiple antiatherogenic functions of HDL is poorly understood. Objective: To establish structure/function relationships of apoA-I mimetic peptides with their antiatherogenic functions. Methods and Results: Twenty-two bihelical apoA-I mimetic peptides were investigated in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells, and inhibition of low-density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face, and angle of the link between the helices are the major factors determining the efficiency and specificity of cholesterol efflux. The peptide with optimal parameters was more effective and specific toward cholesterol efflux than human apoA-I. Charge and size of hydrophobic face were also the major factors affecting antiinflammatory properties, and the presence of cysteine and histidine residues was the main factor determining antioxidant properties. There was no significant correlation between capacities of the peptides to support individual functions; each function had its own optimal set of features. Conclusions: None of the peptides was equally effective in all the antiatherogenic functions tested, suggesting that different functions of HDL may have different mechanisms and different structural requirements. The results do suggest, however, that rationalizing the design of apoA-I mimetic peptides may improve their therapeutic value and may lead to a better understanding of mechanisms of various antiatherogenic functions of HDL.

Effect of Recombinant Human Lecithin Cholesterol Acyltransferase Infusion on Lipoprotein Metabolism in Mice

Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density lipoprotein (HDL) and the presence of an abnormal lipoprotein called lipoprotein X (Lp-X) that contributes to end-stage renal disease. We examined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approximately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal lipoprotein profile, but it had a considerably shorter half-life of approximately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apolipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol efflux to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal lipoprotein phenotype in LCAT-KO mice and increased cholesterol efflux, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency.

Anti-inflammatory and cardioprotective activities of synthetic high-density lipoprotein containing apolipoprotein A-I mimetic peptides.

Apolipoprotein A-I (apoA-I) mimetic peptides may represent an alternative to apoA-I for large-scale production of synthetic high-density lipoproteins (sHDL) as a therapeutic agent. In this study, the cardioprotective activity of sHDL made with either L37pA peptide or its d-stereoisomer, D37pA, was compared to sHDL made with apoA-I. The peptides were reconstituted with palmitoyl-oleoyl-phosphatidylcholine, which yielded sHDL particles comparable to apoA-I sHDL in diameter, molecular weight, and α-helical content. Pretreatment of endothelial cells with either peptide sHDL reduced tumor necrosis factor α-stimulated vascular cell adhesion molecule-1 expression to the same extent as apoA-I sHDL. In an isolated rat heart model of ischemia/reperfusion (I/R) injury, L37pA and D37pA sHDL significantly reduced postischemic cardiac contractile dysfunction compared to the saline control, as indicated by a 49.7 ± 6.4% (L37pA; P < 0.001) and 53.0 ± 9.1% (D37pA; P < 0.001) increase of left ventricular-developed pressure (LVDP) after reperfusion and by a 45.4 ± 3.4% (L37pA; P < 0.001) and 49.6 ± 2.6% (D37pA; P < 0.001) decrease of creatine kinase (CK) release. These effects were similar to the 51.3 ± 3.0% (P < 0.001) increase of LVDP and 51.3 ± 3.0 (P < 0.001) reduction of CK release induced by apoA-I sHDL. Consistent with their cardioprotective effects, all three types of sHDL particles mediated an approximate 20% (P < 0.001) reduction of cardiac tumor necrosis factor α (TNFα) content and stimulated an approximate 35% (P < 0.05) increase in postischemic release of prostacyclin. In summary, L37pA and D37pA peptides can form sHDL particles that retain a similar level of protective activity as apoA-I sHDL on the endothelium and the heart; thus, apoA-I mimetic peptides may be useful therapeutic agents for the prevention of cardiac I/R injury.

High Density Lipoprotein Induces Proliferation and Migration of Human Prostate Androgen Independent Cancer Cells by an ABCA1-dependent Mechanism

Androgen deprivation therapy for prostate cancer leads to a significant increase of high-density lipoprotein (HDL), which is generally viewed as beneficial, particularly for cardiovascular disease, but the effect of HDL on prostate cancer is unknown. In this study, we investigated the effect of HDL on prostate cancer cell proliferation, migration, intracellular cholesterol levels, and the role of cholesterol transporters, namely ABCA1, ABCG1, and SR-BI in these processes. HDL induced cell proliferation and migration of the androgen-independent PC-3 and DU145 cells by a mechanism involving extracellular signal-regulated kinase (ERK) 1/2 and Akt, but had no effect on the androgen-dependent LNCaP cell, which did not express ABCA1 unlike the other cell lines. Treatment with HDL did not significantly alter the cholesterol content of the cell lines. Knockdown of ABCA1 but not ABCG1 or SR-BI by small interfering RNA (siRNA) inhibited HDL-induced cell proliferation, migration, and ERK1/2 and Akt signal transduction in PC-3 cells. Moreover, after treatment of LNCaP cells with charcoal-stripped fetal bovine serum, ABCA1 was induced ∼10-fold, enabling HDL to induce ERK1/2 activation, whereas small interfering RNA knockdown of ABCA1 inhibited HDL-induced ERK1/2 activation. Simvastatin, which inhibited ABCA1 expression in PC-3 and DU145 cells, attenuated HDL-induced PC-3 and DU145 cell proliferation, migration, and ERK1/2 and Akt phosphorylation. In human prostate biopsy samples, ABCA1 mRNA expression was ∼2-fold higher in the androgen deprivation therapy group than in subjects with benign prostatic hyperplasia or pretreatment prostate cancer groups. In summary, these results suggest that HDL by an ABCA1-dependent mechanism can mediate signal transduction, leading to increased proliferation and migration of prostate cancer cells. Mol Cancer Res; 8(9); 1284–94. ©2010 AACR.

Diet-Induced Weight Loss in Overweight or Obese Women and Changes in High-Density Lipoprotein Levels and Function

Diet‐induced weight loss in women may be associated with decreases not only in plasma levels of low‐density lipoprotein cholesterol (LDL‐C), but also in high‐density lipoprotein cholesterol (HDL‐C). Whether a decrease in HDL‐C is associated with altered HDL function is unknown. One hundred overweight or obese women (age 46 ± 11 years, 60 black; 12 diabetic) were enrolled in the 6‐month program of reduced fat and total energy diet and low‐intensity exercise. Serum cholesterol efflux capacity was measured in 3H‐cholesterol‐labeled BHK cells expressing ABCA1, ABCG1, or SR‐B1 transporters and incubated with 1% apolipoprotein B (apoB)‐depleted serum. Antioxidant properties of HDL were estimated by paraoxonase‐1 (PON1) activity and oxygen radical absorbance capacity (ORAC). Endothelial nitric oxide synthase (eNOS) activation was measured by conversion of l‐arginine to l‐citrulline in endothelial cells incubated with HDL from 49 subjects. Participants achieved an average weight loss of 2.2 ± 3.9 kg (P < 0.001), associated with reductions in both LDL‐C (−6 ± 21 mg/dl, P = 0.004) and HDL‐C (−3 ± 9 mg/dl, P = 0.016). Cholesterol efflux capacity by the ABCA1 transporter decreased by 10% (P = 0.006); efflux capacities by the ABCG1 and SR‐B1 transporters were not significantly altered. ORAC decreased by 15% (P = 0.018); neither PON1 activity nor eNOS activation was significantly altered by reduction in HDL‐C. Findings were similar for diabetic and nondiabetic subjects. Diet‐induced weight loss in overweight or obese women is associated with a decrease in HDL‐C levels, but overall HDL function is relatively spared, suggesting that decrease in HDL‐C in this setting is not deleterious to cardiovascular risk.

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.