Human apolipoprotein A-I and A-I mimetic peptides: potential for atherosclerosis reversal

PURPOSE OF REVIEW: Recent publications related to the potential use of apolipoprotein (apo)A-I and apoA-I mimetic peptides in the treatment of atherosclerosis are reviewed. RECENT FINDINGS: A preliminary report indicating that infusion of apoA-IMilano into humans once weekly for 5 weeks caused a significant decrease in coronary artery atheroma volume has sparked great interest in the potential therapeutic use of apoA-I. Recent studies have revealed that HDL quality (e.g. HDL apolipoprotein and lipid content, including oxidized lipids, particle size and electrophoretic mobility, associated enzymatic activities, inflammatory/anti-inflammatory properties, and ability to promote cholesterol efflux) may be more important than HDL-cholesterol levels. Therefore, when developing new strategies to raise HDL-cholesterol concentrations by interfering with HDL metabolism, one must consider the quality of the resulting HDL. In animal models, raising HDL-cholesterol levels by administering oral phospholipids improved both the quantity and quality of HDL and was associated with lesion regression. An apoA-I mimetic peptide, namely 4F synthesized from D-amino acids (D-4F), administered orally to mice did not raise HDL-cholesterol concentrations but promoted the formation of pre-beta HDL containing increased paraoxonase activity, resulting in significant improvements in HDL's anti-inflammatory properties and ability to promote cholesterol efflux from macrophages in vitro. Oral D-4F also promoted reverse cholesterol efflux from macrophages in vivo. SUMMARY: The quality of HDL may be more important than HDL-cholesterol levels. ApoA-I and apoA-I mimetic peptides appear to have significant therapeutic potential in atherosclerosis.     

A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids

Administered subcutaneously, D-4F or L-4F are equally efficacious, but only D-4F is orally efficacious because of digestion of L-4F by gut proteases. Orally administering niclosamide (a chlorinated salicylanilide used as a molluscicide, antihelminthic, and lampricide) in temporal proximity to oral L-4F (but not niclosamide alone) in apoE null mice resulted in significant improvement (P < 0.001) in the HDL-inflammatory index (HII), which measures the ability of HDL to inhibit LDL-induced monocyte chemotactic activity in endothelial cell cultures. Oral administration of L-[113-122]apoJ with niclosamide also resulted in significant improvement (P < 0.001) in HII. Oral administration of niclosamide and L-4F together with pravastatin to female apoE null mice at 9.5 months of age for six months significantly reduced aortic sinus lesion area (P = 0.02), en face lesion area (P = 0.033), and macrophage lesion area (P = 0.02) compared with pretreatment, indicating lesion regression. In contrast, lesions were significantly larger in mice receiving only niclosamide and pravastatin or L-4F and pravastatin (P < 0.001). In vitro niclosamide and L-4F tightly associated rendering the peptide resistant to trypsin digestion. Niclosamide itself did not inhibit trypsin activity. The combination of niclosamide with apolipoprotein mimetic peptides appears to be a promising method for oral delivery of these peptides.     

Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally

To determine if the dose of peptide administered or the plasma level was more important, doses of 0.15, 0.45, 4.5, or 45 mg/kg/day of the peptide D-4F were administered orally or subcutaneously (SQ) to apoliptotein (apo)E null mice. Plasma levels of peptide were ～1,000-fold higher when administered SQ compared with orally. Regardless of the route of administration, doses of 4.5 and 45 mg/kg significantly reduced plasma serum amyloid A (SAA) levels and the HDL inflammatory index (P < 0.0001); doses of 0.15 or 0.45 mg/kg did not. A dose of 45 mg/kg/day administered to apoE null mice on a Western diet reduced aortic atherosclerosis by ～50% (P < 0.0009) whether administered orally or SQ and also significantly reduced plasma levels of SAA (P < 0.002) and lysophosphatidic acid (P < 0.0009). Remarkably, for each dose administered, the concentration and amount of peptide in the feces was similar regardless of whether the peptide was administered orally or SQ. We conclude: i) the dose of 4F administered and not the plasma level achieved determines efficacy; ii) the intestine may be a major site of action for the peptide regardless of the route of administration.     

Apolipoprotein A-I mimetic peptide inhibits atherosclerosis by altering plasma metabolites in hypercholesterolemia

An apolipoprotein A-I mimetic peptide, D-4F, has been shown to improve vasodilation and inhibit atherosclerosis in hypercholesterolemic low-density lipoprotein receptor-null (LDLr(-/-)) mice. To study the metabolic variations of D-4F ininhibiting atherosclerosis, metabonomics, a novel system biological strategy to investigate the pathogenesis, was developed. Female LDLr(-/-) mice were fed a Western diet and injected with or without D-4F intraperitoneally. Atherosclerotic lesion formation was measured, whereas plasma metabolic profiling was obtained on the basis of ultra-high-performance liquid chromatography in tandem with time-of-flight mass spectrometry operating in both positive and negative ion modes. Data were processed by multivariate statistical analysis to graphically demonstrate metabolic changes. The partial least-squares discriminate analysis model was validated with cross-validation and permutation tests to ensure the model's reliability. D-4F significantly inhibited the formation of atherosclerosis in a time-dependent manner. The metabolic profiling was altered dramatically in hypercholesterolemic LDLr(-/-) mice, and a significant metabolic profiling change in response to D-4F treatment was observed in both positive and negative ion modes. Thirty-six significantly changed metabolites were identified as potential biomarkers. A series of phospholipid metabolites, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), phosphatidylcholine (PC), phatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG), particularly the long-chain LysoPC, was elevated dramatically in hypercholesterolemic LDLr(-/-) mice but reduced by D-4F in a time-dependent manner. Quantitative analysis of LysoPC, LysoPE, PC, and DG using HPLC was chosen to validate the variation of these potential biomarkers, and the results were consistent with the metabonomics findings. Our findings demonstrated that D-4F may inhibit atherosclerosis by regulating phospholipid metabolites specifically by decreasing plasma long-chain LysoPC.     

Inhibition of collar-induced carotid atherosclerosis by recombinant apoA-I cysteine mutants in apoE-deficient mice

The previous studies in our laboratory revealed that seven cysteine mutants of apolipoprotein A-I (apoA-I) have different structural features and biological activities in vitro and in vivo. To investigate the potential cardioprotective effects of apolipoprotein A-I(N74C) [apoA-I(N74C)], we examined the anti-inflammatory, antioxidant, and antiatherosclerotic effects of this cysteine mutant in a rapid atherosclerosis model induced by perivascular carotid collar placement in apoE^−/− mice. Lipid-free apoA-I(N74C) showed a significant increased antioxidant potency in low density lipoprotein (LDL) oxidation in vitro and reduced intracellular lipid accumulation in THP-1-derived macrophages, relative to wild-type apoA-I (apoA-Iwt). Mice injected with recombinant HDL (rHDL) reconstituted with apoA-I(N74C) (named rHDL74) through tail veins (40 mg/kg of body weight, three injections) had a significant lower level of serum interleukin-6 (IL-6) and enhanced serum antioxidation compared with mice receiving rHDL reconstituted with apoA-Iwt (named rHDLwt). Moreover, compared with rHDLwt, the rHDL74 in vivo injection resulted in a significant decrease in plaque size, ratio of aorta intima to media, arterial remodeling, and macrophage content in lesions. In summary, intravenous injection with rHDL74 reconstituted with apoA-I cysteine mutant apoA-I (N74C) dramatically delays the development of atherosclerosis induced by perivascular carotid collar placement and reduces vascular remodeling in the carotid artery in apoE^−/− mice.     

Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations ( approximately 35 microM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of approximately 35 microM apoA-I significantly reduced this inflammatory response. We analyzed lipid binding by surface plasmon resonance. The anti-inflammatory 4F peptide bound oxidized lipids with much higher affinity than did apoA-I. Initially, we examined the binding of PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine) and observed that its oxidized products bound 4F with an affinity that was approximately 4-6 orders of magnitude higher than that of apoA-I. This high binding affinity was confirmed in studies with defined lipids and phospholipids. 3F-2 and 3F(14) are also amphipathic alpha-helical octadecapeptides, but 3F-2 inhibits atherosclerosis in mice and 3F(14) does not. Like 4F, 3F-2 also bound oxidized phospholipids with very high affinity, whereas 3F(14) resembled apoA-I. The extraordinary ability of 4F to bind pro-inflammatory oxidized lipids probably accounts for its remarkable anti-inflammatory properties.     

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer''s disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.     

Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer

PURPOSE OF THE REVIEW: This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. RECENT FINDINGS: Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical alpha-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-beta-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. SUMMARY: New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.     

The Apolipoprotein-AI Mimetic Peptide L4F at a Modest Dose Does Not Attenuate Weight Gain,Inflammation, or Atherosclerosis in LDLR-Null Mice

Objective

High density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease risk and associated with a reduced risk of type 2 diabetes. Apolipoprotein A-I (apoA-I; major HDL protein) mimetics have been reported to reduce atherosclerosis and decrease adiposity. This study investigated the effect of L4F mimetic peptide and apoA-I overexpression on weight gain, insulin resistance, and atherosclerosis in an LDL receptor deficient (Ldlr^-/-) model fed a high fat high sucrose with cholesterol (HFHSC) diet.

Methods

Studies in differentiated 3T3-L1 adipocytes tested whether L4F could inhibit palmitate-induced adipocyte inflammation. In vivo studies used male Ldlr^-/- mice fed a HFHSC diet for 12 weeks and were injected daily with L4F (100 µg/mouse) subcutaneously during the last 8 weeks. Wild-type and apoA-I overexpressing Ldlr^-/- mice were fed HFHSC diet for 16 weeks.

Results

Neither L4F administration nor apoA-I overexpression affected weight gain, total plasma cholesterol or triglycerides in our studies. While pre-treatment of 3T3-L1 adipocytes with either L4F or HDL abolished palmitate-induced cytokine expression in vitro, L4F treatment did not affect circulating or adipose tissue inflammatory markers in vivo. Neither L4F administration nor apoA-I overexpression affected glucose tolerance. ApoA-I overexpression significantly reduced atherosclerotic lesion size, yet L4F treatment did not affect atherosclerosis.

Conclusion

Our results suggest that neither L4F (100 µg/day/mouse) nor apoA-I overexpression affects adiposity or insulin resistance in this model. We also were unable to confirm a reduction in atherosclerosis with L4F in our particular model. Further studies on the effect of apoA-I mimetics on atherosclerosis and insulin resistance in a variety of dietary contexts are warranted.     

Evidence for differential effects of apoE3 and apoE4 on HDL metabolism

We present a murine model that examines the effects of macrophage-produced apolipoprotein E3 (apoE3) and apoE4 on VLDL and high density lipoprotein (HDL) metabolism. Mice expressing apoE3 on the Apoe(-/-) background had substantially lower VLDL levels than mice expressing apoE4. In addition, there were differences between the HDL of apoE3- and apoE4-expressing mice. Apoe(-/-) mice have low levels of HDL. Low level expression of either apoE3 or apoE4 was able to restore near-normal HDL levels, which increased dramatically when the mice were challenged with a high-fat diet. ApoE4-expressing mice had smaller HDL than apoE3-expressing mice on both chow and high-fat diets. In addition, plasma from apoE4-expressing mice was less efficient at transferring apoA-I from VLDL to HDL and at generating HDL in vitro than that from apoE3-expressing mice. Thus, we present experimental evidence for differential effects of apoE3 and apoE4 on HDL metabolism that supports epidemiological observations made in humans, which suggested that individual homozygous for the epsilon 4 allele had lower HDL than others.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Atherosclerosis in perlecan heterozygous mice

The hypothesis that lipoprotein association with perlecan is atherogenic was tested by studying atherosclerosis in mice that had a heterozygous deletion of perlecan, the primary extracellular heparan sulfate proteoglycan in arteries. We first studied the expression of perlecan in mouse lesions and noted that this proteoglycan in aorta was found in the subendothelial matrix. Perlecan was also a major component of the lesional extracellular matrix. Mice with a heterozygous deletion had a reduction in arterial wall perlecan expression. Atherosclerosis in these mice was studied after crossing the defect into the apolipoprotein E (apoE) and LDL receptor knockout backgrounds. At 12 weeks, chow-fed apoE null mice with a heterozygous deletion had less atherosclerosis. However, at 24 weeks and in the LDL receptor heterozygous background, the presence of a perlecan knockout allele did not significantly alter lesion size. Thus, it appears that loss of perlecan leads to less atherosclerosis in early lesions. Although this might be attributable to a decrease in lipoprotein retention, it should be noted that perlecan might mediate multiple other processes that could, in sum, accelerate atherosclerosis.     

The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1

Lipoprotein metabolism in the central nervous system (CNS) is based on high-density lipoprotein-like particles that use apoE as their predominant apolipoprotein rather than apoA-I. Although apoA-I is not expressed in astrocytes and microglia, which produce CNS apoE, apoA-I is reported to be expressed in porcine brain capillary endothelial cells and also crosses the blood-brain barrier (BBB). These mechanisms allow apoA-I to reach concentrations in cerebrospinal fluid (CSF) that are approximately 0.5% of its plasma levels. Recently, apoA-I has been shown to enhance cognitive function and reduce cerebrovascular amyloid deposition in Alzheimer's Disease (AD) mice, raising questions about the regulation and function of apoA-I in the CNS. Peripheral apoA-I metabolism is highly influenced by ABCA1, but less is known about how ABCA1 regulates CNS apoA-I. We report that ABCA1 deficiency leads to greater retention of apoA-I in the CNS than in the periphery. Additionally, treatment of symptomatic AD mice with GW3965, an LXR agonist that stimulates ABCA1 expression, increases apoA-I more dramatically in the CNS compared to the periphery. Furthermore, GW3965-mediated up-regulation of CNS apoA-I is independent of ABCA1. Our results suggest that apoA-I may be regulated by distinct mechanisms on either side of the BBB and that apoA-I may serve to integrate peripheral and CNS lipid metabolism. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

D-4F reduces EO6 immunoreactivity, SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a Western diet

LDL receptor-null (LDLR(-/-)) mice on a Western diet (WD) develop endothelial dysfunction and atherosclerosis, which are improved by the apolipoprotein A-I (apoA-I) mimetic peptide D-4F. Focusing on the kidney, LDLR(-/-)mice were fed a WD with D-4F or the inactive control peptide scrambled D-4F (ScD-4F) added to their drinking water. The control mice (ScD-4F) developed glomerular changes, increased immunostaining for MCP-1/CCL2 chemokine, increased macrophage CD68 and F4/80 antigens, and increased oxidized phospholipids recognized by the EO6 monoclonal antibody in both glomerular and tublo-interstitial areas. All of these parameters were significantly reduced by D-4F treatment, approaching levels found in wild-type C57BL/6J or LDLR(-/-) mice fed a chow diet. Sterol-regulatory element binding protein-1c (SREBP-1c) mRNA levels and triglyceride levels were elevated in the kidneys of the control mice (ScD-4F) fed the WD compared with C57BL/6J and LDLR(-/-) mice on chow (P < 0.001 and P < 0.001, respectively) and compared with D-4F-treated mice on the WD (P < 0.01). There was no significant difference in plasma lipids, lipoproteins, glucose, blood pressure, or renal apoB levels between D-4F- and ScD-4F-treated mice. We conclude that D-4F reduced renal oxidized phospholipids, resulting in lower expression of SREBP-1c, which, in turn, resulted in lower triglyceride content and reduced renal inflammation.     

Attenuated atherosclerotic lesions in apoE-Fcγ-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for FcγRs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating FcγR in the progression of atherosclerosis using apolipoprotein E (apoE)-Fcγ-chain double-knockout (DKO) mice. Relative to apoE knockout (KO) mice, arterial lesion formation was significantly decreased in apoE-Fcγ-chain DKO mice. Bone marrow chimera studies showed reduced lesions in apoE KO mice receiving the bone marrow of apoE-Fcγ-chain DKO mice. Compared to apoE KO mice, antioxidized low-density lipoprotein IgG1 (Th2) and IgG2a (Th1), IL-10, and IFN-γ secretion by activated T cells was increased in apoE-Fcγ-chain DKO mice. These findings suggest that reduced atherosclerotic lesion in apoE-Fcγ-chain DKO mice is not due to a Th1/Th2 imbalance. Interestingly, the number of Th17 cells and the secretion of IL-17 by activated CD4(+) cells were decreased in apoE-Fcγ-chain DKO mice. Notably, the number of regulatory T cells, expression of mRNA, and secretion of TGF-β and IL-10 were increased in apoE-Fcγ-chain DKO mice. Furthermore, secretions of IL-6 and STAT-3 phosphorylation essential for Th17 cell genesis were reduced in apoE-Fcγ-chain DKO mice. Importantly, decrease in Th17 cells in apoE-Fcγ-chain DKO mice was due to reduced IL-6 release by APC of apoE-Fcγ-chain DKO mice. Collectively, our data suggest that activating FcγR promotes atherosclerosis by inducing a Th17 response in the hyperlipidemic apoE KO mouse model.     

Apolipoproteins and Apolipoprotein Mimetic Peptides Modulate Phagocyte Trafficking through Chemotactic Activity

The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai^−/− and Apoe^−/− mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.     

Enhanced binding of apolipoprotein A-I variants associated with hypertriglyceridemia to triglyceride-rich particles

Hypertriglyceridemia (HTG) is a common lipid abnormality in humans. However, its etiology remains largely unknown. It was shown that severe HTG can be induced in mice by overexpression of wild-type (WT) apolipoprotein E (apoE) or specific apoA-I mutants. Certain mutations in apoE4 were found to affect plasma triglyceride (TG) levels in mice overexpressing the protein. HTG appeared to positively correlate with the ability of the apoE4 variants to bind to TG-rich particles, protein destabilization, and the exposure of protein hydrophobic surface in solution. Here, we propose that the apoA-I mutations that cause HTG may also lead to changes in the conformation and stability that promote binding of apoA-I to TG-rich lipoproteins. To test this hypothesis, we studied binding to TG-rich emulsion and biophysical properties of the apoA-I mutants that induce HTG, apoA-I[E110A/E111A] and apoA-I[Δ(61-78)], and compared them to those of WT apoA-I and another apoA-I mutant, apoA-I[Δ(89-99)], that does not induce HTG but causes hypercholesterolemia in mice. We found that the apoA-I[E110A/E111A] and apoA-I[Δ(61-78)] mutations lead to enhanced binding of apoA-I to TG-rich particles, destabilization, and greater exposure of the hydrophobic surface of the protein. The apoA-I[Δ(89-99)] mutant did not show enhanced binding to the emulsion or a more exposed hydrophobic surface. Thus, like apoE4, the apoA-I variants that cause HTG in mice have the altered conformation and stability that facilitate their binding to TG-rich lipoproteins and thereby may lead to the reduced level of lipolysis of these lipoproteins. While many factors may be involved in induction of HTG, we suggest that an increased level of association of destabilized loosely folded apolipoproteins with TG-rich lipoproteins may contribute to some cases of HTG in humans.     

Generation of a recombinant apolipoprotein E variant with improved biological functions: hydrophobic residues (LEU-261, TRP-264, PHE-265, LEU-268, VAL-269) of apoE can account for the apoE-induced hypertriglyceridemia

To identify the residues in the carboxyl-terminal region 260-299 of human apolipoprotein E (apoE) that contribute to hypertriglyceridemia, two sets of conserved, hydrophobic amino acids between residues 261 and 283 were mutated to alanines, and recombinant adenoviruses expressing these apoE mutants were generated. Adenovirus-mediated gene transfer of apoE4-mut1 (apoE4 (L261A, W264A, F265A, L268A, V269A)) in apoE-deficient mice (apoE(-/-)) corrected plasma cholesterol levels and did not cause hypertriglyceridemia. In contrast, gene transfer of apoE4-mut2 (apoE4 (W276A, L279A, V280A, V283A)) did not correct hypercholesterolemia and induced mild hypertriglyceridemia. ApoE-induced hyperlipidemia was corrected by co-infection with a recombinant adenovirus expressing human lipoprotein lipase. Both apoE4 mutants caused only a small increase in hepatic very low density lipoprotein-triglyceride secretion. Density gradient ultracentrifugation analysis of plasma and electron microscopy showed that wild-type apoE4 and apoE4-mut2 displaced apoA-I from the high density lipoprotein (HDL) region and promoted the formation of discoidal HDL, whereas the apoE4-mut1 did not displace apoA-I from HDL and promoted the formation of spherical HDL. The findings indicate that residues Leu-261, Trp-264, Phe-265, Leu-268, and Val-269 of apoE are responsible for hypertriglyceridemia and also interfere with the formation of HDL. Substitutions of these residues by alanine provide a recombinant apoE form with improved biological functions.     

Apolipoprotein E is the major physiological activator of lecithin-cholesterol acyltransferase (LCAT) on apolipoprotein B lipoproteins

Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even though apoA-I(-)(/)(-) apoE(-)(/)(-) plasma retained (1)/(3) the amount of B6 LCAT activity. This suggested that lack of LCAT enzyme did not explain the low CER in apoA-I(-)(/)(-) apoE(-)(/)(-) mice and indicated that apoE and apoA-I are the only major activators of LCAT in mouse plasma. Deleting apoE on low-density lipoprotein (LDL) reduced CER (1% free cholesterol (FC) esterified/h) compared to B6 (6% FC esterified/h) and apoA-I(-)(/)(-) (11% FC esterified/h) LDL. Similar sized LDL particles from all four genotypes were isolated by fast protein liquid chromatography (FPLC) after radiolabeling with [(3)H]-free cholesterol (FC). LDLs (1 microg FC) from each genotype were incubated with purified recombinant mouse LCAT; LDL particles from B6 and apoA-I(-)(/)(-) plasma were much better substrates for CE formation (5.7% and 6.3% CE formed/30 min, respectively) than those from apoE(-)(/)(-) and apoE(-)(/)(-) apoA-I(-)(/)(-) plasma (1.2% and 1.1% CE formed/30 min). Western blot analysis showed that the amount of apoA-I on apoE(-)(/)(-) LDLs was higher compared to B6 LDL. Adding apoE to incubations of apoA-I(-)(/)(-) apoE(-)(/)(-) very low density lipoprotein (VLDL) resulted in a 3-fold increase in LCAT CER, whereas addition of apoA-I resulted in a more modest 80% increase. We conclude that apoE is a more significant activator of LCAT than apoA-I on mouse apoB lipoproteins.     

Apolipoprotein E4 in macrophages enhances atherogenesis in a low density lipoprotein receptor-dependent manner

Apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLr) are well recognized determinants of atherosclerosis. In addition to hepatocytes, where both are highly expressed and contribute to plasma lipoprotein clearance, they are expressed in vascular cells and macrophages. In this study, we examined the effects of human apoE isoforms and LDLr levels in atherogenic pathways in primary macrophages ex vivo and atherosclerosis development after bone marrow transfer in vivo using mice expressing human apoE isoforms and different levels of LDLr expression. Increases in LDLr expression significantly increased cholesterol delivery into macrophages in culture, and the effects were more prominent with lipoproteins containing apoE4 than those containing apoE3. Conversely, increased LDLr expression reduced cholesterol efflux in macrophages expressing apoE4 but not in macrophages expressing apoE3. Furthermore, apoE3 protected VLDL from oxidation in vitro more than did apoE4. In LDLr-deficient mice expressing the human apoE4 isoform, Apoe4/4 Ldlr-/-, the replacement of bone marrow cells with those expressing LDLr increased atherosclerotic lesions in a dose-dependent manner compared with mice transplanted with cells having no LDLr. In contrast, atherosclerosis in Apoe3/3 Ldlr-/- mice, expressing the human apoE3 isoform, did not differ by the levels of macrophage LDLr expression. Our results demonstrate that apoE4, but not apoE3, in macrophages enhances atherosclerotic plaque development in mice in an LDLr-dependent manner and suggests that this interaction may contribute to the association of apoE4 with an increased cardiovascular risk in humans.