Novel fatty acyl apoE mimetic peptides have increased potency to reduce plasma cholesterol in mice and macaques.

Ac-hE18A-NH2 is a dual-domain apoE mimetic peptide that possesses the putative receptor binding domain from apoE (LRKLRKRLLR, denoted hE; residues 141-150) covalently attached to lipid-associating peptide 18A. Like apoE, Ac-hE18A-NH2 reduces plasma cholesterol in animal models and exhibits anti-inflammatory properties independent of its cholesterol-reducing effect. Ac-hE18A-NH2 has already undergone phase I clinical trials as a lipid-lowering agent. To explore the therapeutic potential more, we designed and synthesized new analogues by linking ɑ-aminohexanoic acid, octanoic acid, or myristic acid to LRRLRRRLLR-18A-NH2 ([R]hE18A-NH2) and examined the cholesterol-lowering potency in animals. The modified peptides effectively reduced plasma cholesterol in apoE-null mice fed standard chow or a Western diet; the myristyl analogue was the most effective. A single administration of the myristyl analogue reduced plasma total and LDL cholesterol in a dose-dependent manner in hypercholesterolemic cynomolgus macaques for up to 1 week despite the continuation of a cholesterol-supplemented diet. The myristyl peptide (7.4 mg/kg) reduced total and LDL cholesterol at 24 h by 64% and 74%, respectively; plasma HDL levels were modestly reduced and returned to baseline by day 7. These new analogues should exhibit enhanced potency at lower doses than Ac-hE18A-NH2, which may make them attractive therapeutic candidates for clinical trials.

Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for paraoxonase-1 binding and enhancing its activity and stability.

High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.

Cationic peptide mR18L with lipid lowering properties inhibits LPS-induced systemic and liver inflammation in rats.

The cationic single domain peptide mR18L has demonstrated lipid-lowering and anti-atherogenic properties in different dyslipidemic mouse models. Lipopolysaccharide (LPS)-mediated inflammation is considered as one of the potential triggers for atherosclerosis. Here, we evaluated anti-inflammatory effects of mR18L peptide against LPS-mediated inflammation. First, we tested the efficacy and tolerance of 1, 2.5 and 5mg/kg mR18L in normolipidemic rats stimulated with 5mg/kg LPS. LPS and then mR18L were injected in different intraperitoneal regions. By 2h post LPS, mR18L inhibited LPS-mediated plasma TNF-α elevation at all doses, with the effect being stronger for 2.5mg/kg (P<0.05 vs. 1mg/kg, non-significant vs. 5mg/kg). In a similar model, 2.5mg/kg mR18L reduced LPS-mediated inflammation in the liver, as assessed by microscopic examination of liver sections and measurements of iNOS expression in the liver tissue. In plasma, 2.5mg/kg mR18L decreased levels of TNF-α and IL-6, decreased endotoxin activity and enhanced HDL binding to LPS. In another similar experiment, mR18L administered 1h post LPS, prevented elevation of plasma triglycerides by 6h post LPS and increased plasma activity of anti-oxidant enzyme paraoxonase 1, along with noted trends in reducing plasma levels of endotoxin and IL-6. Surface plasmon resonance study revealed that mR18L readily binds LPS. We conclude that mR18L exerts anti-endotoxin activity at least in part due to direct LPS-binding and LPS-neutralizing effects. We suggest that anti-endotoxin activity of mR18L is an important anti-inflammatory property, which may increase anti-atherogenic potential of this promising orally active lipid-lowering peptide.

Regulation of pattern recognition receptors by the apolipoprotein A-I mimetic peptide 4F.

OBJECTIVE: The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages. METHODS AND RESULTS: Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-ß) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts. CONCLUSIONS: These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.

Preservation of biological function despite oxidative modification of the apolipoprotein A-I mimetic peptide 4F.

Myeloperoxidase (MPO)-derived hypochlorous acid induces changes in HDL function via redox modifications at the level of apolipoprotein A-I (apoA-I). As 4F and apoA-I share structural and functional properties, we tested the hypothesis that 4F acts as a reactive substrate for hypochlorous acid (HOCl). 4F reduced the HOCl-mediated oxidation of the fluorescent substrate APF in a concentration-dependent manner (ED(50) ∼ 56 ± 3 µM). This reaction induced changes in the physical properties of 4F. Addition of HOCl to 4F at molar ratios ranging from 1:1 to 3:1 reduced 4F band intensity on SDS-PAGE gels and was accompanied by the formation of a higher molecular weight species. Chromatographic studies showed a reduction in 4F peak area with increasing HOCl and the formation of new products. Mass spectral analyses of collected fractions revealed oxidation of the sole tryptophan (Trp) residue in 4F. 4F was equally susceptible to oxidation in the lipid-free and lipid-bound states. To determine whether Trp oxidation influenced its apoA-I mimetic properties, we monitored effects of HOCl on 4F-mediated lipid binding and ABCA1-dependent cholesterol efflux. Neither property was altered by HOCl. These results suggest that 4F serves as a reactive substrate for HOCl, an antioxidant response that does not influence the lipid binding and cholesterol effluxing capacities of the peptide.

Sidedness of interfacial arginine residues and anti-atherogenicity of apolipoprotein A-I mimetic peptides.

To test the hypothesis that sidedness of interfacial arginine (Arg) in apoA-I mimetic peptides, similar to that observed in apoA-I (Bashtovyy, D. et al. 2011. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function. J. Lipid Res. 52: 435-450.), may be important for biological activity, we compared properties of 4F and analogs, [K4,¹5>R]4F and [K9,¹³>R]4F, with Lys>Arg substitutions on the right and left side, respectively, of the 4F amphipathic helix. Intraperitoneal administration of these peptides into female apoE null mice (n = 13 in each group) reduced en face lesions significantly compared with controls; 4F and [K4,¹5>R]4F were equally effective whereas [K9,¹³>R]4F was less effective. Turnover experiments indicated that [K4,¹5>R]4F reached the highest, whereas [K9,¹³>R]4F had the lowest, plasma peak levels with a similar half life as the [K4,¹5>R]4F analog. The half life of 4F was two times longer than the other two peptides. The order in their abilities to associate with HDL in human plasma, generation of apoA-I particles with pre-ß mobility from isolated HDL, lipid associating ability, and sensitivity of lipid complexes to trypsin digestion was: 4F>[K4,¹5,>R]4F>[K9,¹³>R]4F. These studies support our hypothesis that the sidedness of interfacial Arg residues in the polar face of apoA-I mimetics results in differential biological properties.

Structure and lipid interactions of an anti-inflammatory and anti-atherogenic 10-residue class G(*) apolipoprotein J peptide using solution NMR.

The surprising observation that a 10-residue class G(⁎) peptide from apolipoprotein J, [113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high-resolution structure of [113-122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle, this peptide adopts amphipathic α-helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (-16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (-3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol%) in DPC micelle also results in the lowering of peptide binding enthalpy (-13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α-helical structure of [113-122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.

Oral administration of L-mR18L, a single domain cationic amphipathic helical peptide, inhibits lesion formation in ApoE null mice.

We have shown that Ac-hE18A-NH2, a dual-domain cationic apolipoprotein-mimetic peptide, reduces plasma cholesterol levels in dyslipidemic mice. Two single-domain cationic peptides based on the lytic class L peptide 18L were developed to test the hypothesis that a single-domain cationic amphipathic peptide can reduce atherosclerosis in apolipoprotein (apo)E null mice when orally administered. To incorporate anti-inflammatory properties, aromatic residues were clustered in the nonpolar face similar to peptide 4F, resulting in modified 18L (m18L). To reduce lytic properties, the Lys residues of 18L were replaced with Arg with the resulting peptide called modified R18L (mR18L). Biophysical studies showed that mR18L had stronger interactions with lipids than did m18L. Peptide mR18L was also more effective than m18L in promoting LDL uptake by HepG2 cells. ApoE null mice received normal chow or chow containing m18L or mR18L for six weeks. A significant reduction in plasma cholesterol and aortic sinus lesion area was seen only in the mR18L group. Plasma from mice administered mR18L, unlike those from the control and m18L groups, did not enhance monocyte adhesion to endothelial cells. Thus oral administration of mR18L reduces plasma cholesterol and lesion formation and inhibits monocyte adhesion.

Anti-inflammatory peptides grab on to the whiskers of atherogenic oxidized lipids.

The peptide 4F is known to have potent anti-atherogenic activity. 4F is an 18 residue peptide that has a sequence capable of forming a class A amphipathic helix. Several other class A amphipathic helical, 18 residue peptides with the same polar face but with increasing Phe residues on the nonpolar face have been synthesized with varying degrees of biological activity. In this work we compared the properties of the original 2F peptide, modeled on the consensus sequence of the amphipathic helical segments of the apolipoprotein A-I with the peptide 4F that has two Leu residues replaced with Phe. We demonstrate that the more biologically active 4F peptide has the greatest affinity for binding to several molecular species of oxidized lipids. Lipoprotein particles can be formed by solubilizing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) with peptides. These solubilized lipoprotein particles extract oxidized lipid from liposomes of POPC containing 5 mol% of oxidized lipid. The peptides with the strongest anti-atherogenic activity interact most strongly with the oxidized lipid. The results show that there is a correlation between the biological potency of these peptides and their ability to interact with certain specific cytotoxic lipids, suggesting that this interaction may contribute favourably to their biological properties.

ApoE mimetic peptide reduces plasma lipid hydroperoxide content with a concomitant increase in HDL paraoxonase activity.

ApoE mimetic peptide possesses the putative receptor binding domain 141-150 (LRKLRKRLLR) of apoE covalently linked to the class A amphipathic helical peptide 18A. It dramatically reduces plasma cholesterol in dyslipidemic mouse and rabbit models. Recycling of apoE mimetic peptide increases the duration of preß-HDL formation leading to extended anti-inflammatory and atheroprotective properties.

Bovine HDL and Dual Domain HDL-Mimetic Peptides Inhibit Tumor Development in Mice.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28-2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.

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.

Oral apolipoprotein A-I mimetic peptide improves cognitive function and reduces amyloid burden in a mouse model of Alzheimer's disease.

Recent evidence indicates that inflammation may significantly contribute to the pathogenesis of Alzheimer's disease (AD). Since the apo A-I mimetic peptide D-4F has been shown to inhibit atherosclerotic lesion formation and regress already existing lesions (in the presence of pravastatin) and the peptide also decreases brain arteriole inflammation, we undertook a study to evaluate the efficacy of oral D-4F co-administered with pravastatin on cognitive function and amyloid beta (A beta) burden in the hippocampus of APPSwe-PS1 Delta E9 mice. Three groups of male mice were administered D-4F and pravastatin, Scrambled D-4F (ScD-4F, a control peptide) and pravastatin in drinking water, while drinking water alone served as control. The escape latency in the Morris Water Maze test was significantly shorter for the D-4F+statin administered animals compared to the other two groups. While the hippocampal region of the brain was covered with 4.2+/-0.5 and 3.8+/-0.6% of A beta load in the control and ScD-4F+statin administered groups, in the D-4F+statin administered group A beta load was only 1.6+/-0.1%. Furthermore, there was a significant decrease in the number of activated microglia (p<0.05 vs the other two groups) and activated astrocytes (p<0.05 vs control) upon oral D-4F+statin treatment. Inflammatory markers TNFalpha and IL-1 beta levels were decreased significantly in the D-4F+statin group compared to the other two groups (for IL-1 beta p<0.01 vs the other two groups and for TNF-alpha p<0.001 vs control) and the expression of MCP-1 were also less in D-4F+statin administered group compared to the other two groups. These results suggest that the apo A-I mimetic peptide inhibits amyloid beta deposition and improves cognitive function via exerting anti-inflammatory properties in the brain.

The apoA-I mimetic peptide 4F protects apolipoprotein A-I from oxidative damage.

High density lipoprotein (HDL) is prone to modification by the oxidizing and chlorinating agent hypochlorite anion (OCl-). Oxidation of apolipoprotein (apo) A-I, the major protein in HDL, reduces ABCA-1 mediated cholesterol efflux and other protective responses to HDL. The apoA-I mimetic peptide 4F has been shown to undergo oxidation; however, the ability of the peptide to mediate cholesterol efflux remains intact. Here, we show that 4F protects apoA-I from hypochlorite-mediated oxidation. Mass spectral analysis of apoA-I shows that tyrosine residues that are prone to hypochlorite-mediated chlorination are protected in the presence of 4F. Furthermore, 4F enhances the cholesterol efflux ability of apoA-I to a greater extent than either 4F or apoA-I alone, even after hypochlorite oxidation. These observations suggest that apoA-I in lipid complexes may be protected by the presence of 4F, resulting in the preservation of its anti-inflammatory and anti-atherogenic properties. These studies also form the basis for the future studies of nanoparticles possessing both apoA-I and 4F.

Apolipoprotein A-I mimetics mitigate intestinal inflammation in COX2-dependent inflammatory bowel disease model.

Cyclooxygenase 2 (Cox2) total knockout and myeloid knockout (MKO) mice develop Crohn's-like intestinal inflammation when fed cholate-containing high fat diet (CCHF). We demonstrated that CCHF impaired intestinal barrier function and increased translocation of endotoxin, initiating TLR/MyD88-dependent inflammation in Cox2 KO but not WT mice. Cox2 MKO increased pro-inflammatory mediators in LPS-activated macrophages, and in the intestinal tissue and plasma upon CCHF challenge. Cox2 MKO also reduced inflammation resolving lipoxin A4 (LXA4) in intestinal tissue, while administration of an LXA4 analog rescued disease in Cox2 MKO mice fed CCHF. The apolipoprotein A-I (APOA1) mimetic 4F mitigated disease in both the Cox2 MKO/CCHF and piroxicam-accelerated Il10-/- models of inflammatory bowel disease (IBD) and reduced elevated levels of pro-inflammatory mediators in tissue and plasma. APOA1 mimetic Tg6F therapy was also effective in reducing intestinal inflammation in the Cox2 MKO/CCHF model. We further demonstrated that APOA1 mimetic peptides: i) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (oxPAPC) dependent pro-inflammatory responses in human macrophages and intestinal epithelium; and ii) directly cleared pro-inflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for pro-inflammatory and inflammation resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD.

HDL therapy for cardiovascular diseases: the road to HDL mimetics.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are currently the drug of choice for the clinical management of elevated low-density lipoprotein (LDL) cholesterol. Although statin treatment provides an overall improvement in outcomes, clinical trial data reveal a significant number of cardiac events despite reaching targeted LDL levels. A low serum high-density lipoprotein (HDL) cholesterol level is an independent predictor of cardiovascular risk. Accordingly, there has been interest in determining whether HDL elevation, in addition to LDL lowering, further reduces risk in patients with coronary artery disease. Several commonly prescribed lipid-lowering therapies modestly raise HDL, but their use may be limited by the development of adverse reactions. Emerging data suggest that HDL quality and function may also be significantly reduced by atherosclerosis and other inflammatory diseases. The goal of this review is to discuss the current status of HDL therapeutics, with emphasis on a novel class of agent, the apolipoprotein A-I mimetic peptides, which improve the functional properties of HDL cholesterol.

Oral small peptides render HDL antiinflammatory in mice and monkeys and reduce atherosclerosis in ApoE null mice.

A peptide containing only 4 amino acid residues (KRES) that is too small to form an amphipathic helix, reduced lipoprotein lipid hydroperoxides (LOOH), increased paraoxonase activity, increased plasma HDL-cholesterol levels, rendered HDL antiinflammatory, and reduced atherosclerosis in apoE null mice. KRES was orally effective when synthesized from either L or D-amino acids suggesting that peptide-protein interactions were not required. Remarkably, changing the order of 2 amino acids (from KRES to KERS) resulted in the loss of all biologic activity. Solubility in ethyl acetate and interaction with lipids, as determined by differential scanning calorimetry, indicated significant differences between KRES and KERS. Negative stain electron microscopy showed that KRES formed organized peptide-lipid structures whereas KERS did not. Another tetrapeptide FREL shared many of the physical-chemical properties of KRES and was biologically active in mice and monkeys when synthesized from either L- or D-amino acids. After oral administration KRES and FREL were found associated with HDL whereas KERS was not. We conclude that the ability of peptides to interact with lipids, remove LOOH and activate antioxidant enzymes associated with HDL determines their antiinflammatory and antiatherogenic properties regardless of their ability to form amphipathic helixes.

Inhibition of lipopolysaccharide-induced inflammatory responses by an apolipoprotein AI mimetic peptide.

Previous studies suggest that high-density lipoprotein and apoAI inhibit lipopolysaccharide (LPS)-induced inflammatory responses. The goal of the current study was to test the hypothesis that the apoAI mimetic peptide L-4F exerts antiinflammatory effects similar to apoAI. Pretreatment of human umbilical vein endothelial cells (HUVECs) with LPS induced the adhesion of THP-1 monocytes. Incubation of cells with LPS and L-4F (1 to 50 microg/mL) reduced THP-1 adhesion in a concentration-dependent manner. This response was associated with a significant reduction in the synthesis of cytokines, chemokines, and adhesion molecules. L-4F reduced vascular cell adhesion molecule-1 expression induced by LPS or lipid A, whereas a control peptide (Sc-4F) showed no effect. In contrast to LPS treatment, L-4F did not inhibit IL-1beta- or tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression. The inhibitory effect of L-4F on LPS induction of inflammatory markers was associated with reduced binding of LPS to its plasma carrier molecule, lipopolysaccharide binding protein, and decreased binding of LPS to HUVEC monolayers. LPS and L-4F in HUVEC culture medium were fractionated by fast protein liquid chromatography and were localized to the same fractions, suggesting a physical interaction between these molecules. Proinflammatory responses to LPS are associated with the binding of lipid A to cell surface receptors. The current studies demonstrate that L-4F reduces the expression of inflammatory markers induced by LPS and lipid A and suggest that apoAI peptide mimetics may be useful in the treatment of inflammation associated with endotoxemia.

An oral apoJ peptide renders HDL antiinflammatory in mice and monkeys and dramatically reduces atherosclerosis in apolipoprotein E-null mice.

OBJECTIVE: To determine the properties of a peptide synthesized from D-amino acids corresponding to residues 113 to 122 in apolipoprotein (apo) J. METHODS AND RESULTS: In contrast to D-4F, D- [113-122]apoJ showed minimal self-association and helicity in the absence of lipids. D-4F increased the concentration of apoA-I with pre-beta mobility in apoE-null mice whereas D- [113-122]apoJ did not. After an oral dose D- [113-122]apoJ more slowly associated with lipoproteins and was cleared from plasma much more slowly than D-4F. D- [113-122]apoJ significantly improved the ability of plasma to promote cholesterol efflux and improved high-density lipoprotein (HDL) inflammatory properties for up to 48 hours after a single oral dose in apoE-null mice, whereas scrambled D- [113-122]apoJ did not. Oral administration of 125 microg/mouse/d of D- [113-122]apoJ reduced atherosclerosis in apoE-null mice (70.2% reduction in aortic root sinus lesion area, P=4.3 x 10(-13); 70.5% reduction by en face analysis, P=1.5 x 10(-6)). In monkeys, oral D- [113-122]apoJ rapidly reduced lipoprotein lipid hydroperoxides (LOOH) and improved HDL inflammatory properties. Adding 250 ng/mL of D-[113-122]apoJ (but not scrambled D- [113-122]apoJ) to plasma in vitro reduced LOOH and increased paraoxonase activity. CONCLUSIONS: Oral D- [113-122]apoJ significantly improves HDL inflammatory properties in mice and monkeys and inhibits lesion formation in apoE-null mice.

Surface Density-Induced Pleating of a Lipid Monolayer Drives Nascent High-Density Lipoprotein Assembly.

Biogenesis of high-density lipoproteins (HDL) is coupled to the transmembrane protein, ATP-binding cassette transporter A1 (ABCA1), which transports phospholipid (PL) from the inner to the outer membrane monolayer. Using a combination of computational and experimental approaches, we show that increased outer lipid monolayer surface density, driven by excess PL or membrane insertion of amphipathic helices, results in pleating of the outer monolayer to form membrane-attached discoidal bilayers. Apolipoprotein (apo)A-I accelerates and stabilizes the pleats. In the absence of apoA-I, pleats collapse to form vesicles. These results mimic cells overexpressing ABCA1 that, in the absence of apoA-I, form and release vesicles. We conclude that the basic driving force for nascent discoidal HDL assembly is a PL pump-induced surface density increase that produces lipid monolayer pleating. We then argue that ABCA1 forms an extracellular reservoir containing an isolated pressurized lipid monolayer decoupled from the transbilayer density buffering of cholesterol.