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.

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.

The Apolipoprotein E Mimetic Peptide AEM-2 Attenuates Mitochondrial Injury And Apoptosis In Human THP-1 Macrophages.

Cardiovascular disease, specifically atherosclerosis, is exacerbated by hypercholesterolemia. Current therapies that target lipid lowering, however, are not effective in all patients. Apolipoprotein E (apoE) plays an important role in mediating the clearance of plasma cholesterol and also exerts numerous cytoprotective responses. Our laboratory has synthesized novel therapeutics that mimic the ability of apoE to decrease plasma cholesterol. The apoE mimetic peptide AEM-2 is a dual domain peptide composed of an amphipathic helical region that binds phospholipids and a positively charged region that mediates the hepatic clearance of lipoproteins. Administration of AEM-2 to apoE null mice reduced plasma cholesterol concentration by 80% one hour post-administration. Since apoE is also known to exert anti-inflammatory effects that are independent of its ability to lower cholesterol, we tested effects of AEM-2 on lipopolysaccharide-induced responses in human THP-1 macrophages. Pre-treatment of THP-1 cells with AEM-2 significantly reduced the LPS-induced secretion of IL-6 and TNFα. Since LPS administration is associated with an increase in mitochondrial injury, we monitored effects of AEM-2 on mitochondrial function. AEM-2 significantly reduced mitochondrial superoxide formation, prevented the LPS-induced decrease in mitochondrial membrane potential and attenuated the release of cytochrome c. AEM-2 also inhibited the activities of initiator caspases 8 and 9 and effector caspase 3. The attenuation of apoptosis in AEM-2 treated cells was associated with an increase in cellular autophagy. These data suggest that AEM-2 attenuates cellular injury in LPS-treated THP-1 macrophages and facilitates the removal of cellular debris and damaged organelles via induction of autophagy.

High-Density Lipoprotein Regulation of Mitochondrial Function.

Lipoproteins play a key role in regulating plasma and tissue levels of cholesterol. Apolipoprotein B (apoB)-containing lipoproteins, including chylomicrons, very-low density lipoprotein (VLDL) and low-density lipoprotein (LDL), serve as carriers of triglycerides and cholesterol and deliver these metabolites to peripheral tissues. In contrast, high-density lipoprotein (HDL) mediates Reverse Cholesterol Transport (RCT), a process by which excess cholesterol is removed from the periphery and taken up by hepatocytes where it is metabolized and excreted. Anti-atherogenic properties of HDL have been largely ascribed to apoA-I, the major protein component of the lipoprotein particle. The inflammatory response associated with atherosclerosis and ischemia-reperfusion (I-R) injury has been linked to the development of mitochondrial dysfunction. Under these conditions, an increase in reactive oxygen species (ROS) formation induces damage to mitochondrial structural elements, leading to a reduction in ATP synthesis and initiation of the apoptotic program. Recent studies suggest that HDL-associated apoA-I and lysosphingolipids attenuate mitochondrial injury by multiple mechanisms, including the suppression of ROS formation and induction of autophagy. Other apolipoproteins, however, present in lower abundance in HDL particles may exert opposing effects on mitochondrial function. This chapter examines the role of HDL-associated apolipoproteins and lipids in the regulation of mitochondrial function and bioenergetics.

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.

Bioenergetic programming of macrophages by the apolipoprotein A-I mimetic peptide 4F.

The apoA-I (apolipoprotein A-I) mimetic peptide 4F favours the differentiation of human monocytes to an alternatively activated M2 phenotype. The goal of the present study was to test whether the 4F-mediated differentiation of MDMs (monocyte-derived macrophages) requires the induction of an oxidative metabolic programme. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including PPARγ (peroxisome-proliferator-activated receptor γ) and CD36. Addition of 4F was associated with a significant increase in FA (fatty acid) uptake and oxidation compared with vehicle treatment. Mitochondrial respiration was assessed by measurement of the OCR (oxygen-consumption rate). 4F increased basal and ATP-linked OCR as well as maximal uncoupled mitochondrial respiration. These changes were associated with a significant increase in ΔΨm (mitochondrial membrane potential). The increase in metabolic activity in 4F-treated MDMs was attenuated by etomoxir, an inhibitor of mitochondrial FA uptake. Finally, addition of the PPARγ antagonist T0070907 to 4F-treated MDMs reduced the expression of CD163 and CD36, cell-surface markers for M2 macrophages, and reduced basal and ATP-linked OCR. These results support our hypothesis that the 4F-mediated differentiation of MDMs to an anti-inflammatory phenotype is due, in part, to an increase in FA uptake and mitochondrial oxidative metabolism.

Two apolipoprotein E mimetic peptides with similar cholesterol reducing properties exhibit differential atheroprotective effects in LDL-R null mice.

OBJECTIVE: We investigated two apoE mimetic peptides with similar long-term plasma cholesterol reducing abilities for their effects on atherosclerotic lesions in Western diet-fed female LDL-receptor (LDL-R) null mice. METHODS AND RESULTS: Single doses of peptides Ac-hE18A-NH(2) and mR18L were administered retro-orbitally to LDL-R null mice on Western diet and plasma cholesterol was measured at 10 min, 4 h, and 24 h post administration. Peptide mR18L and not Ac-hE18A-NH(2) reduced plasma cholesterol levels significantly at 4 h post administration. However, multiple administrations (100 µg/mouse twice weekly for 8 weeks) resulted in a similar reduction in plasma cholesterol. Only the plasma from the Ac-hE18A-NH(2) group had significantly reduced reactive oxygen species levels at the end of the treatment protocol. Both mR18L and Ac-hE18A-NH(2) showed reduced atherosclerotic lesion areas. However, peptide Ac-hE18A-NH(2) was significantly more effective in inhibiting atherosclerosis. Both peptides reduced total plaque macrophage load compared to the saline treated animals, with peptide Ac-hE18A-NH(2) having a greater reduction. Incubation of HepG2 cells and THP-1 monocyte-derived macrophages with both peptides in the presence of oxidized phospholipid showed that Ac-hE18A-NH(2) promotes the secretion of apoE from the cells whereas mR18L does not. CONCLUSIONS: Despite similar reductions in plasma cholesterol levels, Ac-hE18A-NH(2) was more effective in inhibiting lesions than mR18L, possibly due to its ability to promote the secretion of apoE from hepatocytes and macrophages.

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.

HDL Mimetic Peptide Administration Improves Left Ventricular Filling and Cardiac output in Lipopolysaccharide-Treated Rats.

AIMS: Cardiac dysfunction is a complication of sepsis and contributes to morbidity and mortality. Since raising plasma apolipoprotein (apo) A-I and high density lipoprotein (HDL) concentration reduces sepsis complications, we tested the hypothesis that the apoA-I mimetic peptide 4F confers similar protective effects in rats treated with lipopolysaccharide (LPS). METHODS AND RESULTS: Male Sprague-Dawley (SD) rats were randomized to receive saline vehicle (n=13), LPS (10 mg/kg: n=16) or LPS plus 4F (10 mg/kg each: n=13) by intraperitoneal injection. Plasma cytokine and chemokine levels were significantly elevated 24 hrs after LPS administration. Echocardiographic studies revealed changes in cardiac dimensions that resulted in a reduction in left ventricular end-diastolic volume (LVEDV), stroke volume (SV) and cardiac output (CO) 24 hrs after LPS administration. 4F treatment reduced plasma levels of inflammatory mediators and increased LV filling, resulting in improved cardiac performance. Chromatographic separation of lipoproteins from plasma of vehicle, LPS and LPS+4F rats revealed similar profiles. Further analyses showed that LPS treatment reduced the agarose electrophoretic mobility of isolated HDL fractions. HDL-associated proteins were characterized by SDSPAGE and mass spectrometry. ApoA-I and apoA-IV were reduced while apoE content was increased in LPStreated rats. 4F treatment in vivo attenuated changes in HDL-associated apolipoproteins and increased the electrophoretic mobility of the particle. CONCLUSIONS: The ability of 4F to reduce inflammation and improve cardiac performance in LPS-treated rats may be due to its capacity to neutralize endotoxin and prevent adverse changes in HDL composition and function.

Two adjacent domains (141-150 and 151-160) of apoE covalently linked to a class A amphipathic helical peptide exhibit opposite atherogenic effects.

OBJECTIVE: We recently described anti-atherogenic properties of the dual domain peptide Ac-hE18A-NH(2) derived by covalently linking the heparin binding domain 141-150 of apoE to 18A, a class A amphipathic helical peptide. In this paper we have compared the properties of Ac-hE18A-NH(2) with the non-heparin binding 151-160 region of apoE linked to 18A (Ac-nhE18A-NH(2)). METHODS AND RESULTS: Both peptides were highly helical in solution and in association with lipids. Ac-hE18A-NH(2) and not Ac-nhE18A-NH(2) enhanced uptake of low density lipoprotein (LDL) in HepG2 cells. While Ac-hE18A-NH(2) retarded the electrophoretic mobility of LDL, Ac-nhE18A-NH(2) slightly enhanced mobility. Ac-hE18A-NH(2) reduced monocyte association with endothelial cells, while Ac-nhE18A-NH(2) increased it. Ac-hE18A-NH(2) also reduced lipid hydroperoxide content of LDL while Ac-nhE18A-NH(2) increased it. A single administration of Ac-hE18A-NH(2) (100 µg/mouse) into apoE null mice dramatically reduced cholesterol (from 600 mg/dL to 180 mg/dL at 5 min and to 60 mg/dL at 5h) while Ac-nhE18A-NH(2) had no effect. Administration (100 µg/mouse/day, three days a week) into apoE null mice for six weeks showed Ac-hE18A-NH(2) group having a moderate aortic sinus lesion reduction compared with the control group (-15.1%), while the Ac-nhE18A-NH(2) administered group had increased lesion area (+33.0% vs controls and 36.1% vs Ac-hE18A-NH(2)). Plasma from mice administered Ac-hE18A-NH(2) for six weeks showed a significant reduction in plasma cholesterol and triglyceride levels and increase in paraoxonase-1 (PON-1) activity compared to controls, while Ac-nhE18A-NH(2) caused no change in plasma cholesterol and decreased PON-1 activity. CONCLUSION: It is proposed that Ac-hE18A-NH(2) reduced lesion progression in apoE null mice due to its anti-inflammatory and lipoprotein clearing properties, while Ac-nhE18A-NH(2) exhibited pro-atherogenic effects.

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.

The apolipoprotein A-I mimetic peptide 4F prevents defects in vascular function in endotoxemic rats.

High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED(50) = 459 +/- 83 nM) compared with controls (ED(50) = 57 +/- 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED(50) = 60 +/- 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of (14)C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.

Apolipoprotein A-I mimetic 4F alters the function of human monocyte-derived macrophages.

HDL and its major protein component apolipoprotein A-I (apoA-I) exert anti-inflammatory effects, inhibit monocyte chemotaxis/adhesion, and reduce vascular macrophage content in inflammatory conditions. In this study, we tested the hypothesis that the apoA-I mimetic 4F modulates the function of monocyte-derived macrophages (MDMs) by regulating the expression of key cell surface receptors on MDMs. Primary human monocytes and THP-1 cells were treated with 4F, apoA-I, or vehicle for 7 days and analyzed for expression of cell surface markers, adhesion to human endothelial cells, phagocytic function, cholesterol efflux capacity, and lipid raft organization. 4F and apoA-I treatment decreased the expression of HLA-DR, CD86, CD11b, CD11c, CD14, and Toll-like receptor-4 (TLR-4) compared with control cells, suggesting the induction of monocyte differentiation. Both treatments abolished LPS-induced mRNA for monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), regulated on activation, normal T-expressed and presumably secreted (RANTES), IL-6, and TNF-alpha but significantly upregulated LPS-induced IL-10 expression. Moreover, 4F and apoA-I induced a 90% reduction in the expression of CD49d, a ligand for the VCAM-1 receptor, with a concurrent decrease in monocyte adhesion (55% reduction) to human endothelial cells and transendothelial migration (34 and 27% for 4F and apoA-I treatments) compared with vehicle treatment. In addition, phagocytosis of dextran-FITC beads was inhibited by 4F and apoA-I, a response associated with reduced expression of CD32. Finally, 4F and apoA-I stimulated cholesterol efflux from MDMs, leading to cholesterol depletion and disruption of lipid rafts. These data provide evidence that 4F, similar to apoA-I, induces profound functional changes in MDMs, possibly due to differentiation to an anti-inflammatory phenotype.

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.

Anti-inflammatory and recycling properties of an apolipoprotein mimetic peptide, Ac-hE18A-NH(2).

Apolipoprotein E (apoE) exerts prominent anti-inflammatory effects and undergoes recycling by target cells. We previously reported that the peptide Ac-hE18A-NH(2), composed of the receptor binding domain (LRKLRKRLLR) of apoE covalently linked to the Class A amphipathic peptide 18A, dramatically lowers plasma cholesterol and lipid hydroperoxides and enhances paraoxonase activity in dyslipidemic animal models. The objective of this study was to determine whether this peptide, analogous to apoE, exerts anti-inflammatory effects and undergoes recycling under in vitro conditions. Pulse chase studies using [(125)I]-Ac-hE18A-NH(2) in THP-1 derived macrophages and HepG2 cells showed greater amounts of intact peptide in the cells at later time points indicating recycling of the peptide. Ac-hE18A-NH(2) induced a 2.5-fold increase in prebeta-HDL in the conditioned media of HepG2 cells. This effect persisted for 3 days after removal of the peptide from culture medium. Ac-hE18A-NH(2) also induced the secretion of cell surface apoE from THP-1 macrophages. In addition, the peptide increased cholesterol efflux from THP-1 cells by an ABCA1 independent mechanism. Moreover, Ac-hE18A-NH(2) inhibited LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression, and reduced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). It also reduced the secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) from THP-1 macrophages even when administered post-LPS and abolished the 18-fold increase in LPS-induced mRNA levels for MCP-1 in THP-1 cells. Taken together, these results suggest that addition of the putative apoE receptor-domain to the Class A amphipathic peptide 18A results in a peptide that, similar to apoE, recycles, thus enabling the potentiation and prolongation of its anti-atherogenic and anti-inflammatory effects. Such a peptide has great potential as a therapeutic agent in the management of atherosclerosis and other inflammatory diseases.

Apolipoprotein A-I mimetic peptide treatment inhibits inflammatory responses and improves survival in septic rats.

Systemic inflammation induces a multiple organ dysfunction syndrome that contributes to morbidity and mortality in septic patients. Since increasing plasma apolipoprotein A-I (apoA-I) and HDL may reduce the complications of sepsis, we tested the hypothesis that the apoA-I mimetic peptide 4F confers similar protective effects in rats undergoing cecal ligation and puncture (CLP) injury. Male Sprague-Dawley rats were randomized to undergo CLP or sham surgery. IL-6 levels were significantly elevated in plasma by 6 h after CLP surgery compared with shams. In subsequent studies, CLP rats were further subdivided to receive vehicle or 4F (10 mg/kg) by intraperitoneal injection, 6 h after sepsis induction. Sham-operated rats received saline. Echocardiographic studies showed a reduction in left ventricular end-diastolic volume, stroke volume, and cardiac output (CO) 24 h after CLP surgery. These changes were associated with reduced blood volume and left ventricular filling pressure. 4F treatment improved blood volume status, increased CO, and reduced plasma IL-6 in CLP rats. Total cholesterol (TC) and HDL were 79 +/- 5 and 61 +/- 4 mg/dl, respectively, in sham rats. TC was significantly reduced in CLP rats (54 +/- 3 mg/dl) due to a reduction in HDL (26 +/- 3 mg/dl). 4F administration to CLP rats attenuated the reduction in TC (69 +/- 4 mg/dl) and HDL (41 +/- 3 mg/dl) and prevented sepsis-induced changes in HDL protein composition. Increased plasma HDL in 4F-treated CLP rats was associated with an improvement in CO and reduced mortality. It is proposed that protective effects of 4F are related to its ability to prevent the sepsis-induced reduction in plasma HDL.

Epithelial cells in fetal intestine produce chemerin to recruit macrophages.

Macrophages are first seen in the fetal intestine at 11-12 wk and rapidly increase in number during the 12- to 22-wk period of gestation. The development of macrophage populations in the fetal intestine precedes the appearance of lymphocytes and neutrophils and does not require the presence of dietary or microbial antigens. In this study, we investigated the role of chemerin, a recently discovered, relatively selective chemoattractant for macrophages, in the recruitment of macrophage precursors to the fetal intestine. Chemerin mRNA/protein expression was measured in jejunoileal tissue from 10- to 24-wk human fetuses, neonates operated for intestinal obstruction, and adults undergoing bariatric surgery. The expression of chemerin in intestinal epithelial cells (IECs) was confirmed by using cultured primary IECs and IEC-like cell lines in vitro. The regulatory mechanisms involved in chemerin expression were investigated by in silico and immunolocalization techniques. IECs in the fetal, but not mature, intestine express chemerin. Chemerin expression peaked in the fetal intestine at 20-24 wk and then decreased to original low levels by full term. During the 10- to 24-wk period, chemerin accounted for most of the macrophage chemotactic activity of cultured fetal IECs. The maturational changes in chemerin expression correlated with the expression of retinoic acid receptor-beta in the intestine. Chemerin is an important mediator of epithelial-macrophage cross talk in the fetal/premature, but not in the mature, intestine. Understanding the regulation of the gut macrophage pool is an important step in development of novel strategies to boost mucosal immunity in premature infants and other patient populations at risk of microbial translocation.

Vasculoprotective Effects of Apolipoprotein Mimetic Peptides: An Evolving Paradigm In Hdl Therapy (Vascular Disease Prevention, In Press.).

Anti-atherogenic effects of high density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) are principally thought to be due to their ability to mediate reverse cholesterol transport. These agents also possess anti-oxidant properties that prevent the oxidative modification of low density lipoprotein (LDL) and anti-inflammatory properties that include inhibition of endothelial cell adhesion molecule expression. Results of the Framingham study revealed that a reduction in HDL levels is an independent risk factor for coronary artery disease (CAD). Accordingly, there has been considerable interest in developing new therapies that specifically elevate HDL cholesterol. However, recent evidence suggests that increasing circulating HDL cholesterol levels alone is not sufficient as a mode of HDL therapy. Rather, therapeutic approaches that increase the functional properties of HDL may be superior to simply raising the levels of HDL per se. Our laboratory has pioneered the development of synthetic, apolipoprotein mimetic peptides which are structurally and functionally similar to apoA-I but possess unique structural homology to the lipid-associating domains of apoA-I. The apoA-I mimetic peptide 4F inhibits atherogenic lesion formation in murine models of atherosclerosis. This effect is related to the ability of 4F to induce the formation of pre-ß HDL particles that are enriched in apoA-I and paraoxonase. 4F also possesses anti-inflammatory and anti-oxidant properties that are independent of its effect on HDL quality per se. Recent studies suggest that 4F stimulates the expression of the antioxidant enzymes heme oxygenase and superoxide dismutase and inhibits superoxide anion formation in blood vessels of diabetic, hypercholesterolemic and sickle cell disease mice. The goal of this review is to discuss HDL-dependent and -independent mechanisms by which apoA-I mimetic peptides reduce vascular injury in experimental animal models.

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.