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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. Cox2MKO increased proinflammatory 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, whereas 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.sup.-/-] models of inflammatory bowel disease (IBD) and reduced elevated levels of proinflammatory 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 (a) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine-dependent (oxPAPC-dependent) proinflammatory responses in human macrophages and intestinal epithelium, and (b) directly cleared proinflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for proinflammatory and inflammation-resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD., Introduction Inflammatory bowel disease (IBD), inclusive of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. IBD consists of a dysregulated [...]

We examined whether reduced levels of Apolipoprotein A-I (apoA-I) in ovarian cancer patients are causal in ovarian cancer in a mouse model. Mice expressing a human apoA-I transgene had (i) increased survival (P < 0.0001) and (ii) decreased tumor development (P < 0.01), when compared with littermates, following injection of mouse ovarian epithelial papillary serous adenocarcinoma cells (ID-8 cells). ApoA-I mimetic peptides reduced viability and proliferation of ID8 cells and cis-platinum-resistant human ovarian cancer cells, and decreased ID-8 cell-mediated tumor burden in C57BL/6J mice when administered subcutaneously or orally. Serum levels of lysophosphatidic acid, a well-characterized modulator of tumor cell proliferation, were significantly reduced (>50% compared with control mice, P < 0.05) in mice that received apoA-I mimetic peptides (administered either subcutaneously or orally), suggesting that binding and removal of lysophosphatidic acid is a potential mechanism for the inhibition of tumor development by apoA-I mimetic peptides, which may serve as a previously unexplored class of anticancer agents. doi/ 10.1073/pnas.1009010107

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. high-density lipoprotein; phagocytosis; adhesion; cytokines; anti-inflammatory doi: 10.1152/ajpcell.00467.2009.

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 [+ or -] 5 and 61 [+ or -] 4 mg/dl, respectively, in sham rats. TC was significantly reduced in CLP rats (54 [+ or -] 3 mg/dl) due to a reduction in HDL (26 [+ or -] 3 mg/dl). 4F administration to CLP rats attenuated the reduction in TC (69 [+ or -] 4 mg/dl) and HDL (41 [+ or -] 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. sepsis; apolipoprotein A-I mimetic peptide; high-density lipoprotein; cardiac function; inflammation

Despite three decades of extensive studies on human apolipoprotein A-I (apoA-I), the major protein component in high-density lipoproteins, the molecular basis for its antiatherogenic function is elusive, in part because of lack of a structure of the full-length protein. We describe here the crystal structure of lipid-free apoA-I at 2.4 [Angstrom]. The structure shows that apoA-I is comprised of an N-terminal four-helix bundle and two C-terminal helices. The N-terminal domain plays a prominent role in maintaining its lipid-free conformation, indicating that mutants with truncations in this region form inadequate models for explaining functional properties of apoA-I. A model for transformation of the lipid-free conformation to the high-density lipoprotein-bound form follows from an analysis of solvent-accessible hydrophobic patches on the surface of the structure and their proximity to the hydrophobic core of the four-helix bundle. The crystal structure of human apoA-I displays a hitherto-unobserved array of positively and negatively charged areas on the surface. Positioning of the charged surface patches relative to hydrophobic regions near the C terminus of the protein offers insights into its interaction with cell-surface components of the reverse cholesterol transport pathway and antiatherogenic properties of this protein. This structure provides a much-needed structural template for exploration of molecular mechanisms by which human apoA-I ameliorates atherosclerosis and inflammatory diseases. helical structure | high-density lipoproteins | x-ray crystal structure atherosclerosis | lipid-free structure

Although oxidatively damaged lipoproteins are implicated in vascular injury, there is little information regarding the role of high-density lipoprotein (HDL) oxidation in atherogenesis. One potential pathway involves hypochlorous acid (HOCl) produced by myeloperoxidase (MPO), a heme protein secreted by phagocytes. We previously showed that 3-chlorotyrosine is a specific product of HOCl. Therefore, to explore the role of oxidized HDL in the pathogenesis of vascular disease, we used MS to quantify 3-chlorotyrosine in HDL isolated from plasma and atherosclerotic tissue. HDL from human aortic atherosclerotic intima had an 8-fold higher level of 3-chlorotyrosine than plasma HDL Tandem MS analysis identified MPO as a component of lesion HDL, suggesting that the two interact in the artery wall. Moreover, immunohistochemical studies found that specific epitopes derived from HOCI colocalized with apolipoprotein A-I, the major protein of HDL. These observations strongly support the hypothesis that MPO promotes HDL oxidation in the human artery wall. Levels of 3-chlorotyrosine were elevated in HDL isolated from the blood of humans with established coronary artery disease, suggesting that circulating levels of oxidized HDL represent a unique marker for clinically significant atherosclerosis. HDL or lipid-free apolipoprotein A-I exposed to HOCl was less able to remove cholesterol from cultured cells by a pathway requiring the cell membrane transporter ATP-binding cassette transporter A1. The detection of 3-chlorotyrosine in HDL isolated from vascular lesions raises the possibility that MPO, by virtue of its ability to form HOCl, may promote atherogenesis by counteracting the established antiatherogenic effects of HDL and the ATP-binding cassette transporter A1 pathway.

A synthesis of the peptide hApoE[141-150]-18A and its end-protected analogue is presented. The possibility that an arginine-rich apo E receptor enhances low-density lipoprotein uptake and clearance is discussed.

Experiments were performed to investigate whether pre-beta-high density lipoproteins (pre-beta-HDL) stimulate human placental lactogen release into the blood stream from normal trophoblast cells and to map the increase in pre-beta-HDL during gestation. Samples were collected from women attending the Obstetrics Clinic at the University of Cincinnati Hospital. The findings showed that pre-beta-HDL plays an important role in regulating the release of human placental lactogen during pregnancy.

Research was conducted to examine the lipid-free structure of apo human apolipoprotein (hA-I) and to gain an insight into the mechanism by which it binds to lipid through various biochemical and structural techniques. The lipid-free structures and lipid-binding characteristics of several deletion mutants were compared with apo delta(1-43)A-I and apo delta(187-243)A-I (25, 26). Results support a hypothetical, multistep structure-based mechanism for apo hA-I lipid binding.

Experiments were performed to investigate the synthetic peptides of human apolipoprotein A-I containing tandem amphipathic alpha-helices. Results showed that peptides one to two and nine to ten exhibited significantly higher lipid affinity than other peptides. It was also observed that except for peptides four to five and five to six, all the peptides possess higher lipid affinities compared to constituent helices. However, the lipid affinities of the peptides was neither equal nor higher to the sum of the lipid affinities of the constituent helices.

Researchers have established that the presence of acidic lipids and of nonbilayer forming lipids has a significant impact on peptide-membrane interactions. This work, which involved studying the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), revealed that 18L peptide destabilizes the membrane in zwitterionic membranes. This leads to the temporary formation of large defects in the membrane. The reciprocal effects of 18L and Ac-18A-NH2 peptides are marked only in lipid systems where there is a tendency towards nonbilayer phase formation.

The N-terminal 43 residues of human apolipoprotein A-I (apo hA-I) are necessary to maintain and stabilize the structure of these amino acids. They also play a crucial role in the conformational switch between the lipid-free and lipid-bound forms. Denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements and far- and near-ultraviolet circular dichroic spectroscopy showed significant structural differences between lipid-free apo (delta)(1-43)A-I and apo hA-I. According to spectral and physical measurements, apo (delta)(1-43)A-I has a folded, tertiary structure, although it is substantially less stable than that of apo hA-I.

The human placental tissue expresses a 22.7 kDa protein that resembles apolipoprotein A-I (apo A-I). The presence of a single apo A-I gene in the human genome indicates that the protein results from an alternative splice of the apo A-I primary transcript. HPLC of partly purified placental extract separates two fractions that react with apo A-I antiserum. Mass spectral analysis reveals that the mass of the fractions is 22,721 daltons. The N-terminal amino acid sequences of the fractions are similar. Northern blot analysis of placental RNA detects only one band smaller than apo A-I mRNA.

Amino acids 141-149 in apolipoprotein E (apoE) present in an amphipathic alpha-helix conformation comprise an immuno-regulatory sequence having either cytostatic or cytotoxic activity. The activity is determined by nonpolar face hydrophobicity and polar face charge density. By mediating removal of cholesterol-rich lipoproteins from the blood, apoE plays a crucial role in the control of cholesterol homeostasis. ApoE is an active suppressor of interlukin 2-dependent T lymphocyte proliferation.

An 18-residue peptide (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeu- LysGluAlaPhe), 18A, binds phospholipds due to its class A amphipathic helix and its NMR spectra shows that these helices prefer antiparallel arrangements even if they are not covalently linked. The Lys present in the polar head of the amphipathic helix are noninteracting and are fully hydrated while those in the Ac-18A-NH2 exist in different microenvironments. The location of the Lys residues with respect to the polar-nonpolar interface of the helix determines their titration behaviour. The NMR spectra is discussed.

Protein/phospholipid complexes are formed during the first interaction of apolipoprotein (apo) AI or similar peptides with cells, and cholesterol efflux follows. Cholesterol and phospholipid efflux increased by 12- and 4-fold respectively from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol.

Magainins lyse only bacterial cells because they contain a Glu residue that interacts with the cholesterol in eukaryotic cells to prevent the formation of a peptide wedge that lyses cell membranes. Experiments with three synthetic class L peptides showed that hemolysis was inhibited by peptides that contained a Glu residue. Many class L venoms are structurally similar to magainins, which are natural antibiotics. But venoms lack the Glu residue and so are able to lyse eukaryotic cells.

The interaction of amphipathic helical peptide 4F with phosphatidylcholine, either in the presence or in the absence of cholesterol and the ability of the peptide to promote the formation of cholesterol-rich domain is studied. The results show that the molecular nature of the interaction of 4F with membranes provides a mechanism for the antiatherogenic actions of the peptide both in inhibiting inflammatory processes in reverse cholesterol transport.