Abstract-When apolipoprotein A-I mimetic peptides synthesized from either D-or L-amino acids were given orally to LDL receptor-null mice, only the peptide synthesized from D-amino acids was stable in the circulation and enhanced the ability of HDL to protect LDL against oxidation. The peptide synthesized from L-amino acids was rapidly degraded and excreted in the urine. When a peptide synthesized from D-amino acids (D-4F) was administered orally to LDL receptor-null mice on a Western diet, lesions decreased by 79%. When added to the drinking water of apoE-null mice, D-4F decreased lesions by approximately 75% at the lowest dose tested (0.05 mg/mL). The marked reduction in lesions occurred independent of changes in total plasma or HDL-cholesterol. Key Words: atherosclerosis Ⅲ HDL Ⅲ apo A-I Ⅲ LDL oxidation I nfusion 1 or transgenic expression 2 of apo A-I, the major apolipoprotein of HDL, protects against atherosclerosis in animals. Proposed mechanisms by which apo A-I protects include reverse cholesterol transport 3 and removal of low levels of oxidized lipids, "seeding molecules" required to oxidize LDL. 4 -6 Apo A-I and class A amphipathic helical peptide analogs of apo A-I remove these "seeding molecules" and prevent LDL oxidation. 4,5 Intraperitoneal administration of an apo A-I mimetic peptide enhanced the ability of HDL to protect LDL against oxidation and protected mice from diet-induced atherosclerosis without changing plasma cholesterol levels. 7 The major limitation for the use of apo A-I or apo A-I mimetic peptides as pharmacological agents has been the need for parenteral administration. Mammalian enzymes such as proteases recognize peptides and proteins synthesized from L-amino acids but rarely recognize those synthesized from D-amino acids. We report here that orally administered apo A-I mimetic peptides synthesized from D-amino acids dramatically inhibit atherosclerosis in mice independent of changes in total plasma or HDL-cholesterol. Methods MiceFemale LDL receptor-null or apoE-null mice on a C57BL/6J background were from Jackson Laboratory, Bar Harbor, Maine. LDL receptor-null mice were maintained on chow diet (Ralston Purina) until they were 4-weeks old when they were switched to a Western diet (Teklad, Madison, WI, diet No. 88137) for 6 weeks. ApoE-null mice were maintained on chow diet throughout the study. LDL receptor-null mice received the test peptide or a vehicle control by gastric gavage twice daily for the periods indicated. At 4-weeks old, the test peptide was added to the drinking water of some of the apoE-null mice and the apoE-null mice were continued on the chow diet. The lyophilized peptide was easily dissolved in a measured quantity of drinking water resulting in a clear solution and was measured and replaced with fresh solution every other day.Mice were bled under anesthesia from the retroorbital venous plexus with Animal Research Committee approval. Atherosclerotic lesions were measured as described. 8 LipoproteinsLDL and HDL were isolated as described. 4 Blood was obtained fr...
Background-These studies were designed to determine the mechanism of action of an oral apolipoprotein (apo) A-I mimetic peptide, D-4F, which previously was shown to dramatically reduce atherosclerosis in mice. Methods and Results-Twenty minutes after 500 g of D-4F was given orally to apoE-null mice, small cholesterolcontaining particles (CCPs) of 7 to 8 nm with pre- mobility and enriched in apoA-I and paraoxonase activity were found in plasma.
Abstract-Despite identical amino acid composition, differences in class A amphipathic helical peptides caused by differences in the order of amino acids on the hydrophobic face results in substantial differences in antiinflammatory properties. One of these peptides is an apolipoprotein A-I (apoA-I) mimetic, D-4F. When given orally to mice and monkeys, D-4F caused the formation of pre- high-density lipoprotein (HDL), improved HDL-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from proinflammatory to antiinflammatory. In apolipoprotein E (apoE)-null mice, D-4F increased reverse cholesterol transport from macrophages. Oral D-4F reduced atherosclerosis in apoE-null and low-density lipoprotein (LDL) receptor-null mice. In vitro when added to human plasma at nanomolar concentrations, D-4F caused the formation of pre- HDL, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. Physical-chemical properties and the ability of various class A amphipathic helical peptides to activate lecithin cholesterol acyltransferase (LCAT) in vitro did not predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. We conclude that the antiinflammatory properties of different class A amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides, which determines the ability of the peptides to sequester inflammatory lipids. These differences appear to be too subtle to predict efficacy based on physical-chemical properties alone. However, understanding these physical-chemical properties provides an explanation for the mechanism of action of the active peptides.
Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called LOCATE that searches amino acid sequences to identify potential amphipathic a-helixes and /3-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular a-helical proteins); motif C (coiled-coil a-helical proteins); and motif B (0 pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by LOCATE using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under P lasma apolipoproteins can be grouped into two general classes, the nonexchangeable apolipoproteins (apolipoprotein [apo] B-100 and apoB-48), and the exchangeable apolipoproteins (all other apolipoproteins).1 The B apolipoproteins, present in chylomicrons, very-low-density lipoprotein, intermediate-density lipoprotein, low-density lipoprotein (LDL), and lipoprotein(a), are highly insoluble in aqueous solutions and thus remain with the lipoprotein particle throughout its metabolism.2 Because of their size and insoluble nature, it has been difficult to deduce the structural motif(s) responsible for the lipid-associating properties of the B apolipoproteins. 34 On the other
Background-We reported that HDL loses its antiinflammatory properties during acute influenza A infection in mice, and we hypothesized that these changes might be associated with increased trafficking of macrophages into the artery wall. The present study tested this hypothesis. Methods and Results-D-4F, an apolipoprotein A-I mimetic peptide, or vehicle in which it was dissolved (PBS) was administered daily to LDL receptor-null mice after a Western diet and after influenza infection. D-4F treatment increased plasma HDL cholesterol and paraoxonase activity compared with PBS and inhibited increases in LDL cholesterol and peak levels of interleukin-6 after infection. Lung viral titers were reduced by 50% in mice receiving D-4F. Injection of female mice with male macrophages, which were detected with real-time polymerase chain reaction to measure the male Sry gene, revealed a marked increase in macrophage traffic into the aortic arch and innominate arteries after infection that was prevented by administration of D-4F. Conclusions-We conclude that loss of antiinflammatory properties of HDL after influenza infection in mice is associated with increased arterial macrophage traffic that can be prevented by administration of
Objectives-We tested for synergy between pravastatin and D-4F by administering oral doses of each in combination that were predetermined to be ineffective when given as single agents. Methods and Results-The combination significantly increased high-density lipoprotein (HDL)-cholesterol levels, apolipoprotein (apo)A-I levels, paraoxonase activity, rendered HDL antiinflammatory, prevented lesion formation in young (79% reduction in en face lesion area; PϽ0.0001) and caused regression of established lesions in old apoE null mice (ie, mice receiving the combination for 6 months had lesion areas that were smaller than those before the start of treatment (Pϭ0.019 for en face lesion area; Pϭ0.004 for aortic root sinus lesion area). After 6 months of treatment with the combination, en face lesion area was 38% of that in mice maintained on chow alone; PϽ0.00004) with a 22% reduction in macrophage content in the remaining lesions (Pϭ0.001), indicating an overall reduction in macrophages of 79%. The combination increased intestinal apoA-I synthesis by 60% (Pϭ0.011). In monkeys, the combination also rendered HDL antiinflammatory. Key Words: atherosclerosis Ⅲ lipoproteins Ⅲ HDL Ⅲ apoA-I mimetic peptides Ⅲ statins S tatins and apolipoprotein (apo)A-I have similar antiinflammatory properties. 1 Ansell et al 2 reported that the inflammatory/antiinflammatory properties of high-density lipoprotein (HDL) identified patients with coronary heart disease (CHD) or CHD equivalents better than HDL-cholesterol levels. Treatment with 40 mg pravastatin daily for 6 weeks significantly improved the inflammatory/antiinflammatory properties of HDL in these patients. 2 However, even after treatment, 40% to 50% of the patients still had frankly pro-inflammatory HDL. 2 Moreover, after pravastatin only 4 of 26 patients achieved HDL that was antiinflammatory to the degree seen in 24 of 26 age-and gender-matched controls. 2 Conclusions-These
Abstract-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 g/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-1-or tumor necrosis factor-␣-induced vascular cell adhesion molecule-1 expression. 1 Approximately 50% of patients in intensive care units develop severe sepsis, and the overall mortality rate of all affected patients is 29%. 1 Mortality is attributable, in large part, to the cytotoxic actions of lipopolysaccharide (LPS) (endotoxin), a component of the outer membrane of Gram-negative bacteria. LPS is composed of a core oligosaccharide, a repeating polysaccharide side chain, and the glycolipid moiety lipid A. 2 Proinflammatory and cytotoxic effects of LPS are mediated by lipid A. 3 LPS is released from bacterial membranes into the circulation where it interacts with lipopolysaccharide binding protein (LBP), a member of the superfamily of phospholipid binding proteins. LBP binds to lipid A and mediates the disaggregation of LPS to form an LBP-LPS complex. 4 LBP directs LPS to membrane-associated CD14 receptors (mCD14) on myeloid cells 5 including monocytes and neutrophils. mCD14 is a cell surface-anchored protein that facilitates the binding of LPS and activation of Toll-like receptor (TLR) 4 which acts as the cellular transducer of LPS action. 2,6 Plasma LPS-LBP may also interact with soluble CD14 to form a complex that activates TLRs on endothelial, epithelial, Kuppfer, and other cells. 7 By activating nuclear factor B-dependent signaling mechanisms, LPS stimulates the synthesis/release of inflammatory cytokines, which play an important role in the innate immune response. 8,9 Dysregulation of this response leads to the development of endothelial dysfunction, intravascular coagulation, pulmonary injury, multiple organ failure, and death.The acute-phase response to bacterial infection induces changes in plasma lipoprotein levels that are characterized by Original
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