Apolipoprotein A-I Mimetic Peptides
http://www.100md.com
动脉硬化血栓血管生物学 2005年第7期
From the David Geffen School of Medicine at UCLA (M.N., S.T.R., S.H., G.H., V.R.G., N.Y., B.J.A., A.M.F.), Los Angeles, Calif; and the Department of Medicine (G.M.A., G.D., D.W.G.), Atherosclerosis Research Unit, University of Alabama, Birmingham.
Correspondence to Dr Mohamad Navab, Room 47-123 CHS, Division of Cardiology, Department of Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte, Avenue, Los Angeles, CA 90095-1679. E-mail mnavab@mednet.ucla.edu
Series Editor: Daniel J. Rader
ATVB In Focus Novel Approaches to the Treatment of Dyslipidemia
Previous Brief Reviews in this Series:
?Chen HC, Farese RV Jr. Inhibition of tgriglyceride synthesis as a treatment strategy for obestiy: lessons from DGAT1-deficient mice. 2005;25:482–486.
?Zalewski A. Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. 2005;25:923–931.
?Rudel LL, Lee RG, Parini P. ACTA2 is a target for treatment of coronary heart disease associated with hypercholesterolemia. 2005;25:1112–1118.
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 pro-inflammatory 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.
The antiinflammatory properties of different class A amphipathic helical peptides such as D-4F depend on subtle differences in the configuration of the hydrophobic face of the peptides that determine the ability of the peptides to sequester inflammatory lipids.
Key Words: HDL ? apoA-I ? peptide mimetics ? D-4F ? atherosclerosis ? inflammation
Introduction
The main protein of high-density lipoprotein (HDL), apolipoprotein A-I (apoA-I), contains 243 amino acids. The ability of apoA-I to remove phospholipids and cholesterol via the ABCA1 pathway1,2 is thought to be a major factor in preventing atherosclerosis. Despite recent evidence that other pathways such as ABCG1 may also be important in HDL-mediated cholesterol efflux,3,4 the importance of apoA-I in providing protection against atherosclerosis in animal models and in humans has been well-documented.5 This review focuses on the search for apoA-I mimetic peptides as therapeutic agents based on physical-chemical and biological properties. As is evident from the review presented here, the physical-chemical properties of these peptides are important but by themselves are not sufficient to predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides is more predictive. Subtle differences in the configuration of the hydrophobic face of apoA-I mimetic peptides determines the ability of the peptides to sequester inflammatory lipids and this seems to be the major determinant of their antiinflammatory properties. 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.
See page 1305
Physical-Chemical Properties of ApoA-I Mimetic Peptides as Predictors of Biologic Activity In Vivo
The search for peptides smaller than apoA-I but with many of the lipid-binding properties of apoA-I led to the synthesis of an 18-amino acid peptide known as 18A.6 The sequence of 18A (D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F) does not have any sequence homology to apoA-I. However, this sequence has the capacity to form a class A amphipathic helix similar to those found in apoA-I and mimics many of the lipid binding properties of apoA-I.7
Modifying the terminal charges of 18A by forming Ac-18A-NH2 (also known as 2F because of the 2 phenylalanine residues at positions 6 and 18) increased lipid affinity.8,9 Many 2F peptide variants have been synthesized, as shown in the Table. The Table also shows the biologic activity of these peptides in cocultures of human artery wall cells and in mouse models of atherosclerosis.10
ApoA-I Mimetic Peptides With Differing Numbers of Phenylalanine Residues Placed at Different Positions
Based on their physical properties, these peptides can be separated into 2 groups. The first group includes the original peptide (2F) containing 2 phenylalanine residues on the hydrophobic face; 2 peptides with 3 phenylalanine residues (3F3 and 3F14); and a peptide with 4 phenylalanine residues on the hydrophobic (4F).
The second group consists of a peptide with 5 phenylalanine residues on the hydrophobic face (5F); a peptide with 6 phenylalanine residues on the hydrophobic face (6F); and a peptide with 7 phenylalanine residues on the hydrophobic face (7F).10
The calculated hydrophobicity of the peptides increased as the number of phenylalanine residues on the nonpolar face increased. The increase in hydrophobicity was reflected in the theoretical lipid affinity ().11 The () value increased gradually from 2F to 4F (from 13.03 to 14.59), and then suddenly increased to 19.07 for 5F.10 From 5F to 7F, there was again a gradual increase in () values.10 The retention time on a C18 reversed-phase high-performance liquid chromatography column, solubility of the peptides, and their ability to penetrate an egg phosphatidylcholine monolayer showed a trend consistent with the theoretical lipid affinity values.10
Circular dichroism data for the peptides in phosphate-buffered saline (PBS) and in the presence of dimyristoyl phosphatidylcholine indicated that some of the peptides had a significant increase in the percent helicity on interacting with dimyristoyl phosphatidylcholine (eg, 2F, 3F3, 3F14, 5F, and 7F), whereas others did not (eg, 4F and 6F).10 The binding of the peptides to dimyristoyl phosphatidylcholine appeared to be similar as determined by differential scanning calorimetry.10 The solubility of the peptides in PBS and their ability to interact with phospholipid monolayers could be divided into 2 groups: 2F, 3F3, 3F14, and 4F had solubility in PBS of >2.0, 1.25, 1.45, and 1.30 mg/mL, respectively, and monolayer exclusion pressures of 38, 38, 39, and 40 dyne/cm, respectively.10 The solubility in PBS for 5F, 6F, and 7F was 0.10, 0.30, and 0.10 mg/mL, respectively, and their monolayer exclusion pressures were 45, 46, and 45 dyne/cm, respectively.10 Thus, in choosing a particular peptide, one must consider the balance between solubility in an aqueous environment and the ability to interact with phospholipid monolayers.
ApoA-I was not able to clarify egg phosphatidylcholine multilamellar vesicles, but all of the peptide analogs were able to do so to different extents. 4F appeared to be most efficient in solubilizing the phospholipid exhibiting kinetics similar to Triton X-100, suggesting that 4F has an optimal hydrophobicity to interact with phospholipids (ie, hydrophobic peptide-peptide interactions favoring self-association were minimized in 4F, thus promoting peptide-lipid interactions).10
All of the peptides activated lecithin cholesterol acyltransferase (LCAT) activity less than apoA-I.10 Moreover, there was no significant difference in LCAT activity between peptides that were found to be biologically inactive (eg, 2F, 3F3, and 3F14) and the biologically active10 peptide 4F.
Thus, all of these physical-chemical parameters and LCAT activity failed to predict peptides that would be found to be biologically inactive versus those that were biologically active.
The Use of Cultured Human Artery Wall Cells as an Assay to Predict In Vivo Efficacy of ApoA-I Mimetic Peptides
It has long been known that circulating low-density lipoprotein (LDL) contains a small amount of lipid hydroperoxides.12 When freshly isolated LDL is added to human artery wall cocultures, it is trapped in the subendothelial space and additional lipid hydroperoxides produced by the artery wall cells are partitioned into the trapped LDL.12,13 When a critical threshold of lipid hydroperoxides within the trapped LDL is reached, arachidonic acid-containing phospholipids in the LDL are oxidized and cause the artery wall cells to produce monocyte chemotactic activity (MCA), which is largely caused by the production of monocyte chemoattractant-1.12,13 When human apoA-I was added to the human artery wall cocultures in a pre-incubation and removed before the addition of human LDL, there was a marked reduction in LDL-induced MCA. However, when human apoA-I was added to these same cocultures together with the LDL (a coincubation), there was no reduction in LDL-induced MCA.13 It was concluded that human apoA-I binds lipid hydroperoxides in such a manner that the lipid hydroperoxides can still participate in the formation of oxidized phospholipids if the apoA-I-lipid hydroperoxide complexes are not removed.13 In contrast, some of the peptide mimetics of apoA-I were effective in inhibiting LDL-induced MCA even in a coincubation, suggesting that the peptides effectively sequestered the lipid hydroperoxides10 and thus acted more like apolipoprotein J (which was shown to be active in a coincubation14) than like apoA-I in this regard.
As noted, the peptide 18A showed enhanced lipid binding when blocking groups were added to produce Ac-18A-NH2. Despite the enhanced lipid binding, Ac-18A-NH2 was relatively weak in its ability to prevent LDL-induced MCA in human artery wall cell cocultures and Ac-18A-NH2 failed to inhibit diet-induced atherosclerosis in mice.10
The ability of the peptides to inhibit LDL-induced MCA in the human artery wall coculture could not be predicted by the differences in the physical-chemical properties of the peptides or the ability of the peptides to activate LCAT.10 When the peptides were added to human artery wall cocultures together with human LDL,10 peptides 3F3 and 3F14 were ineffective in preventing MCA, 2F was weakly effective, and 4F, 5F, and 6F were very effective, whereas 7F was poorly effective (even less effective than 2F). As shown in the Table, the activity in the human artery wall cell cultures were in agreement with the results in mouse models of atherosclerosis, ie, 2F was not effective in vivo, whereas 4F and 5F were quite effective in vivo.
Comparison of 4 Peptides That Are Identical Except for the Position of Their 3 Phenylalanine Residues on the Hydrophobic Face With Peptide 4F
A comparison of 4 peptides, which were identical with each other except for the positions of the phenylalanine residues on the hydrophobic face, was made to further determine the relationship between structure and function of these peptides. Peptides 3F-1, 3F-2, 3F3, and 3F14 were compared with each other and with the 4F peptide15 (Table). The 3F-1, 3F-2, and 4F peptides scavenged lipid hydroperoxides from LDL better than 3F3 or 3F14. Similarly the 3F-1, 3F-2, and 4F peptides were significantly better in preventing oxidized phospholipids from inducing human artery wall cells to produce MCA compared with 3F3 or 3F14. In the presence of lipid, all of these peptides had similar secondary structures and similar hydrophobicities.15 These peptides inserted into monolayers of egg phosphatidylcholine with similar avidity as shown with surface pressure measurements and into bilayers as evidenced by the fluorescence emission from tryptophan. Fluorescence studies suggested that although the peptides bound phospholipids similarly, the tryptophan residue in peptides 4F, 3F-1, and 3F-2 was less motionally restricted than in 3F3 and 3F14. Additionally, fluorescence spectroscopy studies with a fluorescent phospholipid (DPH-PC) revealed increased quenching of DPH-PC, indicating that the active peptides, particularly 4F, allowed a greater penetration of water molecules into the hydrophobic milieu of the membrane.15 Based on the comparison of 4F to the inactive peptides 3F3 and 3F14, it was postulated that the presence of water in the milieu of the hydrophobic region of the peptide-lipid complex would determine the amount of lipid hydroperoxides and oxidized phospholipids transferred from LDL to the sequestered environment of the peptide-containing particles, which in turn would determine the availability of these lipids to stimulate the artery wall cells to produce MCA.15 It should be noted that the differences between 3F-1 and 3F-2 (biologically active) and 3F3 and 3F14 (biologically inactive) were not significant in the quenching of DPH-PC.15 Thus, the measurement of the physical-chemical properties of these peptides leading to the quenching of DPH-PC by 3F-1 and 3F-2 versus 3F3 and 3F14 could not predict their biologic activity.
Other in vitro studies of 4F revealed that it promoted the separation of cholesterol from phospholipid.16 4F penetrated into membranes of pure phosphatidylcholine in the absence of cholesterol better than into bilayers of phosphatidylcholine and cholesterol.16 The circular dichroism spectrum of 4F in buffer indicated that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remained helical over a larger concentration range. On heating, the peptide underwent a thermal transition. Cholesterol had little effect on the secondary structure of the peptide; however, increased tryptophan emission intensity in the absence of cholesterol indicated a deeper penetration of the helix on removal of cholesterol from the membrane. It was hypothesized that the results with these model systems demonstrated changes in peptide-lipid interactions that may relate to the observed biological properties of this peptide.16
Additional studies comparing 3F-2 to 3F14 revealed that both peptides promoted the segregation of cholesterol in membranes containing phosphatidylcholine and cholesterol, but 3F-2 exhibited greater selectivity for partitioning into cholesterol-depleted regions of membrane.17 Magic angle spinning/nuclear magnetic resonance indicated that the aromatic residues of 3F-2 were stacked in the presence of lipid. The aromatic side chains of 3F-2 also penetrated more deeply into membranes of phosphatidylcholine with cholesterol compared with 3F.14 Using the fluorescent probe, 1,3-dipyreneylpropane, it was determined that 3F-2 had a greater effect in altering the hydrocarbon region of the membrane. Based on molecular models in previous studies15 and in these studies,17 it was concluded that the wedge-shaped cross-sectional area of 3F14 has only a minimal effect on the lipid acyl chain packing of membranes, whereas the cylindrical cross sectional area of 3F-2 (and also 4F) causes greater acyl chain perturbations, which facilitate the entry of molecules such as water and lipid hydroperoxides into the hydrophobic milieu of the complex.17 It was postulated that these properties of 3F-2 and 4F allow them to effectively sequester inflammatory lipids, whereas 3F14 is unable to do so. Consistent with these findings, the peptide 3F14 is not antiinflammatory in human artery wall cell cultures, whereas 3F-2 and 4F are highly antiinflammatory.10,15 It should be noted that without previous knowledge of the antiinflammatory properties of 3F-2 and the lack of such properties by 3F14 in human artery wall cell cultures, the differences found in these sophisticated physical-chemical studies17 would likely have been too subtle to have predicted which of these 2 peptides would be biologically active and which would not.
Injection of 5F Inhibits Diet-Induced Atherosclerosis
Garber et al18 provided the first evidence that an apoA-I mimetic peptide could inhibit atherosclerosis in vivo. The peptide 5F was administered by injection at a dose of 20 μg/d for 16 weeks in C57BL/6J mice fed an atherogenic diet. Control mice received either mouse apoA-I at 50 μg/d or PBS. Total plasma cholesterol levels and lipoprotein profiles were not significantly different except that the mice receiving 5F or mouse apoA-I had lower HDL cholesterol when calculated as a percent of total cholesterol.18 No toxicity or production of antibodies to the injected materials was observed. HDL isolated from the mice injected with PBS failed to decrease LDL-induced MCA in the human artery wall coculture, HDL from mice injected with mouse apoA-I was only weakly antiinflammatory, and HDL from mice that were injected with 5F or human apoA-I was significantly antiinflammatory (ie, reduced LDL-induced MCA as much as normal human HDL).18 Atherosclerosis lesion area was significantly reduced in the 5F-treated mice but not in mice receiving mouse apoA-I.18 Thus, after decades of research on apoA-I mimetic peptides,6 the first demonstration in vivo that such peptides could actually inhibit atherosclerosis was achieved.18
Oral D-4F Inhibits Atherosclerosis in Apolipoprotein E-Null and LDL Receptor-Null Mice
In a search for peptides that could be administered orally, it was recalled that mammalian enzymes recognize peptides and proteins synthesized from L-amino acids but rarely recognize peptides synthesized from D-amino acids. When 4F was synthesized from L-amino acids and given orally to mice, it was rapidly degraded.20 However, when 4F was synthesized from D-amino acids and was orally administered, it was found intact in the circulation.20 The oral administration of D-4F rendered HDL from LDL receptor-null mice on a Western diet and HDL from apolipoprotein E (apoE)-null mice on a chow diet antiinflammatory. Oral D-4F also reduced atherosclerotic lesions in these mice by 79% and 75%, respectively, without significantly altering plasma lipid levels.19
One hour after orally administering D-4F to LDL receptor-null mice, the peptide was initially found in fast protein liquid fractions where pre-? HDL would be expected, as well as in those fractions where mature -migrating HDL would be expected. One hour later, the peptide was only seen in the latter fractions, and the peptide was virtually cleared from the plasma by 8 hours (Figure 1).
Figure 1. Plasma distribution of D-4F after oral administration in LDL receptor-null mice. LDL receptor-null mice (n=5 per group) were given 22 μg of N-methyl anthranilyl-D-4F (fluorescent D-4F) by stomach tube. The mice were bled 1, 2, or 8 hours later (A, B, and C, respectively), and plasma was separated by fast protein liquid (FPLC) and analyzed for cholesterol (thin line) and D-4F (thick line).
Addition of D-4F to apoE-null mouse plasma in vitro rapidly caused the movement of apoA-I from -migrating HDL to ?-migrating HDL.20 Twenty minutes after 500 μg of D-4F was given orally as a bolus by stomach tube to apoE-null mice, the plasma contained 138 to 322 ng of D-4F/mL and 85% was associated with HDL.20 Twenty minutes after 500 μg of D-4F was given orally as a bolus by stomach tube to apoE-null mice, small cholesterol-containing particles 7 to 8 nm in size with pre-? mobility and enriched in apoA-I and paraoxonase activity were found in plasma.20 Before the administration of D-4F, mature HDL and fast protein liquid fractions containing the cholesterol-containing particles were pro-inflammatory. Twenty minutes after oral D-4F, both HDL and the cholesterol-containing particles became antiinflammatory and HDL-mediated cholesterol efflux from macrophages in vitro increased.20 Oral D-4F also promoted reverse cholesterol transport from intraperitoneally injected cholesterol-loaded macrophages in vivo.20 After oral D-4F, lipoprotein lipid hydroperoxides decreased in very-low-density lipoprotein/intermediate density lipoprotein, LDL, and in mature HDL, but increased in pre-? HDL.20 Both lipid hydroperoxide content and paraoxonase activity increased in pre-? HDL after oral D-4F. Before D-4F, the pre-? HDL fractions from apoE-null mice were very pro-inflammatory. However, after oral D-4F, despite the increase in lipid hydroperoxide content, the pre-? HDL fractions were antiinflammatory when assayed in the human artery wall coculture system.20 Thus, whereas oral D-4F caused the movement of lipid hydroperoxides to pre-? HDL, the increase in antioxidant enzyme activities such as paraoxonase must have more than compensated to render the pre-? HDL antiinflammatory.
Oral D-4F also caused the formation of pre-? HDL in wild-type C57BL/6J mice on a chow diet and decreased lipid hydroperoxides in HDL while increasing the content of lipid hydroperoxides in pre-? HDL, indicating that the absence of apoE was not required for these actions of D-4F.6
Oral D-4F Increases Paraoxonase Activity and Causes the Formation of Pre-? HDL in Monkeys
Oral administration of D-4F resulted in an increase in paraoxonase activity in monkeys (Figure 2A) and also caused the formation of pre-? HDL (Figure 2B). As previously reported, oral D-4F reduced lipid hydroperoxide levels, converted HDL from pro-inflammatory to antiinflammatory, and enhanced HDL-mediated cholesterol efflux in monkeys.21 Thus, oral D-4F appears to act similarly in mice and monkeys.
Figure 2. Oral D-4F increases paraoxonase activity and causes the formation of pre-? HDL in monkeys. After approval by the UCLA Animal Research Committee blood was obtained from 2 male (4 kg) and 2 female (4 kg) cynomolgus monkeys with conscious sedation (ketamine 10 mg/kg, intramuscularly) before (time zero) and after the administration of a banana shake (by gavage) containing 40 mg of D-4F. A, Paraoxonase activity. B, FPLC fractions immediately after the main peak of HDL analyzed by 2-D gels and probed for apoA-I by Western blot.
Adding Nanomolar Amounts of D-4F to Normal Human Plasma Causes the Formation of Pre-? HDL, Reduces Lipoprotein Lipid Hydroperoxide Content, Increases Paraoxonase Activity, and Converts Proinflammatory HDL to Antiinflammatory
The plasma concentration of D-4F after oral administration of a single dose of 500 μg to apoE-null mice was in the 100 to 300 ng/mL range.20 Adding 250 ng/mL of D-4F to normal human plasma caused the movement of apoA-I to smaller particles (Figure 3A), which had pre-? mobility (Figure 3B). Adding 250 ng/mL of D-4F to human plasma reduced HDL lipid hydroperoxide content (Figure 4A) and increased paraoxonase activity (Figure 4B). Adding 250 ng/mL of D-4F converted pro-inflammatory HDL to antiinflammatory (Figure 5). Thus, adding D-4F to human plasma in vitro produced results similar to that seen in mice and monkeys in vivo.
Figure 3. D-4F (but not scrambled D-4F) causes the movement of apoA-I from larger to smaller particles. A, Normal human plasma was incubated with D-4F or scrambled D-4F at 0.25, 2.5, or 25 μg/mL for 30 minutes at 37°C with gentle mixing. The plasma was then subjected to native PAGE and probed for apoA-I by Western blotting. The experiment shown is representative of 2 of 2 experiments. B, Normal human plasma was incubated with 250 ng/mL of D-4F or the vehicle containing D-4F (Sham) for 30 minutes at 37°C with gentle mixing. The plasma was then subjected to 2-dimensional agarose/native PAGE, and probed for apoA-I by Western blotting. The experiment shown is representative of 2 of 2 experiments.
Figure 4. D-4F decreases HDL lipid hydroperoxides and increases paraoxonase activity. A, Plasma from 2 patients from the study described22 were incubated with 250 ng of D-4F/mL (+D-4F) or the same volume of vehicle in which the D-4F was added (HDL Sham) at 37°C for 30 minutes in a Millipore Amicon Ultra Centrifugal Device with a 100-kDa molecular weight cutoff filter (catalog number UFC 810096), followed by centrifugation at 2100g. After centrifugation, the supernatant was separated by FPLC and analyzed for HDL lipid hydroperoxides (ng HDL LOOH). Patient 1’s HDL became antiinflammatory after treatment with simvastatin.22 Patient 2’s HDL remained pro-inflammatory despite simvastatin treatment.22 *P<0.003. The experiment shown is representative of 3 of 3 experiments. B, D-4F or scrambled D-4F (250 ng/mL) were added to normal human plasma and incubated at 37°C for 30 minutes as described in (A). After centrifugation, the supernatant was fractionated by FPLC and paraoxonase activity was determined in the fractions. The area under the curve after D-4F treatment was 190% of the area under the curve after treatment with scrambled D-4F, which was not different from plasma treated without additions (data not shown). The experiment shown is representative of 6 of 6 experiments.
Figure 5. D-4F converts pro-inflammatory HDL to antiinflammatory. Plasma from a patient with pro-inflammatory HDL that was not on a statin (patient 3), and plasma from patients with pro-inflammatory HDL despite 6 weeks of pravastatin treatment22 (patients 4 5) were incubated with 250 ng of D-4F or with vehicle at 37°C for 30 minutes and centrifuged as described in (A). The supernatants were removed and HDL was isolated by FPLC. Normal human LDL was not (no addition) or was (std LDL) added to human aortic endothelial cells at 100 μg/mL cholesterol with normal human HDL (+control HDL) at 50 μg/mL cholesterol or was added with HDL that was treated with vehicle alone at 50 μg/mL cholesterol (SHAM) or with HDL at 50 μg/mL cholesterol that had been treated with 250 ng/mL of D-4F (D-4F). After 8 hours at 37°C, monocyte chemotactic activity was determined. *P<0.001. The experiment shown is representative of 4 of 4 experiments.
These results raise 2 questions. First, if apoA-I concentrations in normal human plasma are on the order of 1 mg/mL (1000 μg/mL or 1000000 ng/mL), how could adding 25 μg/mL or 2.5 μg/mL or 250 ng/mL of D-4F cause the movement of apoA-I from larger to smaller particles, as occurred in Figure 3A and 3B? The answer probably relates to the ability of 4F to interact with lipids. As noted, 4F, 3F-1, and 3F-2 have the ability to separate cholesterol from phospholipids in membranes and to penetrate into those membranes.15–17 Mature -migrating HDL particles are constantly changing because some portion of apoA-I is always leaving the large HDL particle, generating smaller lipid-poor apoA-I particles. The data reviewed here suggest that apoA-I mimetic peptides such as D-4F can dramatically accelerate this process, probably as a result of their ability to bind to HDL and separate cholesterol from phospholipids, which may facilitate the movement of apoA-I to smaller particles.
Second, how could D-4F decrease HDL lipid hydroperoxide content and increase paraoxonase activity in vitro, as shown in Figure 4A and 4B? Again the physical-chemical properties of 4F, 3F-1, and 3F-2 may provide the explanation. Probably because of the structural characteristics of these peptides when they form lipid-peptide complexes, they allow some water to penetrate the complexes, which may facilitate the ability of the complexes to effectively sequester lipid hydroperoxides.15–17 Forte et al have shown that a number of enzymes including paraoxonase are reversibly inhibited by lipid hydroperoxides.23 The ability of peptides such as 4F to effectively sequester lipid hydroperoxides may lead to the activation of enzymes such as paraoxonase. Navab et al have shown that activated paraoxonase can destroy such lipid hydroperoxides.12,13 Thus, the effective sequestration of a very small quantity of lipid hydroperoxides by peptides such as 4F may activate enzymes such as paraoxonase, leading to further lipid hydroperoxide destruction and providing a positive feedback loop. The physical-chemical characteristics of the peptides that determine their structure after binding lipids, and hence their interaction with the lipid acyl chains of membranes, could lead to a series of events that would appear to be catalytic, whereas in fact the peptides themselves are not catalysts by the usual definition.
Summary
ApoA-I mimetic peptides with similar amino acid compositions may have very subtle differences in their physical-chemical properties that result in very different biologic activities. Physical-chemical properties and the ability of various class A amphipathic helical peptides to activate 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. The antiinflammatory properties of different class A amphipathic helical peptides appear to depend 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.
Acknowledgments
This work was supported in part by USPHS grants HL 30568, HL 34343, the Laubisch, Castera, and M.K. Grey Funds at UCLA.
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Epand RM, Epand RF, Sayer BG, Datta G, Chaddha M, Anantharamaiah GM. Two homologous apolipoprotein AI mimetic peptides. Relationship between membrane interactions and biological activity. J Biol Chem. 2004; 279: 51404–51414.
Garber DW, Datta G, Chaddha M, Palgunachari MN, Hama SY, Navab M, Fogelman AM, Segrest JP, Anantharamaiah GM. A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis. J Lipid Res. 2001; 42: 545–552.
Navab M, Anantharamaiah GM, Hama S, Garber DW, Chaddha M, Hough G, Lallone R, Fogelman AM. Oral administration of an apoA-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol. Circulation. 2002; 105: 290–292.
Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Wagner AC, Frank JS, Datta G, Garber D, Fogelman AM. Oral D-4F causes formation of pre-? High-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice. Circulation. 2004; 109: r120–r125.
Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC, Vahabzadeh K, Hama S, Hough G, Kamranpour N, Berliner JA, Lusis AJ, Fogelman AM. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J Lipid Res. 2004; 45: 993–1007.
Ansell BJ, Navab M, Hama S, Kamranpour N, Fonarow G, Hough G, Rahmani S, Mottahedeh R, Dave R, Reddy ST, Fogelman AM. Inflammatory/antiinflammatory properties of high-density lipoproteins distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment. Circulation. 2003; 108: 2751–2756.
Forte TM, Subbanagounder G, Berliner JA, Blanche PJ, Clermont AO, Jia Z, Oda MN, Krauss RM, Bielicki JK. Altered activities of antiatherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice. J Lipid Res. 2002; 43: 477–485.(Mohamad Navab; G.M. Anant)
Correspondence to Dr Mohamad Navab, Room 47-123 CHS, Division of Cardiology, Department of Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte, Avenue, Los Angeles, CA 90095-1679. E-mail mnavab@mednet.ucla.edu
Series Editor: Daniel J. Rader
ATVB In Focus Novel Approaches to the Treatment of Dyslipidemia
Previous Brief Reviews in this Series:
?Chen HC, Farese RV Jr. Inhibition of tgriglyceride synthesis as a treatment strategy for obestiy: lessons from DGAT1-deficient mice. 2005;25:482–486.
?Zalewski A. Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. 2005;25:923–931.
?Rudel LL, Lee RG, Parini P. ACTA2 is a target for treatment of coronary heart disease associated with hypercholesterolemia. 2005;25:1112–1118.
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 pro-inflammatory 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.
The antiinflammatory properties of different class A amphipathic helical peptides such as D-4F depend on subtle differences in the configuration of the hydrophobic face of the peptides that determine the ability of the peptides to sequester inflammatory lipids.
Key Words: HDL ? apoA-I ? peptide mimetics ? D-4F ? atherosclerosis ? inflammation
Introduction
The main protein of high-density lipoprotein (HDL), apolipoprotein A-I (apoA-I), contains 243 amino acids. The ability of apoA-I to remove phospholipids and cholesterol via the ABCA1 pathway1,2 is thought to be a major factor in preventing atherosclerosis. Despite recent evidence that other pathways such as ABCG1 may also be important in HDL-mediated cholesterol efflux,3,4 the importance of apoA-I in providing protection against atherosclerosis in animal models and in humans has been well-documented.5 This review focuses on the search for apoA-I mimetic peptides as therapeutic agents based on physical-chemical and biological properties. As is evident from the review presented here, the physical-chemical properties of these peptides are important but by themselves are not sufficient to predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides is more predictive. Subtle differences in the configuration of the hydrophobic face of apoA-I mimetic peptides determines the ability of the peptides to sequester inflammatory lipids and this seems to be the major determinant of their antiinflammatory properties. 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.
See page 1305
Physical-Chemical Properties of ApoA-I Mimetic Peptides as Predictors of Biologic Activity In Vivo
The search for peptides smaller than apoA-I but with many of the lipid-binding properties of apoA-I led to the synthesis of an 18-amino acid peptide known as 18A.6 The sequence of 18A (D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F) does not have any sequence homology to apoA-I. However, this sequence has the capacity to form a class A amphipathic helix similar to those found in apoA-I and mimics many of the lipid binding properties of apoA-I.7
Modifying the terminal charges of 18A by forming Ac-18A-NH2 (also known as 2F because of the 2 phenylalanine residues at positions 6 and 18) increased lipid affinity.8,9 Many 2F peptide variants have been synthesized, as shown in the Table. The Table also shows the biologic activity of these peptides in cocultures of human artery wall cells and in mouse models of atherosclerosis.10
ApoA-I Mimetic Peptides With Differing Numbers of Phenylalanine Residues Placed at Different Positions
Based on their physical properties, these peptides can be separated into 2 groups. The first group includes the original peptide (2F) containing 2 phenylalanine residues on the hydrophobic face; 2 peptides with 3 phenylalanine residues (3F3 and 3F14); and a peptide with 4 phenylalanine residues on the hydrophobic (4F).
The second group consists of a peptide with 5 phenylalanine residues on the hydrophobic face (5F); a peptide with 6 phenylalanine residues on the hydrophobic face (6F); and a peptide with 7 phenylalanine residues on the hydrophobic face (7F).10
The calculated hydrophobicity of the peptides increased as the number of phenylalanine residues on the nonpolar face increased. The increase in hydrophobicity was reflected in the theoretical lipid affinity ().11 The () value increased gradually from 2F to 4F (from 13.03 to 14.59), and then suddenly increased to 19.07 for 5F.10 From 5F to 7F, there was again a gradual increase in () values.10 The retention time on a C18 reversed-phase high-performance liquid chromatography column, solubility of the peptides, and their ability to penetrate an egg phosphatidylcholine monolayer showed a trend consistent with the theoretical lipid affinity values.10
Circular dichroism data for the peptides in phosphate-buffered saline (PBS) and in the presence of dimyristoyl phosphatidylcholine indicated that some of the peptides had a significant increase in the percent helicity on interacting with dimyristoyl phosphatidylcholine (eg, 2F, 3F3, 3F14, 5F, and 7F), whereas others did not (eg, 4F and 6F).10 The binding of the peptides to dimyristoyl phosphatidylcholine appeared to be similar as determined by differential scanning calorimetry.10 The solubility of the peptides in PBS and their ability to interact with phospholipid monolayers could be divided into 2 groups: 2F, 3F3, 3F14, and 4F had solubility in PBS of >2.0, 1.25, 1.45, and 1.30 mg/mL, respectively, and monolayer exclusion pressures of 38, 38, 39, and 40 dyne/cm, respectively.10 The solubility in PBS for 5F, 6F, and 7F was 0.10, 0.30, and 0.10 mg/mL, respectively, and their monolayer exclusion pressures were 45, 46, and 45 dyne/cm, respectively.10 Thus, in choosing a particular peptide, one must consider the balance between solubility in an aqueous environment and the ability to interact with phospholipid monolayers.
ApoA-I was not able to clarify egg phosphatidylcholine multilamellar vesicles, but all of the peptide analogs were able to do so to different extents. 4F appeared to be most efficient in solubilizing the phospholipid exhibiting kinetics similar to Triton X-100, suggesting that 4F has an optimal hydrophobicity to interact with phospholipids (ie, hydrophobic peptide-peptide interactions favoring self-association were minimized in 4F, thus promoting peptide-lipid interactions).10
All of the peptides activated lecithin cholesterol acyltransferase (LCAT) activity less than apoA-I.10 Moreover, there was no significant difference in LCAT activity between peptides that were found to be biologically inactive (eg, 2F, 3F3, and 3F14) and the biologically active10 peptide 4F.
Thus, all of these physical-chemical parameters and LCAT activity failed to predict peptides that would be found to be biologically inactive versus those that were biologically active.
The Use of Cultured Human Artery Wall Cells as an Assay to Predict In Vivo Efficacy of ApoA-I Mimetic Peptides
It has long been known that circulating low-density lipoprotein (LDL) contains a small amount of lipid hydroperoxides.12 When freshly isolated LDL is added to human artery wall cocultures, it is trapped in the subendothelial space and additional lipid hydroperoxides produced by the artery wall cells are partitioned into the trapped LDL.12,13 When a critical threshold of lipid hydroperoxides within the trapped LDL is reached, arachidonic acid-containing phospholipids in the LDL are oxidized and cause the artery wall cells to produce monocyte chemotactic activity (MCA), which is largely caused by the production of monocyte chemoattractant-1.12,13 When human apoA-I was added to the human artery wall cocultures in a pre-incubation and removed before the addition of human LDL, there was a marked reduction in LDL-induced MCA. However, when human apoA-I was added to these same cocultures together with the LDL (a coincubation), there was no reduction in LDL-induced MCA.13 It was concluded that human apoA-I binds lipid hydroperoxides in such a manner that the lipid hydroperoxides can still participate in the formation of oxidized phospholipids if the apoA-I-lipid hydroperoxide complexes are not removed.13 In contrast, some of the peptide mimetics of apoA-I were effective in inhibiting LDL-induced MCA even in a coincubation, suggesting that the peptides effectively sequestered the lipid hydroperoxides10 and thus acted more like apolipoprotein J (which was shown to be active in a coincubation14) than like apoA-I in this regard.
As noted, the peptide 18A showed enhanced lipid binding when blocking groups were added to produce Ac-18A-NH2. Despite the enhanced lipid binding, Ac-18A-NH2 was relatively weak in its ability to prevent LDL-induced MCA in human artery wall cell cocultures and Ac-18A-NH2 failed to inhibit diet-induced atherosclerosis in mice.10
The ability of the peptides to inhibit LDL-induced MCA in the human artery wall coculture could not be predicted by the differences in the physical-chemical properties of the peptides or the ability of the peptides to activate LCAT.10 When the peptides were added to human artery wall cocultures together with human LDL,10 peptides 3F3 and 3F14 were ineffective in preventing MCA, 2F was weakly effective, and 4F, 5F, and 6F were very effective, whereas 7F was poorly effective (even less effective than 2F). As shown in the Table, the activity in the human artery wall cell cultures were in agreement with the results in mouse models of atherosclerosis, ie, 2F was not effective in vivo, whereas 4F and 5F were quite effective in vivo.
Comparison of 4 Peptides That Are Identical Except for the Position of Their 3 Phenylalanine Residues on the Hydrophobic Face With Peptide 4F
A comparison of 4 peptides, which were identical with each other except for the positions of the phenylalanine residues on the hydrophobic face, was made to further determine the relationship between structure and function of these peptides. Peptides 3F-1, 3F-2, 3F3, and 3F14 were compared with each other and with the 4F peptide15 (Table). The 3F-1, 3F-2, and 4F peptides scavenged lipid hydroperoxides from LDL better than 3F3 or 3F14. Similarly the 3F-1, 3F-2, and 4F peptides were significantly better in preventing oxidized phospholipids from inducing human artery wall cells to produce MCA compared with 3F3 or 3F14. In the presence of lipid, all of these peptides had similar secondary structures and similar hydrophobicities.15 These peptides inserted into monolayers of egg phosphatidylcholine with similar avidity as shown with surface pressure measurements and into bilayers as evidenced by the fluorescence emission from tryptophan. Fluorescence studies suggested that although the peptides bound phospholipids similarly, the tryptophan residue in peptides 4F, 3F-1, and 3F-2 was less motionally restricted than in 3F3 and 3F14. Additionally, fluorescence spectroscopy studies with a fluorescent phospholipid (DPH-PC) revealed increased quenching of DPH-PC, indicating that the active peptides, particularly 4F, allowed a greater penetration of water molecules into the hydrophobic milieu of the membrane.15 Based on the comparison of 4F to the inactive peptides 3F3 and 3F14, it was postulated that the presence of water in the milieu of the hydrophobic region of the peptide-lipid complex would determine the amount of lipid hydroperoxides and oxidized phospholipids transferred from LDL to the sequestered environment of the peptide-containing particles, which in turn would determine the availability of these lipids to stimulate the artery wall cells to produce MCA.15 It should be noted that the differences between 3F-1 and 3F-2 (biologically active) and 3F3 and 3F14 (biologically inactive) were not significant in the quenching of DPH-PC.15 Thus, the measurement of the physical-chemical properties of these peptides leading to the quenching of DPH-PC by 3F-1 and 3F-2 versus 3F3 and 3F14 could not predict their biologic activity.
Other in vitro studies of 4F revealed that it promoted the separation of cholesterol from phospholipid.16 4F penetrated into membranes of pure phosphatidylcholine in the absence of cholesterol better than into bilayers of phosphatidylcholine and cholesterol.16 The circular dichroism spectrum of 4F in buffer indicated that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remained helical over a larger concentration range. On heating, the peptide underwent a thermal transition. Cholesterol had little effect on the secondary structure of the peptide; however, increased tryptophan emission intensity in the absence of cholesterol indicated a deeper penetration of the helix on removal of cholesterol from the membrane. It was hypothesized that the results with these model systems demonstrated changes in peptide-lipid interactions that may relate to the observed biological properties of this peptide.16
Additional studies comparing 3F-2 to 3F14 revealed that both peptides promoted the segregation of cholesterol in membranes containing phosphatidylcholine and cholesterol, but 3F-2 exhibited greater selectivity for partitioning into cholesterol-depleted regions of membrane.17 Magic angle spinning/nuclear magnetic resonance indicated that the aromatic residues of 3F-2 were stacked in the presence of lipid. The aromatic side chains of 3F-2 also penetrated more deeply into membranes of phosphatidylcholine with cholesterol compared with 3F.14 Using the fluorescent probe, 1,3-dipyreneylpropane, it was determined that 3F-2 had a greater effect in altering the hydrocarbon region of the membrane. Based on molecular models in previous studies15 and in these studies,17 it was concluded that the wedge-shaped cross-sectional area of 3F14 has only a minimal effect on the lipid acyl chain packing of membranes, whereas the cylindrical cross sectional area of 3F-2 (and also 4F) causes greater acyl chain perturbations, which facilitate the entry of molecules such as water and lipid hydroperoxides into the hydrophobic milieu of the complex.17 It was postulated that these properties of 3F-2 and 4F allow them to effectively sequester inflammatory lipids, whereas 3F14 is unable to do so. Consistent with these findings, the peptide 3F14 is not antiinflammatory in human artery wall cell cultures, whereas 3F-2 and 4F are highly antiinflammatory.10,15 It should be noted that without previous knowledge of the antiinflammatory properties of 3F-2 and the lack of such properties by 3F14 in human artery wall cell cultures, the differences found in these sophisticated physical-chemical studies17 would likely have been too subtle to have predicted which of these 2 peptides would be biologically active and which would not.
Injection of 5F Inhibits Diet-Induced Atherosclerosis
Garber et al18 provided the first evidence that an apoA-I mimetic peptide could inhibit atherosclerosis in vivo. The peptide 5F was administered by injection at a dose of 20 μg/d for 16 weeks in C57BL/6J mice fed an atherogenic diet. Control mice received either mouse apoA-I at 50 μg/d or PBS. Total plasma cholesterol levels and lipoprotein profiles were not significantly different except that the mice receiving 5F or mouse apoA-I had lower HDL cholesterol when calculated as a percent of total cholesterol.18 No toxicity or production of antibodies to the injected materials was observed. HDL isolated from the mice injected with PBS failed to decrease LDL-induced MCA in the human artery wall coculture, HDL from mice injected with mouse apoA-I was only weakly antiinflammatory, and HDL from mice that were injected with 5F or human apoA-I was significantly antiinflammatory (ie, reduced LDL-induced MCA as much as normal human HDL).18 Atherosclerosis lesion area was significantly reduced in the 5F-treated mice but not in mice receiving mouse apoA-I.18 Thus, after decades of research on apoA-I mimetic peptides,6 the first demonstration in vivo that such peptides could actually inhibit atherosclerosis was achieved.18
Oral D-4F Inhibits Atherosclerosis in Apolipoprotein E-Null and LDL Receptor-Null Mice
In a search for peptides that could be administered orally, it was recalled that mammalian enzymes recognize peptides and proteins synthesized from L-amino acids but rarely recognize peptides synthesized from D-amino acids. When 4F was synthesized from L-amino acids and given orally to mice, it was rapidly degraded.20 However, when 4F was synthesized from D-amino acids and was orally administered, it was found intact in the circulation.20 The oral administration of D-4F rendered HDL from LDL receptor-null mice on a Western diet and HDL from apolipoprotein E (apoE)-null mice on a chow diet antiinflammatory. Oral D-4F also reduced atherosclerotic lesions in these mice by 79% and 75%, respectively, without significantly altering plasma lipid levels.19
One hour after orally administering D-4F to LDL receptor-null mice, the peptide was initially found in fast protein liquid fractions where pre-? HDL would be expected, as well as in those fractions where mature -migrating HDL would be expected. One hour later, the peptide was only seen in the latter fractions, and the peptide was virtually cleared from the plasma by 8 hours (Figure 1).
Figure 1. Plasma distribution of D-4F after oral administration in LDL receptor-null mice. LDL receptor-null mice (n=5 per group) were given 22 μg of N-methyl anthranilyl-D-4F (fluorescent D-4F) by stomach tube. The mice were bled 1, 2, or 8 hours later (A, B, and C, respectively), and plasma was separated by fast protein liquid (FPLC) and analyzed for cholesterol (thin line) and D-4F (thick line).
Addition of D-4F to apoE-null mouse plasma in vitro rapidly caused the movement of apoA-I from -migrating HDL to ?-migrating HDL.20 Twenty minutes after 500 μg of D-4F was given orally as a bolus by stomach tube to apoE-null mice, the plasma contained 138 to 322 ng of D-4F/mL and 85% was associated with HDL.20 Twenty minutes after 500 μg of D-4F was given orally as a bolus by stomach tube to apoE-null mice, small cholesterol-containing particles 7 to 8 nm in size with pre-? mobility and enriched in apoA-I and paraoxonase activity were found in plasma.20 Before the administration of D-4F, mature HDL and fast protein liquid fractions containing the cholesterol-containing particles were pro-inflammatory. Twenty minutes after oral D-4F, both HDL and the cholesterol-containing particles became antiinflammatory and HDL-mediated cholesterol efflux from macrophages in vitro increased.20 Oral D-4F also promoted reverse cholesterol transport from intraperitoneally injected cholesterol-loaded macrophages in vivo.20 After oral D-4F, lipoprotein lipid hydroperoxides decreased in very-low-density lipoprotein/intermediate density lipoprotein, LDL, and in mature HDL, but increased in pre-? HDL.20 Both lipid hydroperoxide content and paraoxonase activity increased in pre-? HDL after oral D-4F. Before D-4F, the pre-? HDL fractions from apoE-null mice were very pro-inflammatory. However, after oral D-4F, despite the increase in lipid hydroperoxide content, the pre-? HDL fractions were antiinflammatory when assayed in the human artery wall coculture system.20 Thus, whereas oral D-4F caused the movement of lipid hydroperoxides to pre-? HDL, the increase in antioxidant enzyme activities such as paraoxonase must have more than compensated to render the pre-? HDL antiinflammatory.
Oral D-4F also caused the formation of pre-? HDL in wild-type C57BL/6J mice on a chow diet and decreased lipid hydroperoxides in HDL while increasing the content of lipid hydroperoxides in pre-? HDL, indicating that the absence of apoE was not required for these actions of D-4F.6
Oral D-4F Increases Paraoxonase Activity and Causes the Formation of Pre-? HDL in Monkeys
Oral administration of D-4F resulted in an increase in paraoxonase activity in monkeys (Figure 2A) and also caused the formation of pre-? HDL (Figure 2B). As previously reported, oral D-4F reduced lipid hydroperoxide levels, converted HDL from pro-inflammatory to antiinflammatory, and enhanced HDL-mediated cholesterol efflux in monkeys.21 Thus, oral D-4F appears to act similarly in mice and monkeys.
Figure 2. Oral D-4F increases paraoxonase activity and causes the formation of pre-? HDL in monkeys. After approval by the UCLA Animal Research Committee blood was obtained from 2 male (4 kg) and 2 female (4 kg) cynomolgus monkeys with conscious sedation (ketamine 10 mg/kg, intramuscularly) before (time zero) and after the administration of a banana shake (by gavage) containing 40 mg of D-4F. A, Paraoxonase activity. B, FPLC fractions immediately after the main peak of HDL analyzed by 2-D gels and probed for apoA-I by Western blot.
Adding Nanomolar Amounts of D-4F to Normal Human Plasma Causes the Formation of Pre-? HDL, Reduces Lipoprotein Lipid Hydroperoxide Content, Increases Paraoxonase Activity, and Converts Proinflammatory HDL to Antiinflammatory
The plasma concentration of D-4F after oral administration of a single dose of 500 μg to apoE-null mice was in the 100 to 300 ng/mL range.20 Adding 250 ng/mL of D-4F to normal human plasma caused the movement of apoA-I to smaller particles (Figure 3A), which had pre-? mobility (Figure 3B). Adding 250 ng/mL of D-4F to human plasma reduced HDL lipid hydroperoxide content (Figure 4A) and increased paraoxonase activity (Figure 4B). Adding 250 ng/mL of D-4F converted pro-inflammatory HDL to antiinflammatory (Figure 5). Thus, adding D-4F to human plasma in vitro produced results similar to that seen in mice and monkeys in vivo.
Figure 3. D-4F (but not scrambled D-4F) causes the movement of apoA-I from larger to smaller particles. A, Normal human plasma was incubated with D-4F or scrambled D-4F at 0.25, 2.5, or 25 μg/mL for 30 minutes at 37°C with gentle mixing. The plasma was then subjected to native PAGE and probed for apoA-I by Western blotting. The experiment shown is representative of 2 of 2 experiments. B, Normal human plasma was incubated with 250 ng/mL of D-4F or the vehicle containing D-4F (Sham) for 30 minutes at 37°C with gentle mixing. The plasma was then subjected to 2-dimensional agarose/native PAGE, and probed for apoA-I by Western blotting. The experiment shown is representative of 2 of 2 experiments.
Figure 4. D-4F decreases HDL lipid hydroperoxides and increases paraoxonase activity. A, Plasma from 2 patients from the study described22 were incubated with 250 ng of D-4F/mL (+D-4F) or the same volume of vehicle in which the D-4F was added (HDL Sham) at 37°C for 30 minutes in a Millipore Amicon Ultra Centrifugal Device with a 100-kDa molecular weight cutoff filter (catalog number UFC 810096), followed by centrifugation at 2100g. After centrifugation, the supernatant was separated by FPLC and analyzed for HDL lipid hydroperoxides (ng HDL LOOH). Patient 1’s HDL became antiinflammatory after treatment with simvastatin.22 Patient 2’s HDL remained pro-inflammatory despite simvastatin treatment.22 *P<0.003. The experiment shown is representative of 3 of 3 experiments. B, D-4F or scrambled D-4F (250 ng/mL) were added to normal human plasma and incubated at 37°C for 30 minutes as described in (A). After centrifugation, the supernatant was fractionated by FPLC and paraoxonase activity was determined in the fractions. The area under the curve after D-4F treatment was 190% of the area under the curve after treatment with scrambled D-4F, which was not different from plasma treated without additions (data not shown). The experiment shown is representative of 6 of 6 experiments.
Figure 5. D-4F converts pro-inflammatory HDL to antiinflammatory. Plasma from a patient with pro-inflammatory HDL that was not on a statin (patient 3), and plasma from patients with pro-inflammatory HDL despite 6 weeks of pravastatin treatment22 (patients 4 5) were incubated with 250 ng of D-4F or with vehicle at 37°C for 30 minutes and centrifuged as described in (A). The supernatants were removed and HDL was isolated by FPLC. Normal human LDL was not (no addition) or was (std LDL) added to human aortic endothelial cells at 100 μg/mL cholesterol with normal human HDL (+control HDL) at 50 μg/mL cholesterol or was added with HDL that was treated with vehicle alone at 50 μg/mL cholesterol (SHAM) or with HDL at 50 μg/mL cholesterol that had been treated with 250 ng/mL of D-4F (D-4F). After 8 hours at 37°C, monocyte chemotactic activity was determined. *P<0.001. The experiment shown is representative of 4 of 4 experiments.
These results raise 2 questions. First, if apoA-I concentrations in normal human plasma are on the order of 1 mg/mL (1000 μg/mL or 1000000 ng/mL), how could adding 25 μg/mL or 2.5 μg/mL or 250 ng/mL of D-4F cause the movement of apoA-I from larger to smaller particles, as occurred in Figure 3A and 3B? The answer probably relates to the ability of 4F to interact with lipids. As noted, 4F, 3F-1, and 3F-2 have the ability to separate cholesterol from phospholipids in membranes and to penetrate into those membranes.15–17 Mature -migrating HDL particles are constantly changing because some portion of apoA-I is always leaving the large HDL particle, generating smaller lipid-poor apoA-I particles. The data reviewed here suggest that apoA-I mimetic peptides such as D-4F can dramatically accelerate this process, probably as a result of their ability to bind to HDL and separate cholesterol from phospholipids, which may facilitate the movement of apoA-I to smaller particles.
Second, how could D-4F decrease HDL lipid hydroperoxide content and increase paraoxonase activity in vitro, as shown in Figure 4A and 4B? Again the physical-chemical properties of 4F, 3F-1, and 3F-2 may provide the explanation. Probably because of the structural characteristics of these peptides when they form lipid-peptide complexes, they allow some water to penetrate the complexes, which may facilitate the ability of the complexes to effectively sequester lipid hydroperoxides.15–17 Forte et al have shown that a number of enzymes including paraoxonase are reversibly inhibited by lipid hydroperoxides.23 The ability of peptides such as 4F to effectively sequester lipid hydroperoxides may lead to the activation of enzymes such as paraoxonase. Navab et al have shown that activated paraoxonase can destroy such lipid hydroperoxides.12,13 Thus, the effective sequestration of a very small quantity of lipid hydroperoxides by peptides such as 4F may activate enzymes such as paraoxonase, leading to further lipid hydroperoxide destruction and providing a positive feedback loop. The physical-chemical characteristics of the peptides that determine their structure after binding lipids, and hence their interaction with the lipid acyl chains of membranes, could lead to a series of events that would appear to be catalytic, whereas in fact the peptides themselves are not catalysts by the usual definition.
Summary
ApoA-I mimetic peptides with similar amino acid compositions may have very subtle differences in their physical-chemical properties that result in very different biologic activities. Physical-chemical properties and the ability of various class A amphipathic helical peptides to activate 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. The antiinflammatory properties of different class A amphipathic helical peptides appear to depend 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.
Acknowledgments
This work was supported in part by USPHS grants HL 30568, HL 34343, the Laubisch, Castera, and M.K. Grey Funds at UCLA.
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