Oral Small Peptides Render HDL Antiinflammatory in Mice and Monkeys and Reduce Atherosclerosis in ApoE Null Mice
A peptide containing only 4 amino acid residues (KRES) that is too small to form an amphipathic helix, reduced lipoprotein lipid hydroperoxides (LOOH), increased paraoxonase activity, increased plasma HDL-cholesterol levels, rendered HDL antiinflammatory, and reduced atherosclerosis in apoE null mice. KRES was orally effective when synthesized from either L or D-amino acids suggesting that peptide-protein interactions were not required. Remarkably, changing the order of 2 amino acids (from KRES to KERS) resulted in the loss of all biologic activity. Solubility in ethyl acetate and interaction with lipids, as determined by differential scanning calorimetry, indicated significant differences between KRES and KERS. Negative stain electron microscopy showed that KRES formed organized peptide-lipid structures whereas KERS did not. Another tetrapeptide FREL shared many of the physical-chemical properties of KRES and was biologically active in mice and monkeys when synthesized from either L- or D-amino acids. After oral administration KRES and FREL were found associated with HDL whereas KERS was not. We conclude that the ability of peptides to interact with lipids, remove LOOH and activate antioxidant enzymes associated with HDL determines their antiinflammatory and antiatherogenic properties regardless of their ability to form amphipathic helixes.
There has been a long search for peptides smaller than apolipoprotein A-I (apoA-I), but with many of the properties of apoA-I. This search initially led to the synthesis of the peptide 18A.1 18A does not have sequence homology to apoA-I but forms a class A amphipathic helix similar to those found in apoA-I with some of the same lipid binding properties.2
Addition of terminal blocking groups (Ac-18A-NH2) resulted in increased lipid affinity.3 However, Ac-18A-NH2 was relatively weak in preventing LDL-induced monocyte chemotactic activity (MCA) in human artery wall cell cultures and failed to inhibit diet-induced atherosclerosis in mice.4 A series of peptides with increasing phenylalanine residues on the hydrophobic domain were synthesized and 4F was found to be most ideal based on solubility, lipid binding, and biologic activity.4
In addition to its ability to bind cholesterol and phospholipids, apoA-I was found to bind lipid hydroperoxides (LOOH).5 Circulating LDL always contains a small amount of LOOH.5 When freshly isolated LDL is added to human artery wall cocultures, it is trapped in the subendothelial space and additional LOOH produced by the cells partition into the trapped LDL.5,6 When a critical threshold of LOOH within the trapped LDL is reached, arachidonic acid-containing phospholipids in the LDL are oxidized and cause the artery wall cells to produce MCA.5,6 When human apoA-I was added to human artery wall cocultures in preincubation and removed before the addition of LDL, there was a marked reduction in LDL-induced MCA. However, when human apoA-I was added to the cocultures with the LDL (a coincubation) there was no reduction in LDL-induced MCA6 indicating that human apoA-I bound LOOH in a manner that allowed the formation of oxidized phospholipids unless the apoA-I-LOOH complex was removed.6 In contrast some of the peptide mimetics of apoA-I inhibited LDL-induced MCA even in a coincubation, suggesting that the mimetics more effectively sequestered LOOH.4
Oral L-4F (4F synthesized from L-amino acids) was rapidly degraded by proteolytic enzymes and was not biologically active.7 Oral D-4F (4F synthesized from all D-amino acids) was not degraded and dramatically reduced atherosclerosis in mice.7 Subsequent studies demonstrated that D-4F associates with HDL, reduces LOOH, increases the HDL associated enzyme paraoxonase (PON), enhances the formation of pre-β HDL and increases reverse cholesterol transport from macrophages.8
A 10 amino acid sequence from apoJ synthesized from all D-amino acids D-[113–122]apoJ was also recently shown to be able to associate with HDL after an oral dose, reduce LOOH, increase PON activity, render HDL antiinflammatory in mice and monkeys, and dramatically reduce atherosclerosis in apoE null mice without increasing the formation of pre-β HDL.9 Both 4F and D-[113–122]apoJ are predicted by the LOCATE program10 to contain amphipathic helixes (a class A amphipathic helix for D-4F and a class G* amphipathic helix for D-[113–122]apoJ). We asked if the ability to associate with HDL, remove LOOH, increase PON activity, render HDL antiinflammatory, and reduce atherosclerosis in mouse models, requires the formation of an amphipathic helix? To test this question we designed small peptides that have amphipathic properties but which are predicted by the LOCATE program to be too small to form an amphipathic helix.
Materials and Methods
Protected amino acid derivatives, chlorotrityl resin, and condensing reagents were from Nova Biochem, Calif, 14C-labeled amino acids were from American Radiolabeled Chemicals (St Louis, Mo), other materials were from previously identified sources.8,9,11–14
Synthesis of Peptides
Peptides were synthesized by a combination of solid-phase and solution-phase methods,15,16 which are detailed in the online data supplement available at http://circres.ahajournals.org.
ApoE null female mice on a C57BL/6J background were purchased from Jackson Laboratory (Bar Harbor, Me) and maintained on a chow diet (Ralston Purina). Cynomolgous monkeys were from a colony at UCLA. The UCLA Animal Research Committee approved all animal studies.
Plasma Lipoproteins, Artery Wall Cell Cultures, Monocyte Chemotaxis
Lipoproteins, artery wall cell cultures, and monocytes were prepared and determination of MCA was as described previously8 using human (after informed consent) or mouse blood. Plasma was fractionated by FPLC as previously described.8
Quantifying Atherosclerotic Lesions
Aortic root sinus sections and en face aortic preparations were prepared and analyzed as previously described.7,17
Solubility of Peptides in Ethyl Acetate
Peptides were weighed into centrifuge tubes and ethyl acetate (HPLC grade; residue after evaporation <0.0001%) added to give a concentration of 10 mg/mL. Tubes were sealed and kept at room temperature for 30 minutes with vortexing every 10 minutes followed by centrifugation for 5 minutes at 10 000 rpm and the supernatant removed to a previously weighed tube. Ethyl acetate was evaporated under argon and the tubes weighed to determine the amount of peptide in the supernatant.
Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) was performed as described4,12,13 and further detailed in the online data supplement.
Determination of cholesterol, PON activity, lipoprotein-LOOH concentrations, Western analyses, and 2D agarose/native PAGE were as described previously,8 as was the measurement of reverse cholesterol transport from macrophages in vivo.8,18 Interaction of peptides with DMPC4 and negative stain electron microscopy19 were performed as described. Statistical significance was determined by use of model I ANOVA or nonpaired t-test, and significance was defined as a value of P<0.05.
Adding KRES to human artery wall cocultures in a preincubation (but not a coincubation) significantly inhibited LDL-induced MCA (data not shown).
Modifying KRES by substitution of aspartic acid for glutamic acid (ie, KRDS) resulted in a peptide with <10% of the activity of KRES (data not shown).
Administering 200 μg of KRES synthesized from all L-amino acids by gastric gavage, or administering the same amount of peptide in mouse chow rendered HDL antiinflammatory (ie, the HDL from mice that received KRES significantly inhibited LDL-induced MCA) (Figure 1A).
The peptides KRES and KERS differ only in the order of 2 central amino acids. Oral administration of D-KRES (KRES synthesized from all D-amino acids) rendered HDL antiinflammatory (Figure 1B). However, administering the same dose of D-KERS was ineffective (Figure 1B). D-KRES but not D-KERS also significantly reduced the ability of mouse LDL to induce MCA (Figure 1C).
Because both D-4F and D-[113–122]apoJ peptides have been shown to reduce LOOH and increase PON activity when added to plasma in vitro,9,11 we compared the ability of KRES and KERS to reduce LOOH and increase PON activity after addition to apoE null mouse plasma in vitro. The addition of either L- (synthesized from all L-amino acids) or D-KRES to apoE null mouse plasma in vitro reduced LOOH in HDL (Figure 1D) and in LDL (Figure 1E), and increased PON activity (Figure 1F). In contrast addition of L- or D-KERS failed to reduce LOOH or increase PON activity (Figure 1D through 1F).
Two hours after an oral dose L-KRES associated with HDL and the right side of the trailing HDL peak on the FPLC chromatogram where pre-β HDL would be expected but it also associated with smaller particles far to the right of the HDL-containing fractions (Figure 2, Top). After 6 hours there was less L-KRES in the fractions far to the right of HDL and more was found associated with HDL-containing fractions (Figure 2, Top). After 24 hours there was very little L-KRES far to the right of HDL (Figure 2, Top). However, after 24 hours there was almost as much L-KRES associated with HDL-containing fractions as there was at 6 hours. By 48 hours L-KRES was cleared from all fractions (Figure 2, Top). In contrast, after an oral dose L-KERS largely associated with the fractions far to the right of HDL and slightly in the pre-β HDL fractions, but L-KERS was never found in HDL-containing fractions (Figure 2, Bottom). L-KERS absorption appeared to be slower because the values at 6 hours were greater than at 2 hours, but L-KERS was cleared from all fractions by 24 hours (Figure 2, Bottom).
D-KRES but not D-KERS significantly reduced aortic root sinus atherosclerosis (Figure 3A) and aortic atherosclerosis in en face preparations (Figure 3B). The peptides constituted only 0.02% by weight of the diet. There was no significant difference in water or food consumption, body weight, liver weight, and heart weight, and there was no difference in liver function (alkaline phosphatase, AST, ALT, total or conjugated bilirubin, or albumin) or renal function (creatinine) between groups (data not shown). D-KRES significantly raised HDL-cholesterol levels by 27% without significantly altering total plasma cholesterol or triglycerides (Table). D-KRES did not significantly increase the formation of apoA-I with pre-β mobility nor did D-KRES enhance reverse cholesterol from macrophages (data not shown).
KRES is a zwitterionic tetrapeptide with amphipathic properties that is too small to form an amphipathic helix. However, as shown above, it associates with HDL after an oral dose and in vitro reduces LOOH and increases PON activity and it reduces atherosclerosis in apoE null mice. Another zwitterionic tetrapeptide with amphipathic properties that is too small to form an amphipathic helix is FREL. The addition of L-FREL to apoE null mouse plasma in vitro reduced LOOH in HDL (Figure 4⇓A) and in LDL (Figure 4⇓B), and increased PON activity (Figure 4⇓C) similar to the action of KRES (Figure 1D through 1F).
Two hours after oral administration of L-FREL to apoE null mice the peptide was only associated with HDL and was still detectable in HDL after 48 hours (Figure 4⇑D). The results with L-FREL differed from those of L-KRES in that L-FREL did not significantly associate with small particles to the far right of HDL, as was the case for L-KRES (compare Figure 2A and 4⇑⇑D).
FREL administered orally to apoE null mice for 12 weeks significantly decreased LOOH (Figure 4⇑E), increased PON activity (Figure 4⇑F), rendered HDL antiinflammatory (Figure 4⇑G), decreased LDL-induced MCA (Figure 4⇑H), and decreased aortic root sinus lesion area (Figure 4⇑I). Although there was a trend toward decreased en face lesion area in mice treated with L-KRES or D-FREL (data not shown), a statistically significant decrease was only seen with L-FREL (Figure 4⇑J). L-FREL and L-KRES significantly increased HDL-cholesterol without significantly changing total plasma cholesterol or triglycerides (Table).
Based on the data presented above we hypothesized that the interaction with lipids of KRES and FREL would be similar and different from that of KERS. As shown in Figure 5A KRES and FREL were readily soluble in ethyl acetate whereas KERS was not.
Figure 5B shows that in the absence of added peptide, DMPC formed monotonous vesicles of 12.5 to 14 nm in diameter, which were not altered by the addition of KERS suggesting that KERS did not interact with the DMPC. In contrast, the addition of KRES to DMPC caused the formation of stacked lipid-peptide bilayers with a bilayer dimension of 3.4 to 4.1 nm with the spacing between the bilayers of ≈2 nm (Figure 5C). KRES also caused the formation of peptide–lipid complexes with a diameter of 7.5 nm (Figure 5C). Not shown, KRES also caused the formation of peptide–lipid complexes with a diameter of 38 nm. Figure 5D demonstrates that the addition of FREL to DMPC caused the formation of stacked lipid-peptide bilayers similar to those formed by KRES.
KRES reduced the highly cooperative gel to liquid-crystalline phase transition of DMPC multilamelar vesicles to a significantly larger extent than did KERS (Figure 5E). Thus, KRES interacts more avidly with DMPC to change DMPC phase transition properties. These results are in agreement with the results obtained by negative stain electron microscopy.
To determine whether these tetrapeptides were active in another species, we administered FREL to monkeys. Oral FREL significantly reduced LOOH and rendered monkey HDL antiinflammatory (Figure 6).
Our previous studies5,6 indicated that the ability of apoA-I to modulate inflammatory processes relates in part to its ability to bind and remove inflammatory lipids.20 Because all peptides to date with these properties (4F, 5F, D-[113–122]apoJ) have the potential to form an amphipathic helix, we asked if the formation of a helix was required.
Preliminary studies with molecular modeling suggested that KRES is zwitterionic with segregated polar and nonpolar faces consistent with an amphipathic molecule, which could not form a helix because of its small size (data not shown). KRES was active in vitro and in vivo in apoE null mice regardless of whether it was synthesized from L- or from D-amino acids strongly suggesting that its activity was not dependent on peptide-protein interactions. Reversing the order of the 2 middle amino acids yielded KERS, which was biologically inactive. Preliminary molecular modeling of KERS also showed it to be zwitterionic with segregation of polar and nonpolar faces consistent with an amphipathic molecule, which could not form a helix because of its small size (data not shown).
Both KRES and KERS are soluble in water as would be expected for peptides that are zwitterionic (data not shown) but solubility in ethyl acetate was dramatically different between KRES and KERS (Figure 5A). KRES formed peptide-lipid structures with DMPC, which were not formed by KERS (Figure 5B and 5C), and DSC confirmed that KRES interacted more avidly with DMPC than KERS (Figure 5E).
In vitro, KRES reduced LOOH (Figure 1D and 1E) and increased PON activity (Figure 1F). In vivo, there were substantial differences in the plasma distribution of the 2 peptides after an oral dose (Figure 2). KRES associated with HDL but KERS did not.
Another tetrapeptide, FREL, was also readily soluble in ethyl acetate (Figure 5A) and formed structures similar to KRES with DMPC (Figure 5D). Similar to KRES (and different from KERS) FREL reduced LOOH and increased PON activity in vitro (Figure 4⇑A through 4C). Similar to KRES (and different from KERS), FREL associated with HDL after an oral dose, but FREL was only associated with HDL (Figure 4⇑D) whereas KRES was also associated with fractions far to the right of HDL in the FPLC chromatogram (Figure 2). Similar to KRES (and different from KERS) (Figure 3A and 3B) FREL inhibited atherosclerosis in apoE null mice (Figure 4I and 4⇑J). Therefore, the interaction of the peptides with lipids in vitro predicted their biologic activity in vivo.
Residues 39 to 42 of human lactorferrin are KRDS. The addition of hydrophobic alkyl groups to the terminal ends of KRDS synthesized from all L-amino acids yielded a peptide known as PEP 1261, which inhibited platelet aggregation in vitro.21 Injection of PEP 1261 intraperitoneally in 10% DMSO at a dose of 10 mg/kg/d reduced adjuvant induced arthritis in rats.22 In our hands this peptide was only weakly active in vitro in inhibiting LDL-induced MCA compared with KRES (data not shown).
The increase in PON activity in vitro by KRES (Figure 1F) and FREL (Figure 4⇑C) was associated with a decrease in LOOH (Figure 1D and 1E, 4A and 4⇑⇑B) and likely reflects the activation of HDL enzymes as a result of the removal of inhibitory oxidized lipids.23 Based on our previously published work,5,6,8,20 the reduced ability of LDL to induce MCA, and the improved antiinflammatory properties of HDL after oral administration of KRES or FREL is consistent with the removal of LOOH from LDL and HDL and the activation of HDL-associated antioxidant enzymes.
The reduction in lesions with KRES and FREL was less dramatic than that seen7 with D-4F or D-[113–122]apoJ.9 The difference in apparent potency may have multiple causes. D-4F and D-[113–122]apoJ effectively sequester inflammatory lipids such that they inhibit LDL-induced MCA in both a preincubation and a coincubation.4–6,9 The tetrapeptides were active only in a preincubation but not in a coincubation (data not shown) indicating a relative decreased ability to sequester inflammatory lipids.
Also unlike D-4F, the tetrapeptides did not increase the formation of pre-β HDL or enhance reverse cholesterol transport from macrophages in apoE null mice (data not shown) despite causing a significant increase in plasma HDL-cholesterol (Table).
Regardless of the relative potency of the tetrapeptides compared with the larger helix forming peptides, the studies presented here demonstrate that peptides which are too small to form amphipathic helixes can still bind lipids, associate with HDL, reduce LOOH, and increase PON activity and that these properties result in antiinflammatory activity in both mice and monkeys.
This work was supported by National Institutes of Health grants HL-30568 to A.M.F., HL-34343 to G.M.A., and the Laubisch, Castera, and M.K. Gray Funds at UCLA to A.M.F.
M.N., G.M.A., S.T.R., S.H., and A.M.F. are principals in Bruin Pharma.
Original received March 15, 2005; revision received July 11, 2005; accepted August 2, 2005.
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