Mimetic peptides of human apoA-I helix 10 get together to lower lipids and ameliorate atherosclerosis: is the action in the gut?1

This issue of the Journal of Lipid Research contains an intriguing paper from the Scripps Research Institute by Zhao and colleagues (1) investigating the in vivo anti-atherosclerotic properties of HDL-like nanoparticles containing α-helical amphipathic peptides. The group uses 23-amino acid peptides representing a modified tenth helix of human apoA-I (amino acid residues 221-241, modified sequence = CGVLESFKASFLSALEEWTKKLQ). This last helix of apoA-I is associated with initial lipid binding and cholesterol efflux by the apoprotein (2, 3). The two conservative amino acid substitutions were designed to increase amphiphilicity and spectral properties. The use of the tenth helix sequence as a peptide model by Zhao et al. is novel and is in contrast to the development of the 18A peptide and subsequently the 4F peptide, which were designed as idealized class A amphipathic helices without sequence homology to a natural apoprotein (4). Zhao et al. formed HDL-like nanoparticles with their peptide mimetics of human apoA-I helix 10 by incubation with DMPC multilamellar vesicles without utilizing cholate. The HDL-like nanoparticles come in two flavors, those with monomeric 23-mer peptide and those with peptide trimers; that is, three 23-mer peptide monomers each linked to a small uncharged organic scaffold to generate a multivalent construct. To contrast this report with the majority of work in the apoA-I mimetic peptide field, peptide mimetics such 18A and 4F have generally been investigated as monomers and administered to animals as bare peptide without associated lipid (5) with some notable exceptions (6, 7). While tandem linked dimeric peptides have previously been investigated (5), there is no previous description of the anti-atherosclerotic properties of trimeric peptides. The Scripps group had previously shown that these nanoparticles were 9–12 nm discs that incorporated into and remodeled HDL in vitro and mediated cholesterol efflux in culture. It similarly remodeled HDL in vivo following intraperitoneal injection (8). The complexing of peptide with DMPC provided a degree of protease protection in vitro relative to lipid-free peptide. The nanoparticles with trimeric peptides were more effective at incorporating into HDL, mediating cholesterol efflux, and also were more resistant to proteolysis than the nanoparticles with monomeric peptide. The improved ability of nanoparticles with peptide multimers to interact with HDL and efflux cholesterol mirrors what our group and others have reported with tandem symmetric or asymmetric dimeric apoA-I mimetic peptides with proline linkers (9–11). Using the LDL-receptor null mouse model of atherosclerosis, the mice were switched to Western-type diet and simultaneously treated with the nanoparticles. The Scripps group shows striking efficacy in decreasing both nonHDL-C (plasma total cholesterol reduced ∼40% after 2 weeks of treatment) and aortic atherosclerosis (whole aorta and sinus lesion reductions ∼50% after 10 weeks of treatment) by either the intraperitoneal or oral route of administration of the nanoparticles. This is in striking contrast to the effect of the well-described 4F and other apoA-I mimetic peptides, which can decrease atherosclerotic lesion size without a consistent significant effect on plasma lipids (5). The use of this human apoA-I tenth helix mimetic seems to provide lipid lowering abilities not typically seen in apoA-I mimetic peptides. Of note, previous mimetic reports generally used lipid-free peptide rather than the peptide-lipid particles reported here. Prior to the current report, mimetic peptides based on apoE (12–14), which contain motifs for binding the LDL-receptor or scavenger receptors, were the exception in the mimetic peptide field in that they show both lipid lowering and anti-atherosclerotic effect. However, lipid lowering may not be the primary mechanism by which the nanoparticles are anti-atherogenic. Zhao et al. show that their HDL-like nanoparticles have oral efficacy in lipid lowering only at a younger age (12 weeks, i.e., two weeks of treatment) but not after a more extended treatment (at 20 weeks). At 20 weeks of age, the mice given nanoparticles by the oral route did not exhibit significantly lower plasma lipids. Nevertheless these mice had notably reduced atherosclerotic lesions. Of note, despite these nanoparticles being atheroprotective by the oral route, they could not be detected by pharmacokinetic studies of the plasma using sensitive LC-MS selected ion monitoring techniques. Thus plasma concentrations and lipid lowering do not seem to be the major bases for the anti-atherogenicity of PO-administered nanoparticles. Additionally, nanoparticles with monomeric peptide showed similar in vivo potency as those with trimeric peptide, despite previous work by the Scripps group showing improved plasma pharmacokinetics and in vivo HDL remodeling by the trimeric peptide nanoparticles (8). What then might be the basis for the anti-atherogenicity of the nanoparticles? The effectiveness of 4F peptides at preventing atherosclerosis is generally thought to be due to binding oxidized lipid (15) or reverse cholesterol transport (16) as opposed to plasma lipid lowering. In the current study, plasma 15(S)-HETE levels were not influenced by the nanoparticle treatment and in vivo macrophage to feces reverse cholesterol transport was not investigated, although these nanoparticles were previously shown to mediate efficient cholesterol efflux from macrophages in culture (8). However, oral administration shows no detectable nanoparticle in plasma so there likely is not significant lipoprotein association/remodeling or reverse cholesterol transport promoted by the nanoparticles in this set of mice. In addition, nanoparticles with the monomeric peptide are more susceptible to proteolysis but are still effective orally. Overall, the current study suggests that the in vitro and short-term in vivo nanoparticle properties investigated previously by the authors are unlikely to play a significant role in the in vivo atheroprotection. All these findings lend further credence to the hypothesis that the gut is the site of action for apoA-I mimetic peptides (17). This has been highlighted recently by the Fogelman group (18, 19) in experiments using transgenic tomato feeds as a source of peptide administration where anti-inflammatory, lipid lowering, and anti-atherosclerotic effects were seen but intact 6F peptide was not found in the plasma. Further investigations of the effect of nanoparticle treatment on gut lipid absorption, lysophosphatidic acid accumulation, enterocyte lipid esterification and transporter function, and chyle composition obviously are called for. Perhaps work by the Fogelman group with 6F-transgenic tomato feeds and the current work showing equivalency of peptide via IP and oral administration (without detectable peptide plasma levels after oral dosing) should hasten workers in the field of atherosclerosis to pay as much attention to the gut as they do to other tissues where atheroprotective mechanisms may be operating. The basis for the resistance of these peptides to gut proteases also needs exploration. The Scripps group point out several ways in which their peptides behave similarly to the 4F/6F peptide studies, despite their differences in primary amino acid sequence, including that they are effective in vivo despite the lack of detection of the peptides in the plasma. However, the use of an α-helix involved in initial apoA-I lipid binding would otherwise be expected to behave very differently from the 4F-type peptides. For instance, nanoparticle treatment did not effectively reduce plasma 15-S-HETE and showed mixed effectiveness at reducing plasma serum amyloid A, two features commonly reported for the 4F and 6F peptides (15, 18, 20). The binding of oxidized lipids by these nanoparticles has not been studied by the Scripps group, but lipid-peptide complexes may not be expected to bind oxidized lipid with as high affinity as lipid-free peptides. The current study illustrates that apoA-I mimetic peptides with different structural properties may exert their protective effects via different mechanisms. While it is well known that apoA-I has many putative functions in atheroprotection, it is becoming apparent that this is also the case for mimetic peptides. Additionally, this work points out a possible disconnect between apparently anti-atherogenic functionality in vitro (such as cholesterol efflux in culture) and the actual atheroprotective effect in vivo. This work also continues to show the dispensability of lipid lowering for atheroprotection by apoA-I mimetic peptides. Intravenous infusions of HDL, HDL-like particles, and delipidated apoA-I have been reported to be effective in reducing atherosclerotic lesions in animal models (21) although the results have been less robust in human studies (22). The current report and work by the Fogelman group have raised the exciting possibility of a future orally available agent targeted at improving HDL function, likely through a gut-mediated mechanism of action. While recent therapeutic trials of pharmacologic HDL-C raising agents have been disappointing (23, 24), the ongoing work with apoA-I peptides continues to show promise.