A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice

Abstract

Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol efflux with K(m) molar efficiency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR(-/-) and apolipoprotein (apo)E(-/-) mice approximately 5-7 months of age, following 13-18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR(-/-) mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 +/- 2 vs.11.3 +/- 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 +/- 5.8 vs. 33 +/- 4.9% control, P = 0.014). In apoE(-/-) mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by approximately 45% (lesion area = 15 +/- 7 vs. 25 +/- 8% control, P = 0.00016; plaque lipid-content = 20 +/- 6 vs. 32 +/- 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24-48 h. In summary, relatively short-term treatment of mice with the potent cholesterol efflux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal injection.

Fig. 1.

Creation of α-helix peptide ATI-5261. Top bar represents the CT domain of apoE (aa216-299). The aa238-270 segment possesses features useful for constructing a model class A α-helix. These features include numerous sites for intra-helical ionic interactions (i+4) at the lipid-water interface (35) and potential for designing a favorable nonpolar surface for binding lipid. Underlined qlutamine (Q) and alanine (A) residues were replaced with underlined tryptophan (W) and phenylalanine (F) to accommodate strongly hydrophobic amino acids at these sites within a shorter sequence. The polar Q21 was replaced with leucine (L) to expand nonpolar surface-area (ATI-5261-wheel and –net diagrams) and I13 replaced with phenylalanine (F) to further increase hydrophobicity. Q residues on the polar surface were replaced with smaller alanine (A) residues, generally good for α-helices. The A4 to serine (S) substitution was intended to minimize hydrophobic character of the polar surface. Glutamate (E) was used at positions 15 and 19 to create an alignment of acidic residues down the center of the polar surface, thereby endowing class A structure. E19 also created an additional site for salt-bridge formation with R23. Right side of the figure compares the original apoE CT sequence with that of ATI-5261 (amino acid changes in bold). Net-charge of the original aa238-266 sequence = 0 and net-charge of ATI-5261 = -1. Also shown are Far-UV CD spectra and % α-helicity for the original apoE CT seq. aa236-266 (top scan) vs. ATI-5261 (bottom scan, mean ± SD, n = 3), using 62 μM concentrations of peptides in 10 mM phosphate buffer (pH = 7.4).

Fig. 2.

In vitro cholesterol efflux activity of ATI-5261. A: Cholesterol efflux activity (8 h) of ATI-5261 versus original segment derived from apoE CT domain aa238-266. J774 cells were treated with (shaded bars) and without (open bars) a cAMP analog to modulate ABCA1 expression. Both peptides were capped with N-terminal acetyl and C-terminal amide groups and used in lipid-free form in serum-free medium. Results are from two experiments (performed in triplicate), comparing ATI-5261 (30 μg/ml) to the original segment at either 30 or 100 μg/ml, respectively. B: Time-course of [3H]cholesterol efflux to the lipid-free ATI-5261 (30 μg/ml), using J774 macrophages treated with (closed circles) and without (open) a cAMP analog. C: ABCA1-dependent cholesterol efflux using J774 cells treated with (shaded bars) and without (open) cAMP. A-I, E3 and CT refer to full-length apoA-I, apoE3 and the CT domain of apoE. Each acceptor was used in lipid-free form at 30 μg/ml serum-free medium. Values are means ± SD, n = 5. D: Cholesterol efflux (cAMP treated J774 cells) to increasing concentrations of lipid-free ATI-5261 and apoA-I. Values are means ± SD, n = 3. E: Nondenaturing gradient (4–20%) gel electrophoresis of ATI-5261:POPC complexes following standard (left gel) and short run-times (right); lane 1, standards (nm); lane 2 and 3, ATI-5261:POPC complexes (4 μg); lane 4, lipid-free ATI-5261 (4 μg). F: Cholesterol efflux activity of ATI-5261:POPC complexes versus lipid-free peptide, using each at 50 and 100 μg/ml (based on peptide mass). Values are means ± SD, n = 3. G: Dependence of cholesterol efflux on concentration of ATI-5261:POPC complexes, using cAMP treated J774 macrophages. Results are from a single experiment using triplicate wells (error bars smaller than symbols when not seen) and are representative of two experiments.

Fig. 3.

Anti-atherosclerosis effects of ATI-5261 in LDLR^−/− mice. Male mice (8 weeks of age) were fed a HFWD for 7 weeks. The mice subsequently received daily intraperitoneal injections of saline (vehicle control) or lipid-free ATI-5261 peptide (30 mg/kg) for 6 weeks in the continued presence of HFWD. A: Area of whole-aorta covered with plaque-lesions at termination. B: Lipid content of aortic-sinus plaque using Oil Red O. Individual data points shown with means ± SD, n = 8 mice per group.

Fig. 4.

ATI-5261 enhances macrophage RCT and reduces atherosclerosis in apoE^−/− mice. A: To assess RCT, atherosclerotic (6 months) male mice were injected intraperitoneally with [³H]cholesterol-labeled J774 macrophage foam-cells plus lipid-free ATI-5261 at a dose of 20 mg/kg (shaded bars) or with the [3H]foam-cells alone (open bars). For the former, [3H]foam- cells and peptide were injected together. Left: Appearance of [³H]cholesterol in plasma following treatment. Right: [³H]sterol per gram of feces collected between 0-24 h (24 h) and 24-48 h (48 h) intervals. Values are means ± SD (n = 5). Vehicle-control plasma dpm/ml corresponded to 5.4 ± 0.82 and 8.9 ± 1.6% of injected [3H] at 24 and 48 h, respectively; ATI-5261 plasma dpm/ml to 8.9 ± 1.1 and 11.6 ± 1.9%; control versus ATI-5261 [3H]sterol 1.11 ± 0.16 versus 1.65 ± 0.28% of total injected [3H] at 48 h. Results representative of two experiments. B: Impact of ATI-5261 on atherosclerosis: male apoE^−/− mice were fed HFWD for 18 weeks, then injected intraperitoneally with ATI-5261 once daily (left) or on alternate days (right) to maintain equivalent peptide delivery (18 mg) over 6 weeks. Each treatment group had its own vehicle control, as the number of total injections differed (42 vs. 21 injections, respectively). Lipid content of aortic-sinus plaque is shown versus vehicle controls. Values are means ± SD, n = 8 mice per group. P = 0.0017 for 30 mg/kg dose every other day versus 15 mg/kg daily.

Fig. 5.

Anti-atherosclerosis effects of ATI-5261 and ATI-5261:POPC complexes. Male apoE^−/− mice (7 weeks of age) were fed a HFWD for 18 weeks. Mice then received intraperitoneal injections of either saline (control, n = 18), ATI-5261 (n = 19), or ATI-5261:POPC complexes (n = 14) on alternate days for 6 weeks (chow diet). A dose of 30 mg/kg was used per injection for both lipid-free ATI-5261 and ATI-5261:POPC complexes, based on peptide mass. A: Percentage of whole aorta covered with plaque lesions. B: Lipid-content of aortic-sinus plaque at termination of treatment, shown as data from individual mice with means ± SD. Representative cross-sections of aortic tissue stained with Oil Red O shown vertically on right (top to bottom as saline controls, lipid-free ATI-5261 peptide and ATI-5621:POPC complexes).