5A apolipoprotein mimetic peptide promotes cholesterol efflux and reduces atherosclerosis in mice

Abstract

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

Fig. 1.

Effect of 5A on cholesterol efflux. Free 5A (A and C) or 5A-POPC (B and D) at the indicated doses were used to efflux cholesterol from ABCA1-transfected (A and B) or ABCG1-transfected (C and D) BHK cells. Contribution of cholesterol efflux from the transporter (▵) was calculated after subtraction of cholesterol efflux from the transfected cell line (■) from the mock-transfected control cell line (●). Results are expressed as the mean ± 1 S.D. of triplicates.

Fig. 2.

Effect of ex vivo incubation of 5A-POPC on plasma lipoproteins after separation by FPLC. A, protein absorbance FPLC profile of 5A reconstituted with POPC (1:7 M ratio). B, phospholipid FPLC profile of 5A reconstituted with POPC (1:7 M ratio). C, FPLC profile of biotinylated 5A-POPC (5 μg /ml final concentration) incubated with plasma for 45 min at 37°C and separated by FPLC. D, apoA-I FPLC profile of plasma before (dashed line) and after (solid line) incubation with 5A-POPC (5 μg /ml final concentration) for 45 min at 37°C. E, phospholipid FPLC profile of plasma before (dashed line) and after (solid line) incubation with 5A-POPC (5 μg/ml final concentration) for 45 min at 37°C. F, total cholesterol FPLC profile of plasma before (dashed line) and after (solid line) incubation with 5A-POPC (5 μg /ml final concentration) for 45 min at 37°C.

Fig. 3.

Effect of ex vivo incubation of 5A-POPC on plasma lipoproteins after separation by electrophoresis. 5A-POPC (5 μg/ml final dose) was incubated for 45 min at 37°C with either HDL or LDL isolated by density gradient ultracentrifugation or with human serum and then separated by electrophoresis on the 2100 Bioanalyzer. Lanes 1 and 8, 5A-POPC; lane 2, HDL; lane 3, HDL plus 5A-POPC; lane 4, LDL; lane 5, LDL plus 5A-POPC; lane 6, human serum; lane 7, human serum plus 5A-POPC. Horizontal lines indicate location of internal control.

Fig. 4.

Effect of in vivo treatment of 5A-POC on plasma lipid and lipoproteins. A, C, and E, apoE-KO mice (n = 5) were injected intravenously with 5A-POPC (30 mg/kg), and pooled plasma collected at baseline (dashed lines), 3 h after injection (gray solid lines), and 24 h after injection (black solid lines) was separated by FPLC and analyzed for total cholesterol (A), cholesteryl esters (C), and phospholipids (E). B, D, and F, enlargements of the HDL peak from A, C, and E, respectively.

Fig. 5.

Effect of in vivo treatment of 5A-POC on cholesterol efflux by plasma. apoE-KO mice (n = 6) were injected intravenously with 5A-POPC (30 mg/kg), and pooled plasma collected at baseline (black bars) and 1 h (white bars), 6 h (gray bars), and 24 h (striped bars) after injection were tested for cholesterol efflux from control mock-transfected cells, ABCA1-transfected cells, or ABCG1-transfected cells. Results represent the mean ± 1 S.D. of quadruplicates. ∗, p < 0.05 compared with baseline result.

Fig. 6.

Effect of 5A-POPC treatment on in vivo cholesterol efflux from total tissues. A, cholesterol efflux from tissues in rats (n = 4) injected intravenously with the indicated dose of 5A-POPC was determined from isotope dilution curves. B, fecal sterol excretion was determined for neutral sterols (open bars) and bile acids (solid bars) for rats treated with the indicated dose of 5A-POPC. Results represent the mean ± 1 S.D. of triplicates. ∗, p < 0.05 compared with the 0-dose treatment result.

Fig. 7.

Effect of 5A-POPC treatment on in vivo cholesterol efflux from macrophages. C57BL/6 mice (n = 7) were injected intraperitoneally with RAW 264.7 macrophage radiolabeled with ³H-cholesterol. 5A-POPC (30 mg/kg) was injected intraperitoneally 8 h before and within 1 h before cell injection. Twenty-four hours after cell injection, plasma was monitored for radioactive counts (A), and stool was monitored for fecal sterols (B) and fecal bile acids (C). Results represent the mean ± 1 S.D. of seven replicates. ∗, p < 0.01 compared with the untreated control group.

Fig. 8.

Effect of 5A-POPC treatment on atherosclerosis. Each set of data are represented by a scattered dot plot (left) and a box and whiskers plot (right). A and B, 4-month-old apoE-KO mice (n = 8) treated intravenously with POPC or 5A-POPC (30 mg/kg) (A) or 8-month old apoE-KO mice (n = 8) treated intravenously with saline or 5A-POPC (30 mg/kg) (B) on a normal chow diet were injected every Monday, Wednesday, and Friday for 13 weeks and then analyzed for percentage of surface area coverage of aortic plaque. C, 4-month old apoE-KO mice (n = 8) on a normal chow diet were injected intraperitoneally with 5A-SM/DPCC (30 mg/kg) or saline every Monday, Wednesday, and Friday for 13 weeks and then analyzed for percentage of surface area coverage of aortic plaque.