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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2009 Dec 3;30(2):246–252. doi: 10.1161/ATVBAHA.109.200196

The 5A apolipoprotein A-I mimetic peptide displays anti-inflammatory and antioxidant properties in vivo and in vitro

Fatiha Tabet 1, Alan T Remaley 2, Aude I Segaliny 1, Jonathan Millet 1, Ling Yan 1, Shirley Nakhla 1, Philip J Barter 1,4, Kerry-Anne Rye 1,3,4, Gilles Lambert 1,5
PMCID: PMC2828392  NIHMSID: NIHMS172804  PMID: 19965776

Abstract

Objectives

The apolipoprotein (apo) A-I mimetic peptide 5A is highly specific for ABCA1-transporter mediated cholesterol efflux. We investigated whether the 5A peptide shares other beneficial features of apoA-I, such as protection against inflammation and oxidation.

Methods

New-Zealand White rabbits received an infusion of apoA-I, reconstituted HDL containing apoA-I ((A-I)rHDL) or the 5A peptide complexed with phospholipids (PLPC), prior to inserting a collar around the carotid artery. Human coronary artery endothelial cells (HCAECs) were incubated with (A-I)rHDL or 5A/PLPC prior to TNFa stimulation.

Results

ApoA-I, (A-I)rHDL and 5A/PLPC reduced the collar mediated increase in (i) endothelial expression of cell adhesion molecules VCAM-1 and ICAM-1, (ii) O2− production as well as the expression of the Nox4 catalytic subunits of the NADPH oxidase, and (iii) infiltration of circulating neutrophils into the carotid intima-media. In HCAECs, both 5A/PLPC and (A-I)rHDL inhibited TNFa induced ICAM-1 and VCAM-1 expression as well as the NF-κB signalling cascade and O2− production. The effects of the 5A/PLPC complex were no longer apparent in HCAECs knocked down for ABCA1.

Conclusion

Like apoA-I, the 5A peptide inhibits acute inflammation and oxidative stress in rabbit carotids and HCAECs. In vitro, the 5A peptide exerts these beneficial effects through interaction with ABCA1.

Keywords: HDL, apoA-I, mimetic peptide, inflammation, oxidation

Introduction

HDL protect against the development of atherosclerosis by mediating reverse cholesterol transport (RCT), a pathway by which excess cholesterol is removed from peripheral cells such as plaque macrophages, to the liver for excretion1, 2. The pathogenesis of atherosclerosis, however, is not limited to impaired lipid metabolism and flux. Inflammatory and oxidative damage also play a pivotal role in atherogenesis and HDL have been shown to inhibit these processes3. In vitro and in vivo studies have demonstrated that HDL inhibit endothelial expression of adhesion molecules, thereby preventing monocyte recruitment into the arterial wall4. HDL also remove and/or inactivate oxidized lipids from LDL particles5. In addition, HDL decrease O2− production and inactivate neutrophil NADPH oxidase, a respiratory burst enzyme, which is an important source of ROS in the vessel wall6.

Acute intravenous infusions of HDL7 have been shown to have rapid and beneficial effects on the arterial wall. Infusion of reconstituted HDL (rHDL) containing apoA-I8 or its potentially more effective variant apoA-I Milano9, 10 in clinical trials respectively promoted the regression of coronary atherosclerosis, reversed remodeling of the external elastic membrane, and reduced the atheroma volume. Because of the relatively large quantities of apoA-I that are used during these infusions, the cost of generating recombinant apoA-I or purified apoA-I from human serum that is free from endotoxin is a limitation of this approach. In contrast, synthetic peptide analogs of the amphipathic helixes of apoA-I, which are considerably easier and less costly to produce, offer an alternative approach for rHDL therapy11, 12. ApoA-I mimetic peptides also offer several other possible advantages: namely they can be administered orally, they can be readily complexed with phospholipids and various structural variants with different functional properties and potentially improved atheroprotective properties can be easily engineered.

We recently designed a series of asymmetric variants of the prototypical 37pA bi-helical apoA-I mimetic peptide, which contains two identical class A amphipatic helices linked by a proline residue13. This was achieved by substituting non-polar amino acids on the hydrophobic face of the COOH terminal helix with alanine14. The 5A variant, which contains a high lipid affinity helix paired with a low lipid affinity helix with 5 alanine substitutions, showed the greatest specificity for ABCA1 mediated cholesterol efflux, as well as the lowest cytotoxicity14.

The aim of this study was to investigate whether, in addition to mediating ABCA1-specific efflux, the 5A peptide shares other features of full-length apoA-I, such as anti-inflammatory and antioxidant properties. We now report that like apoA-I, the 5A peptide reduces acute inflammation and oxidative stress both in rabbit carotid arteries (in vivo) and in primary human coronary artery endothelial cells (HCAECs). We also showed in HCAECs that the 5A peptide exerts these beneficial effects through interactions with ABCA1.

Methods

For a complete methods section see the supplemental materials

Peptide Synthesis and Solubilisation

The 5A bi-helical peptide (DWLKAFYDKVAEKLKEAF-P-DWAKAAYDKAAEKAKEAA) was synthesized by a solid-phase procedure using Fmoc/DIC/HOBt chemistry and purified to greater than 99% by reverse-phase HPLC. Purity was assessed by MALDI-TOF-MS (Bruker Ultraflex)14. The 5A peptide was reconstituted with 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) (Avanti Polar Lipids, Alabaster, AL) at a 1:8 molar ratio, using a co-lyophilization procedure14. The peptide:PLPC complexes were dissolved in NaCl 0.9% and sterilized using 0.2mm filters. The peptide and phospholipid content of the 5A/PLPC complexes were quantitated using the bicinchoninic acid (BCA) protein assay (Pierce) and phospholipid assay (Wako) kits, respectively. The final 5A:PLPC ratio was 1:7.

Isolation of apoA-I and preparation of reconstituted HDL

Blood samples from normal healthy donors (Gribbles Transfusion, South Australia) were collected in EDTA-Na2 tubes and pooled. HDL were separated by sequential ultracentrifugation (1.063 < d < 1.21 g/ml), delipidated and apoA-I was purified by anion exchange chromatography on a Q Sepharose Fast Flow column attached to an FPLC (Äkta) system (GE Healthcare, Chalfont, UK). The purity of the apoA-I was judged to be >95% on a 20% SDS-Phast gel (GE Healthcare). Discoidal reconstituted HDL containing apoA-I and PLPC, referred to as (A-I)rHDL, were prepared (initial PLPC:apoA-I molar ratio 100:1) by the cholate dialysis method15. Lipid-free apoA-I and discoidal (A-I)rHDL were dialyzed extensively against endotoxin free PBS, pH 7.4 before use. The final PLPC:apoA-I molar ratio for the discoidal (A-I)rHDL was 80:1.

Results

Plasma analysis

Plasma samples collected (i) before infusion, (ii) after collar implantation, and (iii) before sacrifice were assayed for lipids. Pre-infusion values were 0.79±0.09 mM total-cholesterol, 0.52±0.08 mM HDL-cholesterol, and 0.91±0.09 mM triglycerides. The saline, lipid free apoA-I, (A-I)rHDL, and the 5A/PLPC complex infusions had no effect on plasma lipid levels either at the time of collar implantation (24 hours later) or at the time of sacrifice (48 hours later).

Anti-inflammatory effects of the 5A/PLPC complex in rabbit arteries

Compared with non-collared arteries, the collared arteries of the saline-infused rabbits had markedly increased endothelial expression of VCAM-1 and ICAM-1, as well as increased infiltration of circulating neutrophils into the intima-media (Fig.1). This is consistent with what has been reported previously4. ICAM-1 is expressed constitutively by endothelial cells but at levels that could not be detected by immunohistochemistry in non-collared arteries, as reported previously16. As anticipated 4, 16, when the animals were infused with lipid-free apoA-I, endothelial expression of VCAM-1 and ICAM-1 decreased by 72±15% (p<0.05) and 59±11% (p<0.05) respectively. Neutrophil infiltration was reduced by 65±7% (p<0.05) (Fig. 1). Likewise, when the animals were infused with (A-I)rHDL, endothelial expression of VCAM-1 and ICAM-1 was decreased by 61±6% (p<0.05) and 64±8% (p<0.05) respectively, and neutrophil infiltration was reduced by 52±10% (p<0.05), compared with saline infused rabbits (Fig. 1). Infusion of the 5A/PLPC complex was associated with less staining for both VCAM-1 and ICAM-1, respectively 57±9% (p<0.05) and 71±5% (p<0.05) less than saline. Neutrophil infiltration was reduced by 62±8% (p<0.05), compared with saline infused rabbits (Fig. 1). Thus, a single 20mg/kg infusion of the 5A/PLPC complex was as effective (p>0.85, all) as a single 8mg/kg infusion of lipid-free apoA-I or (A-I)rHDL at reducing collar-induced acute inflammation in NZW rabbit carotid arteries. In addition, we isolated mRNA from sections of collared and non-collared arteries from the saline, 5A/PLPC complex and lipid-free apoA-I infused rabbits. Compared with the non-collared arteries, the collared arteries from the saline-treated animals had higher VCAM-1 mRNA levels normalized to 18S (100±30 vs. 20±4 Arbitrary Units (A.U.), p<0.05). Lipid-free apoA-I and the 5A/PLPC complex inhibited the collar-induced increase of endothelial VCAM-1 mRNA expression by 74% (to 24±10 AU, p<0.05) and 69% (to 31±17 AU, p<0.05), respectively.

Figure 1.

Figure 1

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Immunohistochemical staining for VCAM-1, ICAM-1 and CD18 in representative non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg), lipid-free apoA-I (8mg/kg), or (A-I)rHDL 24 hours prior to collar implantation. Histograms: endothelial expression of VCAM-1, ICAM-1 and infiltration of neutrophils (CD18) into the intima-media were quantified as described in Methods. Results are expressed as mean ±SEM (*p<0.05 vs. saline, ** p<0.01 vs. no collar, n=6 per group).

Anti-oxidant effects of the 5A/PLPC complex in rabbit arteries

To evaluate the anti-oxidant properties of the 5A mimetic peptide, we measured Nox2 and Nox4 mRNA levels in the non-collared and collared arteries of saline, 5A/PLPC complex and lipid-free apoA-I infused rabbits. Compared with non-collared carotid arteries, the collared arteries from saline-treated animals had increased Nox2 (100±20 vs. 23±7, p<0.05) and Nox4 (100±17 vs. 26±9, p<0.05) (Fig. 2A) mRNA levels. The collar-induced Nox2 mRNA expression was inhibited by 69% (from a 100±20 to 31±16) and 70% (100±20 to 30±12) by the single lipid-free apoA-I and 5A/PLPC complex infusions, respectively. It is possible that reduced Nox2 mRNA expression is simply a consequence of the reduced influx of neutrophils in the artery wall rather than a specific anti-oxidant effect. To test this, we incubated human neutrophils with PBS, (A-I)rHDL or the 5A/PLPC complex for 16 hours. The neutrophils were then stimulated for 3 hours with phorbol myristate acetate (PMA). Nox2 protein expression was increased upon PMA stimulation by 50%. Neither (A-I)rHDL or the 5A/PLPC complex altered Nox2 expression significantly in either the stimulated or the non-stimulated conditions (Suppl Fig 1). In vivo, the collar-induced increase in Nox4 mRNA expression was inhibited by 64% (from 100±17 to 36±12) and 71% (from 100±17 to 29±18) by the infusion of lipid-free apoA-I and the 5A/PLPC complex (p<0.05 for all). To investigate whether Nox4 inhibition as well as reduced neutrophil influx in the arterial wall by the 5A/PLPC complex or lipid-free apoA-I was associated with an inhibition in ROS expression, we used DHE nuclear fluorescence to measure ROS levels in the collared and non-collared arteries (Fig. 2B). Compared with non-collared arteries, DHE fluorescence was increased in the collared arteries from the saline infused rabbits, which was consistent with increased ROS expression (22±0.8 vs. 100±11, respectively, p<0.05). The 5A/PLPC complex was as potent as lipid-free apoA-I at inhibiting the collar-induced ROS expression (100±11 to 45±4 and 100±11 to 40±5, respectively) (p<0.05 for all vs. saline-infused animals; p=0.92 for 5A/PLPC vs. lipid-free apoA-I).

Figure 2.

Figure 2

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(A) mRNA levels of Nox 2 and Nox 4 in non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg of 5A) or lipid-free apoA-I (8mg/kg), 24 hours prior to collar implantation. (B) ROS generation was detected using dihydroethidum (DHE) fluorescence in non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg of 5A) or lipid-free apoA-I (8mg/kg), as described in Methods. (*p<0.05 vs. non-collar, **p<0.05 vs. saline, n=6 per group). Results are expressed as mean expression levels of Nox 2, or Nox 4, normalized to 18S, ±SEM (*p<0.01 vs. Non-Collar, **p<0.05 vs. saline, n=6 per group).

Anti-inflammatory effects of the 5A/PLPC complex in human artery endothelial cells

We investigated by flow cytometry whether the 5A/PLPC complex modulates the cell surface expression of ICAM-1 and VCAM-1 in human coronary artery endothelial cells (HCAECs) stimulated with TNFα. Both (A-I)rHDL and the 5A/PLPC complex dose dependently reduced the TNFα induced expression of VCAM-1 (Fig 3A) and ICAM-1 (Fig 3B), reaching a maximal response at 1mg/mL. We showed in a time course study that 2 to 24 hours incubation of either (A-I)rHDL or the 5A/PLPC complex significantly reduced the TNFα-induced expression of VCAM-1 (Fig 3C) and ICAM-1 (Fig 3D) in HCAECs. We next assessed by western blot the modulation by the 5A/PLPC complex and (A-I)rHDL (1 mg/mL - 16 hours) of total ICAM-1 and VCAM-1 protein expression in HCAECs stimulated with TNFα (Fig. 4A). As judged by Evans blue staining, this had no effect on cell viability (not shown). The addition of the pro-inflammatory cytokine, TNFα, induced both ICAM-1 and VCAM-1 protein expression by 162% (from 38±1 to 100±22) and 361% (from 21.5±1 to 100±3), respectively (p<0.05, all). The 5A/PLPC complex reduced the TNFα-induced ICAM-1 and VCAM-1 protein expression by 57% (to 43±10) and 85% (to 15±4), compared to 53% (to 47±4) and 77% (to 23±8), respectively, for the cells that were incubated with discoidal (A-I)rHDL (p<0.05 vs. PBS, for all).

Figure 3.

Figure 3

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(A-B) Flow cytometry analysis of TNFα-induced VCAM-1 (panel A) and ICAM-1 (panel B) expression in HCAECs exposed to either PBS, or increasing concentrations of (A-I)rHDL, or increasing concentrations of the 5A/PLPC complex for 16 hours. (C-D) Flow cytometry analysis of TNFα-induced VCAM-1 (panel C) and ICAM-1 (panel D) expression in HCAECs exposed to either PBS, or 1mg/mL (A-I)rHDL, or 1mg/mL 5A/PLPC for 2, 12, 16 or 24 hours. Changes in mean fluorescence of cell surface VCAM-1 and ICAM-1 expression are expressed as mean±SEM of 4 independent experiments (#p<0.01 vs. control (C), *p<0.05 vs. PBS).

Figure 4.

Figure 4

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(panels A & B) Immunoblot analysis of TNFα-induced ICAM-1 and VCAM-1 expression in HCAECs exposed to PBS, (A-I)rHDL (final apoA-I concentration 1mg/mL), or the 5A/PLPC complex (final peptide concentration 1mg/mL) for 16 hours. Relative quantification of ICAM-1 and VCAM-1 normalized to β-actin is expressed as mean±SEM of 3 independent experiments performed in triplicate (*p<0.05 vs. control (C), **p<0.05 vs. PBS). (panels C&D) Immunoblot analysis of the nuclear translocation of NF-κB p65 subunit and of the phosphorylation of IκB in HCAEC cells exposed to PBS, 1mg/mL (A-I)rHDL, or 1mg/mL 5A/PLPC for 16 hours and stimulated with TNFα for 10 min. Relative quantification of NF-κB and P-IκB normalized to total IκB is expressed as mean±SEM of 3 independent experiments performed in triplicate (*p<0.05 vs. control (C), **p<0.05 vs. PBS). Experiments shown in panels B &D were performed in HCAECs knocked down for ABCA1.

Since the 5A mimetic peptide is highly specific for ABCA1 mediated cellular cholesterol efflux14, we next assessed whether the anti-inflammatory properties of the 5A/PLPC complex are also mediated through interactions with the ABCA1 transporter. We transiently transfected HCAECs with specific siRNAs to downregulate ABCA1 protein expression by >80% (24-48 hours after transfection) in our cellular model (Suppl Fig 2). In HCAECs knocked down for ABCA1, the 5A/PLPC complex failed to inhibit TNFα-induced ICAM-1 and VCAM-1 protein expression, whereas the discoidal (A-I)rHDL inhibited ICAM-1 (from 100±27 to 48±9) and VCAM-1 (from 100±32 to 39±13) protein expression to an extent comparable to what was observed for non-transfected cells (Fig. 4B). To further investigate whether ABCA1 plays a role in the inflammatory cascade, we have measured the activation of the nuclear factor-κB (NF-κB), a transcription factor that modulates VCAM-1 and ICAM-1 gene expression upon inflammatory insult17. This was performed in both non-transfected HCAECs as well as in HCAECs knocked-down for ABCA1. NF-κB is retained in the cytoplasm as part of a complex with IkB. Phosphorylation of IκB (P-IκB) leads to its degradation, thereby releasing NF-κB to translocate to the nucleus. We measured the expression of the NF-κB p65 subunit in nuclear extracts of HCAECs cells 10 minutes after stimulation with TNFα. We also measured the expression of both IκB and P-IκB in these cells by western blot. As shown in Fig 4C, TNFα treatment was associated with a 8-fold increased phosphorylation of IκB and a 2-fold increased nuclear tranlocation of NF-κB p65 (p<0.01, all). Pre-incubation of the cells for 16 hours with either 1mg/mL (A-I)rHDL or the 5A/PLPC complex reduced the TNFα induced phosphorylation of IκB by 43% (to 57±8) and 40% (to 60±5), and NF-κB p65 nuclear translocation by 21% (to 79±4) and 47% (to 53±9), respectively (p<0.05, all). In HCAECs cells knocked down for ABCA1, the 5A/PLPC complex failed to inhibit TNFα-induced phosphorylation of IκB and NF-κB p65 nuclear translocation, whereas (A-I)rHDL significantly inhibited phosphorylation of IκB by 39% (to 61±10) and NF-κB p65 nuclear translocation by 26% (to 74±7) (Fig 4D). Experiments performed in HCAECs cells transfected with a control non-silencing siRNA yielded results similar to those obtained in non-transfected cells (not shown).

Anti-oxidant effects of the 5A/PLPC complex in human artery endothelial cells

We next determined whether the 5A/PLPC complex and discoidal (A-I)rHDL were also able to inhibit TNFα-induced ROS, and whether this was dependent on the expression of ABCA1. HCAECs were loaded with dihydroethidium and exposed to TNFα (Fig. 5A). In line with previous reports18, 19, this resulted in a 42±5% increase in ethidium fluorescence (p<0.05), which was reduced by the presence of (A-I)rHDL (from 142±5% to 99±7%, p<0.05) and by the presence of the 5A/PLPC complex (from 142±5% to 110±6%, p<0.05) (Fig. 5A). We performed a similar series of experiments in HCAECs knocked down for ABCA1 and found that the 5A/PLPC complex failed to inhibit TNFα-mediated ROS expression, whereas (A-I)rHDL significantly inhibited it, although not back to baseline levels (Fig. 5B). Experiments performed in HCAECs cells transfected with a control non-silencing siRNA yielded results similar to those obtained in non-transfected cells (not shown).

Figure 5.

Figure 5

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TNFα induced ROS production in HCAECs exposed to PBS, (A-I)rHDL (Final apoA-I concentration 1mg/mL), or the 5A/PLPC complex (final peptide concentration 1mg/mL) for 16 hours. ROS generation was detected using dihydroethidum (DHE) fluorescence. (A) Nuclear DHE fluorescence in HCAECs. (B) Nuclear DHE fluorescence in HCAECs transfected with ABCA1 siRNA (50nM). Results are expressed as mean±SEM of three independent experiments performed in triplicates (*p<0.05 vs. control (C), **p<0.05 vs. PBS).

Discussion

The 5A peptide, like apoA-I, contains high and low lipid affinity helixes, and has been shown to specifically remove cellular cholesterol from lipid microdomains formed by the transporter ABCA114. Here we investigated whether this peptide also shares other potentially atheroprotective functions of apoA-I. We found that the 5A peptide displays antioxidant and anti-inflammatory properties similar to those of apoA-I in both (i) an animal model of acute vascular oxidation and inflammation and (ii) a model of agonist-induced oxidation and inflammation in human coronary artery endothelial cells. Moreover, we showed in the in vitro model that the anti-inflammatory and antioxidant effects of the 5A peptide are mediated via the ABCA1 transporter and NF-κB signalling pathways.

Our laboratory has previously reported that the application of a non-occlusive silastic collar around the carotid arteries of normolipemic rabbits promotes an acute inflammatory response characterized by extensive neutrophil infiltration in the intima-media associated with a dramatic up-regulation of ICAM-1 and VCAM-1 expression on the luminal surface of the endothelium4, 16. In the present study, lipid-free apoA-I, (A-I)rHDL and the 5A/PLPC complex reduced the inflammatory response to this insult and inhibited VCAM-1 gene expression by 60-70%. This is consistent with an inhibition of VCAM-1 at the transcriptional level, because the primary regulator of VCAM-1 expression is NF-κB17. Indeed, our cellular experiments indicate that (A-I)rHDL and the 5A/PLPC complex prevent TNFα induced NF-κB activation. Since NF-κB is extremely sensitive to cellular redox status, it may be speculated that the anti-inflammatory and antioxidant effects of both apoA-I and the 5A peptide are coupled, at least in part20.

Among the major sources of ROS, which contribute to the pathogenesis of atherosclerosis, is the NADPH oxidase, a multi-subunit family of enzymes with each member being distinguished by the specific Nox catalytic subunit. Nox2-containing NADPH oxidase is mainly expressed in phagocytic cells, including neutrophils, whereas Nox4-containing NADPH oxidase is expressed in vascular cells, such as smooth muscle cells and endothelial cells21-23. Using DHE staining, we showed that both apoA-I and the 5A/PLPC complex are able to inhibit ROS generation in the vessel wall. The expression of Nox4 within the endothelium was reduced in the collared arteries of apoA-I and 5A/PLPC infused rabbits at the mRNA level. The expression of Nox2 was also decreased primarily as a result of reduced neutrophil recruitment within the arterial wall. Thus, the ability of apoA-I and the 5A mimetic peptide to act at multiple sites in preventing oxidation may account for their potency in reducing atherosclerosis [Remaley AT, manuscript in preparation].

Only a few apoA-I mimetics have been shown to have direct and/or indirect antioxidant effects. The 4F peptide, for example, inhibits the formation of oxidized lipids in LDL24, 25, prevents the association of the oxidant myeloperoxidase with HDL in high-fat fed LDLr-knockout mice24, and increases HDL antioxidant paraoxonase activity in atherosclerosis-prone apoE knockout mice26. Another peptide that only contains 4 amino acids (KRES) was also found to reduce lipoprotein lipid hydroperoxide content and increase paraoxonase activity in HDL27. The mechanisms by which the 5A/PLPC complex displays antioxidant properties appear to be different from those displayed by the 4F and KRES peptides. Unlike the 4F peptide, the hydrophobicity of the 5A peptide is reduced compared with the prototypical 18A/37pA peptide 12. In that respect, it is not surprising that the 4F peptide acts as a scavenger of oxidized lipids from cells or circulating lipoproteins25, 27, 28. The 5A peptide, by contrast, seems to exert its anti-inflammatory and antioxidant effects mainly through interactions with endothelial cells via ABCA1.

Interestingly, HCAEC cells knocked down for ABCA1 had increased baseline VCAM-1 and ICAM-1 expression. This is in line with a recent report showing that lipopolysaccharide-treated ABCA1 deficient mouse primary macrophages not only accumulate free cholesterol but also secrete more pro-inflammatory cytokines, and exhibit enhanced activation of NF-κB, compared with wild-type macrophages29. Indeed, we did not detect any P-IκB in non-stimulated and non-transfected HCAECs, whereas P-IκB could be detected in non-stimulated HCAECs knocked down for ABCA1 (Fig. 4C&D). Since the 5A peptide has been shown to promote cellular cholesterol efflux specifically via ABCA114, it may be speculated that the ability of the 5A peptide to reduce the cholesterol content of cells that accounts for its anti-inflammatory and antioxidant properties. But the effect of apoA-I on various parameters of inflammation were not blocked by ABCA1 silencing in our cellular model, indicating that other transporters (e.g. ABCG1) and/or receptors (e.g. SR-B1) may also be involved in mediating the anti-inflammatory and antioxidant effects of the full-length apoA-I. However, our results do not indicate that these properties of the 5A peptide are cholesterol driven since cholesterol efflux is also reduced toward (A-I)rHDL upon ABCA1 knock-down and yet the anti-inflammatory and antioxidant properties are not altered.

In summary, we have shown that the 5A peptide displays antioxidant and anti-inflammatory properties similar to these of apoA-I, both in acute in vivo and in vitro models, and that in vitro these effects of the 5A peptide are mediated primarily via ABCA1 and the NF-κB signalling pathway. The 5A peptide was recently found to reduce en face atherosclerosis lesions by 60% in apoE −/− mice as has been observed with other similar peptides [Remaley AT, manuscript in preparation]. Future studies relating the in vitro properties of this peptide and other peptides with their in vivo effect on atherosclerosis in animal models will enhance our understanding of the mechanisms by which they inhibit atherosclerosis. This information will be valuable in providing a rationale for the design of apoA-I mimetic peptides for use as therapeutic agents in humans12. It will also likely lead to a better understanding of the anti-atherogenic properties of HDL and perhaps to better diagnostic tests based on HDL for assessing cardiovascular risk.

Condensed Abstract.

In addition to cellular cholesterol efflux, the 5A mimetic peptide also displays the anti-oxidant and anti-inflammatory properties of apoA-I, as shown in rabbit arteries and in vascular endothelial cells in culture. These properties are mediated via the ABCA1 transporter and NF-κB signalling pathways.

Supplementary Material

Supp1

Acknowledgements

We thank K. Berry, L. Hou, F. Charlton, F. Moheimani and I. Sotirchos for excellent technical assistance.

Funding - This work was funded in part by a program grant from the National Health and Medical Research Council (NHMRC) of Australia and by a grant in aid G08S3700 to KAR and GL from the National Heart Foundation of Australia.

Footnotes

Disclosure - None

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