The 5A Apolipoprotein A-I Mimetic Peptide Displays Antiinflammatory and Antioxidant Properties In Vivo and In Vitro
Objectives— The apolipoprotein (apo)A-I mimetic peptide 5A is highly specific for ATP-binding cassette transporter (ABC)A1-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 high-density lipoprotein (HDL) containing apoA-I ([A-I]rHDL), or the 5A peptide complexed with phospholipids (1-palmitoyl-2-linoleoyl phosphatidylcholine [PLPC]), before inserting a collar around the carotid artery. Human coronary artery endothelial cells (HCAECs) were incubated with (A-I)rHDL or 5A/PLPC before stimulation with tumor necrosis factor α.
Results— ApoA-I, (A-I)rHDL, and 5A/PLPC reduced the collar-mediated increase in (1) endothelial expression of cell adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1; (2) production, as well as the expression of the Nox4 catalytic subunits of the NADPH oxidase; and (3) infiltration of circulating neutrophils into the carotid intima–media. In HCAECs, both 5A/PLPC and (A-I)rHDL inhibited tumor necrosis factor-α–induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, as well as the nuclear factor κB signaling cascade and 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.
High-density lipoprotein (HDL) protects against the development of atherosclerosis by mediating reverse cholesterol transport, a pathway by which excess cholesterol is removed from peripheral cells such as plaque macrophages, to the liver for excretion.1,2 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 has been shown to inhibit these processes.3 In vitro and in vivo studies have demonstrated that HDL inhibits endothelial expression of adhesion molecules, thereby preventing monocyte recruitment into the arterial wall.4 HDL also removes and/or inactivates oxidized lipids from low-density lipoprotein (LDL) particles.5 In addition, HDL decreases production and inactivates neutrophil NADPH oxidase, a respiratory burst enzyme that is an important source of reactive oxygen species (ROS) in the vessel wall.6
Acute intravenous infusions of HDL7 have been shown to have rapid and beneficial effects on the arterial wall. Infusion of reconstituted HDL (rHDL) containing apolipoprotein (apo)A-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 or endotoxin is a limitation of this approach. In contrast, synthetic peptide analogs of the amphipathic helices of apoA-I, which are considerably easier and less costly to produce, offer an alternative approach for rHDL therapy.11,12 ApoA-I mimetic peptides also offer several other possible advantages: namely they can be administered orally and 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 asymmetrical variants of the prototypical 37pA bihelical apoA-I mimetic peptide, which contains 2 identical class A amphipathic helices linked by a proline residue.13 This was achieved by substituting nonpolar amino acids on the hydrophobic face of the COOH terminal helix with alanine.14 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 ATP-binding cassette transporter (ABC)A1-mediated cholesterol efflux, as well as the lowest cytotoxicity.14
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 antiinflammatory 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.
Materials and Methods
For a detailed description of the methods in this study, see the supplemental Materials and Methods section, available online at http://atvb.ahajournals.org.
Peptide Synthesis and Solubilization
The 5A bihelical 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 high-performance liquid chromatography. 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, Ala) at a 1:8 molar ratio, using a colyophilization procedure.14 The peptide: PLPC complexes were dissolved in NaCl 0.9% and sterilized using 0.2-mm filters. The peptide and phospholipid content of the 5A/PLPC complexes were quantitated using the bicinchoninic acid 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 was separated by sequential ultracentrifugation (1.063<d<1.21 g/mL) and delipidated, and apoA-I was purified by anion exchange chromatography on a Q Sepharose Fast Flow column attached to an fast protein liquid chromatography (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, was prepared (initial PLPC:apoA-I molar ratio 100:1) by the cholate dialysis method.15 Lipid-free apoA-I and discoidal (A-I)rHDL was 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.
Plasma samples collected (1) before infusion, (2) after collar implantation, and (3) before euthanasia were assayed for lipids. Preinfusion values were 0.79±0.09 mmol/L total cholesterol, 0.52±0.08 mmol/L HDL-cholesterol, and 0.91±0.09 mmol/L 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 euthanasia (48 hours later).
Antiinflammatory Effects of the 5A/PLPC Complex in Rabbit Arteries
Compared with noncollared arteries, the collared arteries of the saline-infused rabbits had markedly increased endothelial expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, as well as increased infiltration of circulating neutrophils into the intima–media (Figure 1). This is consistent with what has been reported previously.4 ICAM-1 is expressed constitutively by endothelial cells but at levels that could not be detected by immunohistochemistry in noncollared arteries, as reported previously.16 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) (Figure 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 (Figure 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 (Figure 1). Thus, a single 20 mg/kg infusion of the 5A/PLPC complex was as effective (P>0.85, all) as a single 8 mg/kg infusion of lipid-free apoA-I or (A-I)rHDL at reducing collar-induced acute inflammation in New Zealand white (NZW) rabbit carotid arteries. In addition, we isolated mRNA from sections of collared and noncollared arteries from the saline-, 5A/PLPC complex–, and lipid-free apoA-I–infused rabbits. Compared with the noncollared arteries, the collared arteries from the saline-treated animals had higher VCAM-1 mRNA levels normalized to 18S (100±30 versus 20±4 arbitrary units, 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 arbitrary units, P<0.05) and 69% (to 31±17 arbitrary units, P<0.05), respectively.
Antioxidant Effects of the 5A/PLPC Complex in Rabbit Arteries
To evaluate the antioxidant properties of the 5A mimetic peptide, we measured Nox2 and Nox4 mRNA levels in the noncollared and collared arteries of saline-, 5A/PLPC complex–, and lipid-free apoA-I–infused rabbits. Compared with noncollared carotid arteries, the collared arteries from saline-treated animals had increased Nox2 (100±20 versus 23±7, P<0.05) and Nox4 (100±17 versus 26±9, P<0.05) (Figure 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 antioxidant 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 on PMA stimulation by 50%. Neither (A-I)rHDL nor the 5A/PLPC complex altered Nox2 expression significantly in either the stimulated or the nonstimulated conditions (supplemental Figure I). 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 dihydroethidium (DHE) nuclear fluorescence to measure ROS levels in the collared and noncollared arteries (Figure 2B). Compared with noncollared arteries, DHE fluorescence was increased in the collared arteries from the saline infused rabbits, which was consistent with increased ROS expression (22±0.8 versus 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 versus saline-infused animals; P=0.92 for 5A/PLPC versus lipid-free apoA-I).
Antiinflammatory Effects of the 5A/PLPC Complex in Human Artery Endothelial Cells
By flow cytometry, we investigated 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 tumor necrosis factor (TNF)α. Both (A-I)rHDL and the 5A/PLPC complex dose-dependently reduced the TNFα induced expression of VCAM-1 (Figure 3A) and ICAM-1 (Figure 3B), reaching a maximal response at 1 mg/mL. We showed in a time course study that 2 to 24 hours of incubation of either (A-I)rHDL or the 5A/PLPC complex significantly reduced the TNFα-induced expression of VCAM-1 (Figure 3C) and ICAM-1 (Figure 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 (Figure 4A). As judged by Evans blue staining, this had no effect on cell viability (not shown). The addition of the proinflammatory 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 versus PBS for all).
Because the 5A mimetic peptide is highly specific for ABCA1-mediated cellular cholesterol efflux,14 we next assessed whether the antiinflammatory properties of the 5A/PLPC complex are also mediated through interactions with the ABCA1 transporter. We transiently transfected HCAECs with specific small interfering (si)RNAs to downregulate ABCA1 protein expression by >80% (24 to 48 hours after transfection) in our cellular model (supplemental Figure II). 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 nontransfected cells (Figure 4B). To further investigate whether ABCA1 plays a role in the inflammatory cascade, we have measured the activation of the nuclear factor (NF)-κB, a transcription factor that modulates VCAM-1 and ICAM-1 gene expression on inflammatory insult.17 This was performed in both nontransfected HCAECs, as well as in HCAECs knocked-down for ABCA1. NF-κB is retained in the cytoplasm as part of a complex with IκB. 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 Figure 4C, TNFα treatment was associated with a 8-fold increased phosphorylation of IκB and a 2-fold increased nuclear translocation of NF-κB p65 (P<0.01, all). Preincubation of the cells for 16 hours with either 1 mg/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) (Figure 4D). Experiments performed in HCAECs cells transfected with a control nonsilencing siRNA yielded results similar to those obtained in nontransfected cells (not shown).
Antioxidant 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 DHE and exposed to TNFα (Figure 5A). In line with previous reports,18,19 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) (Figure 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 to baseline levels (Figure 5B). Experiments performed in HCAECs cells transfected with a control nonsilencing siRNA yielded results similar to those obtained in nontransfected cells (not shown).
The 5A peptide, like apoA-I, contains high and low lipid-affinity helices and has been shown to specifically remove cellular cholesterol from lipid microdomains formed by the transporter ABCA1.14 Here, we investigated whether this peptide also shares other potentially atheroprotective functions of apoA-I. We found that the 5A peptide displays antioxidant and antiinflammatory properties similar to those of apoA-I in both (1) an animal model of acute vascular oxidation and inflammation and (2) a model of agonist-induced oxidation and inflammation in human coronary artery endothelial cells. Moreover, we showed in the in vitro model that the antiinflammatory and antioxidant effects of the 5A peptide are mediated via the ABCA1 transporter and NF-κB signaling pathways.
Our laboratory has previously reported that the application of a nonocclusive 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 upregulation of ICAM-1 and VCAM-1 expression on the luminal surface of the endothelium.4,16 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% to 70%. This is consistent with an inhibition of VCAM-1 at the transcriptional level, because the primary regulator of VCAM-1 expression is NF-κB.17 Indeed, our cellular experiments indicate that (A-I)rHDL and the 5A/PLPC complex prevent TNFα-induced NF-κB activation. Because NF-κB is extremely sensitive to cellular redox status, it may be speculated that the antiinflammatory and antioxidant effects of both apoA-I and the 5A peptide are coupled, at least in part.20
Among the major sources of ROS, which contribute to the pathogenesis of atherosclerosis, is the NADPH oxidase, a multisubunit 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 cells.21–23 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 (A.T.R., 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 LDL,24,25 prevents the association of the oxidant myeloperoxidase with HDL in high-fat fed LDL receptor knockout mice,24 and increases HDL antioxidant paraoxonase activity in atherosclerosis-prone apoE knockout mice.26 Another peptide that only contains 4 amino acids (KRES) was also found to reduce lipoprotein lipid hydroperoxide content and increase paraoxonase activity in HDL.27 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 lipoproteins.25,27,28 The 5A peptide, by contrast, seems to exert its antiinflammatory 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 proinflammatory cytokines and exhibit enhanced activation of NF-κB, compared with wild-type macrophages.29 Indeed, we did not detect any P-IκB in nonstimulated and nontransfected HCAECs, whereas P-IκB could be detected in nonstimulated HCAECs knocked down for ABCA1 (Figure 4C and 4D). Because the 5A peptide has been shown to promote cellular cholesterol efflux specifically via ABCA1,14 it may be speculated that the ability of the 5A peptide to reduce the cholesterol content of cells that accounts for its antiinflammatory 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 (eg, ABCG1) and/or receptors (eg, SR-B1) may also be involved in mediating the antiinflammatory 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 because cholesterol efflux is also reduced toward (A-I)rHDL on ABCA1 knockdown and yet the antiinflammatory and antioxidant properties are not altered.
In summary, we have shown that the 5A peptide displays antioxidant and antiinflammatory 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 signaling 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 (A.T.R., 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 humans.12 It will also likely lead to a better understanding of the antiatherogenic properties of HDL and perhaps to better diagnostic tests based on HDL for assessing cardiovascular risk.
We thank K. Berry, L. Hou, F. Charlton, F. Moheimani, and I. Sotirchos for excellent technical assistance.
Sources of Funding
This work was funded in part by a program grant from the National Health and Medical Research Council (NHMRC) of Australia and by grant-in-aid G08S3700 from the National Heart Foundation of Australia (to K.-A.R. and G.L.).
Received June 4, 2009; revision accepted November 25, 2009.
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