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The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins
Atherosclerosis 217 (2011) 395–400
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The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins Belinda A. Di Bartolo a , Stephen J. Nicholls b , Shisan Bao c , Kerry-Anne Rye a,f,g , Alison K. Heather d,e , Philip J. Barter a,f , Christina Bursill a,∗ a
Lipid Research Group, Heart Research Institute, Sydney, Australia Department of Cell Biology, Cleveland Clinic, Cleveland, OH, USA c Discipline of Pathology, University of Sydney, Australia d Gene Regulation Group, Heart Research Institute, Sydney, Australia e Department of Medical and Molecular Biosciences, University of Technology, Sydney, Australia f Department of Medicine, University of Sydney, Australia g Department of Medicine, University of Melbourne, Australia b
a r t i c l e
i n f o
Article history: Received 9 August 2010 Received in revised form 31 March 2011 Accepted 1 April 2011 Available online 16 April 2011 Keywords: Apolipoprotein A-I Apolipoprotein A-I mimetic peptides Inflammation Adhesion molecules Chemokines
a b s t r a c t Objectives: Mimetic peptides of apolipoprotein A-I (apoA-I) present a new strategy for promoting the biological activity of high density lipoproteins (HDL). This study aimed to compare the anti-inflammatory effects of ETC-642, a new apoA-I mimetic peptide, with discoidal reconstituted HDL (rHDL). Methods: New Zealand White rabbits (n = 42) received daily infusions of saline, rHDL or discoidal complexes of an amphipathic peptide, ETC-642 (1–30 mg/kg), prior to insertion of non-occlusive carotid collars. Human coronary artery endothelial cells (HCAECs) were pre-incubated with ETC-642 or rHDL before TNF-␣ stimulation. Monocyte adhesion was investigated by pre-incubating HCAECs with rHDL or ETC-642, stimulating with TNF-␣ and incubating with THP-1 monocytes. Results: Infusion of ETC-642 resulted in dose-dependent reductions of collar-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the artery wall (p < 0.05). Pre-incubation of HCAECs with ETC-642 and rHDL reduced TNF-␣-induced THP-1 monocyte adhesion (p < 0.01). Furthermore, ETC-642 and rHDL treatment reduced TNF-␣ induced mRNA levels of inflammatory markers VCAM-1, fractalkine, MCP-1 and the p65 subunit of NF-B (p < 0.05). Conclusion: These studies demonstrate that ETC-642 exhibits anti-inflammatory properties that are comparable to apoA-I both in vivo and in vitro and that these effects are mediated via the NF-B signaling pathway. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The atheroprotective nature of high-density lipoproteins (HDL) has been well established [1]. In addition to its well-characterized role in the promotion of reverse cholesterol transport [2], HDL also possesses antioxidant and antithrombotic properties and, in particular, anti-inflammatory properties [3]. In cell culture systems, HDL inhibits endothelial cell expression of proinflammatory adhesion molecules and chemokines and, as a consequence, reduces monocyte chemotaxis [4]. Animal models have also shown that transgenic over-expression of the main protein component of HDL, apolipoprotein (apo)A-I and the infusion of high-dose apoA-IMilano reduces the inflammatory
∗ Corresponding author at: The Heart Research Institute, 7 Eliza St, Newtown, NSW 2050, Australia. Tel.: +61 2 8208 8900; fax: +61 2 9565 5584. E-mail address: [email protected] (C. Bursill). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.04.001
composition of experimental atheroma by reducing infiltrating macrophages and plaque lipid in apoE knockout mice [5]. ApoA-I, with a chain length of 243 amino acids, is the most abundant HDL apolipoprotein. The size of this protein limits its usefulness as a therapeutic agent. However, peptides as short as 18 amino acids, based on the apoA-I structure, have been synthesised and shown to possess some properties of full length apoA-I. Animal studies demonstrate that mimetic peptides reduce atherosclerotic lesions, improve endothelial dysfunction, decrease proinflammatory lipoproteins and diminish monocyte recruitment, all independent of any increase in HDL cholesterol levels [6–9]. Together these studies suggest that apoA-I mimetic peptides may confer benefits comparable to those of rHDL containing full length apoA-I. Given that mimetic peptides are easier to synthesize than full-length apoA-I, there is considerable interest in developing them as potential therapeutic agents, particularly as apoA-I has been shown in human
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trials to cause plaque regression and reduce inflammatory markers [10,11]. The apoA-I mimetic peptide used in our study contains a 22-amino acid synthetic amphipathic peptide based on the sequence of L-amino acid residues: P-V-L-D-L-F-R-E-L-L-N-E-L-LE-A-L-K-Q-K-L-K (ESP 24218; unpublished, Pfizer). This peptide was combined with two naturally occurring phospholipids, sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The resulting peptide/phospholipid complex (named ETC642) has a molecular weight of 2623 and a molar ratio of ESP 24218 to sphingomyelin to DPPC of 1:3.75:3.75. The peptide, ESP 24218 mimics the structure of the amphipathic ␣-helices of apoA-I when combined with lipids. The anti-inflammatory properties of ETC-642 remain unknown. These studies demonstrate for the first time that intravenous infusion of ETC-642 is as effective as apoA-I-containing rHDL at inhibiting acute vascular inflammation in a rabbit carotid collar model. In vitro studies also find that ETC-642 reduces inflammatory markers VCAM-I, MCP-1, fractalkine and the NF-B p65 subunit as effectively as rHDL. These studies demonstrate the efficacy of ETC-642 as a potential agent for reducing acute vascular inflammation.
White rabbits (n = 7/group). The rabbits were infused intravenously with either saline, rHDL containing 25 mg of apoA-I (8 mg/kg) or ETC-642 at concentrations of 1, 3, 10 or 30 mg/kg, immediately and at 24 h after collar placement. Rabbits were sacrificed 48 h after collar placement. VCAM-1 and ICAM-1 endothelial expression were assessed on carotid arteries and plasma chemistries were determined as described in Supplementary Information (SI). In vitro, the anti-inflammatory properties of ETC-642 were compared to rHDL in human coronary endothelial cells (HCAECs). Monocyte adhesion was assessed as described in SI. Total mRNA was extracted from treated cells and mRNA levels of VCAM-1, ICAM-1, MCP-1, fractalkine and NF-B were measured by real-time PCR. 3. Results 3.1. Plasma lipid concentrations
HDLs were isolated from pooled samples of donated human plasma (Gribbles Pathology, Adelaide, SA, Australia) by sequential ultracentrifugation in the 1.063–1.21 g/mL density range. The HDL were delipidated and apoA-I isolated by anion chromatography on a Q-Sepharose Fast Flow column (GE Healthcare Biosciences, Waukesha, WI, USA) attached to an fast protein liquid chromatography system [12].
Plasma concentrations of triglyceride, phospholipid and free and total cholesterol are presented in Supplementary Table 1. Compared to baseline values, infusions of saline, rHDL and ETC642 at 1, 3 and 10 mg/kg had no effect on the concentration of triglyceride, phospholipid, free or total cholesterol in either the post-treatment sample (collected 1 h after the first dosing) or in the sample collected at time of sacrifice, 48 h after insertion of the collar. At 1 h post-treatment, 30 mg/kg of ETC-642 increased plasma triglyceride levels by 0.84 ± 0.32 mmol/L (p < 0.05), plasma total cholesterol by 1.41 ± 0.53 mmol/L (p < 0.05), plasma free cholesterol by 1.16 ± 0.44 mmol/L (p < 0.05) and plasma phospholipids by 1.8 ± 0.85 mmol/L (p < 0.05) relative to baseline levels. At study end, neither triglycerides, total cholesterol, free cholesterol or phospholipids were significantly elevated compared to baseline in animals receiving 30 mg/kg of ETC-642.
2.2. Preparation of rHDLs containing apoA-I and PLPC
3.2. Lipoprotein analysis
Discoidal rHDL containing apoA-I complexed to 1-palmitoyl2-linoleoyl phosphatidylcholine (PLPC) (Avanti Polar Lipids, Alabaster, AL, USA) were prepared using the cholate dialysis method [13]. The phospholipid:apoA-I molar ratio was 100:1. The resulting rHDL were dialysed extensively against endotoxin-free phosphate buffered saline (PBS) (pH 7.4) before use.
HDL and LDL cholesterol concentrations were analysed at the commencement of the study, 1 h post treatment and at the time of sacrifice (Table 1). There were no changes in lipoprotein levels in animals treated with saline, rHDL or ETC642 at doses of 1 and 3 mg/kg. Animals receiving 10 mg/kg of ETC-642 exhibited a significant increase in HDL cholesterol 1 h after treatment from 0.37 ± 0.04 mmol/L at baseline to 0.66 ± 0.05 mmol/L (p < 0.05), which subsequently returned to baseline levels at sacrifice. A similar change was observed with infusions of ETC-642 at 30 mg/kg, where at 1 h post-treatment, HDL cholesterol levels had increased from 0.54 ± 0.09 mmol/L at baseline to 1.73 ± 0.1 mmol/L (p < 0.05). At the conclusion of the study HDL cholesterol had returned to baseline levels. LDL cholesterol concentrations were significantly increased from 0.22 ± 0.08 mmol/L at baseline to 0.42 ± 0.09 mmol/L at sacrifice in animals receiving 30 mg/kg of ETC-642 (p < 0.05). In all other conditions and at all other time points LDL cholesterol remained unchanged.
2. Materials and methods 2.1. Isolation of apolipoprotein A-I
2.3. Preparation of ETC-642 The ETC-642 was provided as a lyophilized solid by Esperion Therapeutics, Pfizer (Groton, MI, USA). The complex consists of ESP-2418, a peptide containing 22 amino acid residues as a synthetic amphipathic peptide based on the sequence of L-amino acid residues: P-V-L-D-L-F-R-E-L-L-N-E-L-L-E-A-L-K-Q-KL-K. ESP-2418 is complexed to two phospholipids, sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The resulting peptide/phospholipid complex is called ETC-642. The lyophilized solid was rehydrated with 50 mL of a sterile bicarbonate saline solution (Esperion Therapeutics, Pfizer, Groton, MI, USA) gently swirled to mix. Three cycles of warming at 50 ◦ C and cooling at room temperature was performed to ensure the mimetic peptide was sufficiently reconstituted. The final concentration of ETC-642 was 10 mg/mL and was stored at 4 ◦ C before use. 2.4. Animal study and in vitro work Non-occlusive peri-arterial collars were applied to the left common carotid artery of normo-cholesterolaemic New Zealand
3.3. ETC-642 reduces endothelial adhesion molecule expression The ability of ETC-642 to reduce adhesion molecule expression in the carotid endothelium was investigated. The collar-induced changes to ICAM-1 are shown in Fig. 1. Collar insertion resulted in a significant increase in ICAM-1 from 1.98 ± 0.35 image units in non-collared arteries to 17.5 ± 1.72 image units (p < 0.001). The increased expression of ICAM-1 in collared arteries was reduced by 94% after infusion of rHDL (1.1 ± 3.38 image units, p < 0.05). No effect was seen at 1 mg/kg, but infusions of ETC-642 at 3 mg/kg
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Table 1 Plasma lipoprotein profiles of animals infused with saline, rHDL (8 mg/kg) and ETC-642 at concentrations of 1, 3, 10 and 30 mg/kg at baseline, post-treatment and at time of sacrifice (n = 7 in each group). Results are expressed as mean ± SEM. Treatment
HDL – C (mmol/L) Baseline
Saline rHDL ETC-642 1 mg/kg ETC-642 3 mg/kg ETC-64210 mg/kg ETC-64230 mg/kg *
0.47 0.42 0.54 0.46 0.37 0.54
± ± ± ± ± ±
0.03 0.05 0.13 0.06 0.04 0.09
LDL – C (mmol/L) Post-treatment 0.41 0.46 0.48 0.48 0.66 1.73
± ± ± ± ± ±
0.03 0.04 0.1 0.06 0.05* 0.1*
Time of sacrifice 0.42 0.43 0.4 0.42 0.34 0.4
± ± ± ± ± ±
0.03 0.07 0.05 0.33 0.04 0.08
Baseline 0.14 0.12 0.25 0.16 0.19 0.22
± ± ± ± ± ±
0.02 0.02 0.05 0.04 0.04 0.08
Post-treatment 0.1 0.1 0.26 0.15 0.23 0.19
± ± ± ± ± ±
0.01 0.02 0.08 0.04 0.04 0.06
Time of sacrifice 0.17 0.16 0.15 0.18 0.22 0.42
± ± ± ± ± ±
0.03 0.04 0.02 0.03 0.04 0.09*
p < 0.05 compared to baseline.
reduced expression by 60% (6.30 ± 2.74 image units; p = 0.05), 10 mg/kg reduced expression by 57% (6.77 ± 3.05 image units) and 30 mg/kg reduced the collar-induced expression of ICAM-1 by 91% (1.26 ± 0.4 image units, p < 0.05) compared to saline-infused animals with carotid collars. As was the case with ICAM-1 expression, collar insertion resulted in a significant increase in VCAM-1 compared to noncollared carotid arteries (0.12 ± 0.02 image units in non-collared arteries compared to 1.78 ± 0.19 image units, p < 0.001). The increased expression of VCAM-1 in collared arteries was decreased by 98% after infusion of rHDL (0.02 ± 0.09 image units, p < 0.05). Infusions of ETC-642 decreased VCAM-1 expression at all treatment concentrations. The dose of 1 mg/kg decreased collar-induced expression by 89% (0.18 ± 0.22, p < 0.05), 3 mg/kg reduced expression by 71% (0.59 ± 0.13 image units, p < 0.05), and 10 mg/kg, reduced expression by 94% (0.09 ± 0.07 image units, p < 0.05), compared to collar-induced control animals. Infusion of ETC-642 at 30 mg/kg also significantly decreased the collar-induced expression by 92% (0.14 ± 0.07 image units, p < 0.05) compared to control animals.
endothelial cells increased by 2.4-fold, compared to unstimulated HCAECs (p < 0.05, Fig. 2). When HCAECs were pre-incubated with rHDL, prior to stimulation with TNF-␣, THP-1 monocyte adhesion was reduced by 41% (p < 0.05), compared to stimulated HCAEC controls. Furthermore, pre-treatment of HCAECs with ETC-642 reduced the TNF-␣-induced monocyte adhesion to HCAECs by 60% (p < 0.01). 3.5. Effect of ETC-642 on adhesion molecule and chemokine gene expression Stimulation with TNF-␣ substantially increased the mRNA levels of VCAM-1, ICAM-1, fractalkine and MCP-1 (5.6-fold, 7.5-fold, 36fold and 10-fold respectively), p < 0.001 (Fig. 3). The TNF-␣-induced increase in VCAM-1 was reduced in cells that were pre-incubated with either rHDL (52%; p < 0.001) or ETC-642 (20%; p < 0.01), when compared to the stimulated control cells. Pre-incubation of cells with rHDL or ETC-642, prior to TNF-␣ stimulation, also reduced fractalkine mRNA levels by 44% (p < 0.001) and 25% (p < 0.01), respectively and MCP-1 levels by 45% (p < 0.001) and 46% (p < 0.001). Finally, we demonstrate that neither rHDL nor ETC-642 were able to reduce the TNF-␣ induced increase in ICAM-1
3.4. Monocyte adhesion 3.6. ETC-642 reduces NF-B gene expression Here we investigated the ability of rHDL and ETC-642 to alter the adherence of monocytes to endothelial cells. When HCAECs were incubated with TNF-␣, adhesion of THP-1 monocytes to the
NF-B regulates a host of inflammatory genes including VCAM1, MCP-1 and fractalkine. To determine if ETC-642 and rHDL
Fig. 1. ETC-642 reduces endothelial adhesion molecule expression. Panel A: Immunohistochemical staining for ICAM-1 and VCAM-1 in representative collared carotid artery sections from NZW rabbits infused with either saline, rHDL or ETC-642 (30 mg/kg). Panel B: Endothelial expression of ICAM-1 and VCAM-1 were quantified as described in Section 2. Results are expressed as mean ± SEM. *p < 0.05 compared to saline; **p < 0.01 compared to saline.
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Fig. 4. ETC-642 reduces NF-B. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC-642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. mRNA levels of the p65 subunit of NF-B were determined by real time PCR. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0001.
Fig. 2. ETC-642 reduces monocyte adhesion to endothelial cells. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC-642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. HCAECs were washed with PBS and incubated with fluorescently labelled THP-1 monocytes (1 × 106 cell/well) for 1 h. Fluorescence was measured using 490 nm (excitation) and 540 nm (emission). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
decrease the expression of these genes via NF-B, we measured mRNA levels of the p65 subunit. Results show that stimulation with TNF-␣ increased the level of p65 by 3.4-fold, p < 0.001. Preincubation of HCAECs with rHDL and ETC-642 significantly reduced mRNA levels of the p65 subunit (45% and 52%, respectively; p < 0.001), when compared to stimulated controls (Fig. 4).
4. Discussion Due to the multiple beneficial properties of HDL, there has been considerable interest in developing HDL-related therapeutics. ApoA-I is a large protein, containing 243 amino acids arranged in 10 amphipathic helices, thereby making its synthesis as a therapeutic agent difficult. This has driven the impetus to develop smaller peptides that are easily synthesised but still exhibit the same beneficial properties as apoA-I. A number of investigators have developed short peptides that bear no amino acid homology to apoA-I but retain the ability to efflux cholesterol from cells, one of the main functions of HDL [14]. The fact that short peptide sequences are considerably easier and cheaper to prepare than full length apoA-I make this an attractive option in the development of new therapies for treatment of cardiovascular disease [15].
Fig. 3. Effect of ETC-642 on chemokine and adhesion molecule gene expression. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. VCAM-1, ICAM-1, fractalkine and MCP-1 mRNA levels were quantified by real time PCR. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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In this article, we compare the anti-inflammatory properties of a new apoA-I mimetic peptide, ETC-642, with those of rHDL in order to validate its efficacy as an anti-inflammatory agent. ETC-642 is a complex consisting of an apoA-I mimetic peptide combined with phospholipids. In this study we found that administration of ETC642 significantly decreases adhesion molecule expression (VCAM1, ICAM-1) in collared rabbit carotid arteries and causes transient elevations in plasma lipid levels. Also, in vitro studies in HCAECs show that ETC-642 reduces monocyte adhesion, adhesion molecule expression and chemokine expression, via inhibition of NF-B, a pivotal regulator of inflammation. As reported previously, infusions of rHDL into NZW rabbits did not alter their lipid profiles [16]. In contrast, 1 h post-infusion with 30 mg/kg ETC-642 total cholesterol were significantly elevated. This appeared to be predominantly due to a significant increase in free cholesterol. Interestingly, analysis of the lipoprotein fractions revealed that there was a significant increase in HDL cholesterol at this time point, indicating that the extra free cholesterol was in the HDL fraction. This suggests that ETC-642 is promoting the efflux of free cholesterol from cells to HDL particles. The exact mechanism for these effects is not able to be delineated in the current study but is consistent with previous observations in which ETC-642 was found to mobilize cholesterol in pre-clinical studies and to transiently elevate circulating HDL cholesterol levels in patients [17,18]. Furthermore, another study found that a single injection of ETC-642 transiently elevated plasma cholesterol levels, 45 min after administration in rats [19]. In the current study it was found that the LDL cholesterol fraction was elevated at the time of sacrifice, 48 h after the administration of ETC-642. This implies that cholesterol was transferred from HDL to LDL via cholesterol ester transport protein [20]. An increase in LDL cholesterol could be considered adverse and suggests that a lower dose of ETC-642, such as 10 mg/kg, is potentially more beneficial as it still exhibits the same anti-inflammatory properties without the unwanted rise in LDL cholesterol. However, it is likely that this elevation in LDL cholesterol is transient. Human intervention studies have found that weekly infusions for 5 weeks of mimetic peptide ETC-216, did not change LDL cholesterol levels [10]. Application of peri-arterial collars resulted in a substantial increase in endothelial expression of both VCAM-1 and ICAM1 in the carotid artery. This increased expression was reduced by infusion of apoA-I-containing rHDL, consistent with previous studies [4,16]. The expression of VCAM-1 and ICAM-1 was also significantly and dose-dependently reduced by administration of ETC-642. Another mimetic peptide 5A, that interacts specifically with the ATP binding cassette transporter, ABCA-1, has also recently been found to reduce both ICAM-1 and VCAM-1 in collared rabbit carotid arteries [21], further supporting an anti-inflammatory role for mimetic peptides. Circulating monocytes adhere to endothelial cells after stimulation by proinflammatory cytokines. This process is mediated through a complex co-ordination of numerous proteins, including adhesion molecules and chemokines, on both the endothelial cells and the monocytes. It is likely that the decreased expression of VCAM-1, MCP-1 and fractalkine by rHDL and ETC-642, contributed to the inhibition of monocyte adhesion to endothelial cells. Interestingly, when cells are pre-treated with rHDL or ETC642, it leads to a reduction in endothelial cell VCAM-1 expression in vitro, even when removed from cells prior to cytokine stimulation [23]. This implies that exposure of cells to rHDL and ETC-642 modifies the endothelial cells making them resistant to stimulation for a prolonged period of time [24]. A possible mechanism for the rHDL/ETC-642-mediated suppression of inflammatory markers is via inhibition of the NF-B activation pathway. NF-B regulates VCAM-1, MCP-1 and fractalkine [25,26]. We found that both rHDL and ETC-642 reduced the p65 NF-B subunit. Consistent with this,
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the apoA-I mimetic peptide, 5A, was found to also suppress VCAM1 and ICAM-1 via a reduction in NF-B [21]. Additional studies with the 5A peptide delineated this mechanism further. The 5A peptide was found to also suppress the phosphorylation of IB␣, an upstream mediator of the NF-B pathway. Furthermore, siRNA studies revealed that inhibition of p65 by 5A was mediated through ABCA1 [21], thereby raising the possibility that ETC-642 may reduce p65 via this same pathway. The expression of endothelial cell adhesion proteins is increased in vitro in response to proinflammatory cytokines. HDL has been shown to inhibit cytokine-induced expression of cell surface adhesion molecules VCAM-1 and ICAM-1 in activated endothelial cells both in vitro [27,28] and in vivo [16,21,29]. In the current study we found that the apoA-I mimetic peptide, ETC-642, also reduces VCAM-1 and ICAM-1 expression on the endothelial surface of the arterial wall in vivo. In vitro studies also found that both rHDL and ETC-642 reduce the expression of VCAM-1 and the proinflammatory chemokines MCP-1 and fractalkine. This study provides further support for an anti-inflammatory role of rHDL and now also ETC-642. We did not, however, find that ETC-642 reduced ICAM1 expression in vitro, which is in contrast to our in vivo findings where ICAM-1 was significantly reduced. This maybe explained via post-translational modification of the ICAM-1 protein. Furthermore, ICAM-1 expression is less dependent on NF-B signaling than VCAM-1 or chemokines, which all have NF-B response elements upstream of their promoter regions [26]. ICAM-1 is also regulated by the ERK and JAK/STAT signaling pathways, which are not upstream of NF-B [30]. The lack of effect of rHDL and ETC642 on ICAM-1 maybe due to its regulation via these alternative signaling systems. One of the limitations of this study is that it is impossible to conclude whether the mimetic peptide or the phospholipid complexes are responsible for the anti-inflammatory properties of ETC-642. However, previous investigations, using the same rabbit model of acute inflammation have demonstrated that the phospholipid DPPC, as used for ETC-642, had no effect on adhesion molecule expression [16]. Furthermore, in vitro studies have found that lipidfree apoA1 was not as effective at reducing inflammation as rHDL (apoA1 + phospholipid) and that phospholipid vesicles alone had no effect [22]. Take together, it appears that whilst phospholipids enhance the anti-inflammatory properties of apoA1, they are ineffective on their own. This suggests that the anti-inflammatory properties of ETC-642 are likely to be maximal when the peptide and phospholipids are complexed together and that the peptide or phospholipids alone are likely to have either no effect or modest affects on inflammation. Consistent with this, in the optimisation of ETC-642 by Esperion, DPPC, sphingomyelin and the peptide were tested for their ability to mobilise and esterify cholesterol, two of the main steps in the reverse cholesterol transport pathway. Following injection into rabbits the peptide and the phospholipids were found to individually have no effect on mobilising cholesterol but when tested as a combination, achieved maximal effects on both cholesterol mobilisation and esterification (unpublished data). Despite the immense potential of mimetic peptides to treat a range of cardiovascular disorders, their translation to clinic has not been entirely successful due to findings that they cause adverse effects [10]. For example, patients receiving the mimetic peptide ETC-216 over a 5-week period were reported to have increased gastrointestinal problems, nausea and headaches. One other clinical study however, found that oral administration of D-4F to high-risk patients was tolerated well but only a single dose was given [31]. Overall, further studies on mimetic peptides seem warranted to facilitate their translation into clinic. In conclusion the apoA-I mimetic peptide, ETC-642, exhibits anti-inflammatory properties that are comparable to rHDL in vitro
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and in vivo. These studies highlight the therapeutic potential of mimetic peptides for the prevention of vascular inflammation and cardiovascular disease. Acknowledgements This study was supported by a Heart Research Institute PhD scholarship (Dr Di Bartolo), a Heart Foundation Career Development Fellowship (CR07S3331) and a Bushell Foundation grant (Dr Bursill). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.04.001. References [1] Förstermann U, Pollock JS, Schmidt HHHW, Heller M, Murad F. Calmodulindependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991;88:1788–92. [2] Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Annu Rev Physiol 1991;53:161–77. [3] Plautz G, Nabel EG, Nabel GJ. Introduction of vascular smooth muscle cells expressing recombinant genes in vivo. Circulation 1991;83:578–83. [4] Dinerman JL, Lawson DL, Mehta JL. Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation. Am J Physiol 1991;260:H698–701. [5] Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487–91. [6] Gibbs RA. DNA amplification by the polymerase chain reaction. Anal Chem 1990;62:1202–14. [7] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1989. [8] Ausubel FM, Brent R, Kingston RE, et al. Current protocols in molecular biology; 1991. [9] Luscher TF, Richard V, Tanner FC. Endothelium-derived vasoactive factors and their role in the coronary circulation. Trends Cardiovasc Med 1991;1:179–85. [10] Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J 1990;4:2868–80. [11] Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, Gewert D. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucleic Acids Res 1991;19:184. [12] Cercek B, Fishbein MC, Forrester JS, Helfant RH, Fagin JA. Induction of insulinlike growth factor I messanger RNA in rat aorta after balloon denudation. Circ Res 1990;66:1755–60.
[13] Lim CS, Chapman GD, Gammon RS, et al. Direct in vivo gene transfer into the coronary and peripheral vasculatures of the intact dog. Circulation 1991;83:2007–11. [14] Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 1987;51:987–1000. [15] Schwartz SM, Foy L, Bowen-Pope DF, Ross R. Derivation and properties of platelet-derived growth factor-independent rat smooth muscle cells. Am J Pathol 1990;136:1417–27. [16] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156–9. [17] Epstein SE, Siegall CB, Biro S, Fu Y-M, FitzGerald D, Pastan I. Cytotoxic effects of a recombinant chimeric toxin on rapidly proliferating vascular smooth muscle cells. Circulation 1991;84:778–87. [18] Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol 1990;15:1667–87. [19] Leinwand LA, Leiden JM. Gene transfer into cardiac myocytes in vivo. Trends Cardiovasc Med 1991;1:271–6. [20] Schmidt HHHW, Murad F. Purification and characterization of a human NO synthetase. Biochem Biophys Res Commun 1991;181:1372–7. [21] Conti MA, Sellers JR, Adelstein RS. Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains. Biochemistry 1991;30:966–70. [22] Baker PW, Rye KA, Gamble JR, Vadas MA, Barter PJ. Ability of reconstituted high density lipoproteins to inhibit cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilical vein endothelial cells. J Lipid Res 1999;40:345–53. [23] Timmermans PBMWM, Carini DJ, Chiu AT, et al. Angiotensin II receptor antagonists from discovery to antihypertensive drugs. Hypertension 1991;18:136– 42. [24] Nabel EG, Plautz G, Nabel GJ. Gene transfer into vascular cells. JACC 1991;17:189B–94B. [25] Vanhoutte PM. Vascular effects of serotonin and ischemia. J Cardiovasc Pharmacol 1990;16:S15–9. [26] Schwartz SM, Heimark RL, Majesky MW. Develomental mechanisms underlying pathology of arteries. Physiol Rev 1990;70:1177–209. [27] Califf RM, Fortin DF, Frid DJ, et al. Restenosis after coronary angioplasty: an overview. J Am Coll Cardiol 1991;17:2B–13B. [28] Willerson JT, Eidt JF, McNatt J, et al. Role of thromboxane and serotonin as mediators in the development of spontaneous alterations in coronary blood flow and neointimal proliferation in canine models with chronic coronary artery stenoses and endothelial injury. JACC 1991;17:101B–10B. [29] Ferns GAA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science 1991;253:1129–32. [30] Clowes AW, Clowes MM, Fingerle J, Reidy MA. Regulation of smooth muscle growth in injured artery. J Cardiovasc Pharmacol 1989;14(Suppl 6):S12– 5. [31] Chapman GD, Lim CS, Gammon RS, et al. Gene transfer into coronary arteries of intact animals with a percutaneous balloon catheter. Circ Res 1992;71:27– 33.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins Belinda A. Di Bartolo a , Stephen J. Nicholls b , Shisan Bao c , Kerry-Anne Rye a,f,g , Alison K. Heather d,e , Philip J. Barter a,f , Christina Bursill a,∗ a
Lipid Research Group, Heart Research Institute, Sydney, Australia Department of Cell Biology, Cleveland Clinic, Cleveland, OH, USA c Discipline of Pathology, University of Sydney, Australia d Gene Regulation Group, Heart Research Institute, Sydney, Australia e Department of Medical and Molecular Biosciences, University of Technology, Sydney, Australia f Department of Medicine, University of Sydney, Australia g Department of Medicine, University of Melbourne, Australia b
a r t i c l e
i n f o
Article history: Received 9 August 2010 Received in revised form 31 March 2011 Accepted 1 April 2011 Available online 16 April 2011 Keywords: Apolipoprotein A-I Apolipoprotein A-I mimetic peptides Inflammation Adhesion molecules Chemokines
a b s t r a c t Objectives: Mimetic peptides of apolipoprotein A-I (apoA-I) present a new strategy for promoting the biological activity of high density lipoproteins (HDL). This study aimed to compare the anti-inflammatory effects of ETC-642, a new apoA-I mimetic peptide, with discoidal reconstituted HDL (rHDL). Methods: New Zealand White rabbits (n = 42) received daily infusions of saline, rHDL or discoidal complexes of an amphipathic peptide, ETC-642 (1–30 mg/kg), prior to insertion of non-occlusive carotid collars. Human coronary artery endothelial cells (HCAECs) were pre-incubated with ETC-642 or rHDL before TNF-␣ stimulation. Monocyte adhesion was investigated by pre-incubating HCAECs with rHDL or ETC-642, stimulating with TNF-␣ and incubating with THP-1 monocytes. Results: Infusion of ETC-642 resulted in dose-dependent reductions of collar-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the artery wall (p < 0.05). Pre-incubation of HCAECs with ETC-642 and rHDL reduced TNF-␣-induced THP-1 monocyte adhesion (p < 0.01). Furthermore, ETC-642 and rHDL treatment reduced TNF-␣ induced mRNA levels of inflammatory markers VCAM-1, fractalkine, MCP-1 and the p65 subunit of NF-B (p < 0.05). Conclusion: These studies demonstrate that ETC-642 exhibits anti-inflammatory properties that are comparable to apoA-I both in vivo and in vitro and that these effects are mediated via the NF-B signaling pathway. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The atheroprotective nature of high-density lipoproteins (HDL) has been well established [1]. In addition to its well-characterized role in the promotion of reverse cholesterol transport [2], HDL also possesses antioxidant and antithrombotic properties and, in particular, anti-inflammatory properties [3]. In cell culture systems, HDL inhibits endothelial cell expression of proinflammatory adhesion molecules and chemokines and, as a consequence, reduces monocyte chemotaxis [4]. Animal models have also shown that transgenic over-expression of the main protein component of HDL, apolipoprotein (apo)A-I and the infusion of high-dose apoA-IMilano reduces the inflammatory
∗ Corresponding author at: The Heart Research Institute, 7 Eliza St, Newtown, NSW 2050, Australia. Tel.: +61 2 8208 8900; fax: +61 2 9565 5584. E-mail address: [email protected] (C. Bursill). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.04.001
composition of experimental atheroma by reducing infiltrating macrophages and plaque lipid in apoE knockout mice [5]. ApoA-I, with a chain length of 243 amino acids, is the most abundant HDL apolipoprotein. The size of this protein limits its usefulness as a therapeutic agent. However, peptides as short as 18 amino acids, based on the apoA-I structure, have been synthesised and shown to possess some properties of full length apoA-I. Animal studies demonstrate that mimetic peptides reduce atherosclerotic lesions, improve endothelial dysfunction, decrease proinflammatory lipoproteins and diminish monocyte recruitment, all independent of any increase in HDL cholesterol levels [6–9]. Together these studies suggest that apoA-I mimetic peptides may confer benefits comparable to those of rHDL containing full length apoA-I. Given that mimetic peptides are easier to synthesize than full-length apoA-I, there is considerable interest in developing them as potential therapeutic agents, particularly as apoA-I has been shown in human
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trials to cause plaque regression and reduce inflammatory markers [10,11]. The apoA-I mimetic peptide used in our study contains a 22-amino acid synthetic amphipathic peptide based on the sequence of L-amino acid residues: P-V-L-D-L-F-R-E-L-L-N-E-L-LE-A-L-K-Q-K-L-K (ESP 24218; unpublished, Pfizer). This peptide was combined with two naturally occurring phospholipids, sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The resulting peptide/phospholipid complex (named ETC642) has a molecular weight of 2623 and a molar ratio of ESP 24218 to sphingomyelin to DPPC of 1:3.75:3.75. The peptide, ESP 24218 mimics the structure of the amphipathic ␣-helices of apoA-I when combined with lipids. The anti-inflammatory properties of ETC-642 remain unknown. These studies demonstrate for the first time that intravenous infusion of ETC-642 is as effective as apoA-I-containing rHDL at inhibiting acute vascular inflammation in a rabbit carotid collar model. In vitro studies also find that ETC-642 reduces inflammatory markers VCAM-I, MCP-1, fractalkine and the NF-B p65 subunit as effectively as rHDL. These studies demonstrate the efficacy of ETC-642 as a potential agent for reducing acute vascular inflammation.
White rabbits (n = 7/group). The rabbits were infused intravenously with either saline, rHDL containing 25 mg of apoA-I (8 mg/kg) or ETC-642 at concentrations of 1, 3, 10 or 30 mg/kg, immediately and at 24 h after collar placement. Rabbits were sacrificed 48 h after collar placement. VCAM-1 and ICAM-1 endothelial expression were assessed on carotid arteries and plasma chemistries were determined as described in Supplementary Information (SI). In vitro, the anti-inflammatory properties of ETC-642 were compared to rHDL in human coronary endothelial cells (HCAECs). Monocyte adhesion was assessed as described in SI. Total mRNA was extracted from treated cells and mRNA levels of VCAM-1, ICAM-1, MCP-1, fractalkine and NF-B were measured by real-time PCR. 3. Results 3.1. Plasma lipid concentrations
HDLs were isolated from pooled samples of donated human plasma (Gribbles Pathology, Adelaide, SA, Australia) by sequential ultracentrifugation in the 1.063–1.21 g/mL density range. The HDL were delipidated and apoA-I isolated by anion chromatography on a Q-Sepharose Fast Flow column (GE Healthcare Biosciences, Waukesha, WI, USA) attached to an fast protein liquid chromatography system [12].
Plasma concentrations of triglyceride, phospholipid and free and total cholesterol are presented in Supplementary Table 1. Compared to baseline values, infusions of saline, rHDL and ETC642 at 1, 3 and 10 mg/kg had no effect on the concentration of triglyceride, phospholipid, free or total cholesterol in either the post-treatment sample (collected 1 h after the first dosing) or in the sample collected at time of sacrifice, 48 h after insertion of the collar. At 1 h post-treatment, 30 mg/kg of ETC-642 increased plasma triglyceride levels by 0.84 ± 0.32 mmol/L (p < 0.05), plasma total cholesterol by 1.41 ± 0.53 mmol/L (p < 0.05), plasma free cholesterol by 1.16 ± 0.44 mmol/L (p < 0.05) and plasma phospholipids by 1.8 ± 0.85 mmol/L (p < 0.05) relative to baseline levels. At study end, neither triglycerides, total cholesterol, free cholesterol or phospholipids were significantly elevated compared to baseline in animals receiving 30 mg/kg of ETC-642.
2.2. Preparation of rHDLs containing apoA-I and PLPC
3.2. Lipoprotein analysis
Discoidal rHDL containing apoA-I complexed to 1-palmitoyl2-linoleoyl phosphatidylcholine (PLPC) (Avanti Polar Lipids, Alabaster, AL, USA) were prepared using the cholate dialysis method [13]. The phospholipid:apoA-I molar ratio was 100:1. The resulting rHDL were dialysed extensively against endotoxin-free phosphate buffered saline (PBS) (pH 7.4) before use.
HDL and LDL cholesterol concentrations were analysed at the commencement of the study, 1 h post treatment and at the time of sacrifice (Table 1). There were no changes in lipoprotein levels in animals treated with saline, rHDL or ETC642 at doses of 1 and 3 mg/kg. Animals receiving 10 mg/kg of ETC-642 exhibited a significant increase in HDL cholesterol 1 h after treatment from 0.37 ± 0.04 mmol/L at baseline to 0.66 ± 0.05 mmol/L (p < 0.05), which subsequently returned to baseline levels at sacrifice. A similar change was observed with infusions of ETC-642 at 30 mg/kg, where at 1 h post-treatment, HDL cholesterol levels had increased from 0.54 ± 0.09 mmol/L at baseline to 1.73 ± 0.1 mmol/L (p < 0.05). At the conclusion of the study HDL cholesterol had returned to baseline levels. LDL cholesterol concentrations were significantly increased from 0.22 ± 0.08 mmol/L at baseline to 0.42 ± 0.09 mmol/L at sacrifice in animals receiving 30 mg/kg of ETC-642 (p < 0.05). In all other conditions and at all other time points LDL cholesterol remained unchanged.
2. Materials and methods 2.1. Isolation of apolipoprotein A-I
2.3. Preparation of ETC-642 The ETC-642 was provided as a lyophilized solid by Esperion Therapeutics, Pfizer (Groton, MI, USA). The complex consists of ESP-2418, a peptide containing 22 amino acid residues as a synthetic amphipathic peptide based on the sequence of L-amino acid residues: P-V-L-D-L-F-R-E-L-L-N-E-L-L-E-A-L-K-Q-KL-K. ESP-2418 is complexed to two phospholipids, sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The resulting peptide/phospholipid complex is called ETC-642. The lyophilized solid was rehydrated with 50 mL of a sterile bicarbonate saline solution (Esperion Therapeutics, Pfizer, Groton, MI, USA) gently swirled to mix. Three cycles of warming at 50 ◦ C and cooling at room temperature was performed to ensure the mimetic peptide was sufficiently reconstituted. The final concentration of ETC-642 was 10 mg/mL and was stored at 4 ◦ C before use. 2.4. Animal study and in vitro work Non-occlusive peri-arterial collars were applied to the left common carotid artery of normo-cholesterolaemic New Zealand
3.3. ETC-642 reduces endothelial adhesion molecule expression The ability of ETC-642 to reduce adhesion molecule expression in the carotid endothelium was investigated. The collar-induced changes to ICAM-1 are shown in Fig. 1. Collar insertion resulted in a significant increase in ICAM-1 from 1.98 ± 0.35 image units in non-collared arteries to 17.5 ± 1.72 image units (p < 0.001). The increased expression of ICAM-1 in collared arteries was reduced by 94% after infusion of rHDL (1.1 ± 3.38 image units, p < 0.05). No effect was seen at 1 mg/kg, but infusions of ETC-642 at 3 mg/kg
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Table 1 Plasma lipoprotein profiles of animals infused with saline, rHDL (8 mg/kg) and ETC-642 at concentrations of 1, 3, 10 and 30 mg/kg at baseline, post-treatment and at time of sacrifice (n = 7 in each group). Results are expressed as mean ± SEM. Treatment
HDL – C (mmol/L) Baseline
Saline rHDL ETC-642 1 mg/kg ETC-642 3 mg/kg ETC-64210 mg/kg ETC-64230 mg/kg *
0.47 0.42 0.54 0.46 0.37 0.54
± ± ± ± ± ±
0.03 0.05 0.13 0.06 0.04 0.09
LDL – C (mmol/L) Post-treatment 0.41 0.46 0.48 0.48 0.66 1.73
± ± ± ± ± ±
0.03 0.04 0.1 0.06 0.05* 0.1*
Time of sacrifice 0.42 0.43 0.4 0.42 0.34 0.4
± ± ± ± ± ±
0.03 0.07 0.05 0.33 0.04 0.08
Baseline 0.14 0.12 0.25 0.16 0.19 0.22
± ± ± ± ± ±
0.02 0.02 0.05 0.04 0.04 0.08
Post-treatment 0.1 0.1 0.26 0.15 0.23 0.19
± ± ± ± ± ±
0.01 0.02 0.08 0.04 0.04 0.06
Time of sacrifice 0.17 0.16 0.15 0.18 0.22 0.42
± ± ± ± ± ±
0.03 0.04 0.02 0.03 0.04 0.09*
p < 0.05 compared to baseline.
reduced expression by 60% (6.30 ± 2.74 image units; p = 0.05), 10 mg/kg reduced expression by 57% (6.77 ± 3.05 image units) and 30 mg/kg reduced the collar-induced expression of ICAM-1 by 91% (1.26 ± 0.4 image units, p < 0.05) compared to saline-infused animals with carotid collars. As was the case with ICAM-1 expression, collar insertion resulted in a significant increase in VCAM-1 compared to noncollared carotid arteries (0.12 ± 0.02 image units in non-collared arteries compared to 1.78 ± 0.19 image units, p < 0.001). The increased expression of VCAM-1 in collared arteries was decreased by 98% after infusion of rHDL (0.02 ± 0.09 image units, p < 0.05). Infusions of ETC-642 decreased VCAM-1 expression at all treatment concentrations. The dose of 1 mg/kg decreased collar-induced expression by 89% (0.18 ± 0.22, p < 0.05), 3 mg/kg reduced expression by 71% (0.59 ± 0.13 image units, p < 0.05), and 10 mg/kg, reduced expression by 94% (0.09 ± 0.07 image units, p < 0.05), compared to collar-induced control animals. Infusion of ETC-642 at 30 mg/kg also significantly decreased the collar-induced expression by 92% (0.14 ± 0.07 image units, p < 0.05) compared to control animals.
endothelial cells increased by 2.4-fold, compared to unstimulated HCAECs (p < 0.05, Fig. 2). When HCAECs were pre-incubated with rHDL, prior to stimulation with TNF-␣, THP-1 monocyte adhesion was reduced by 41% (p < 0.05), compared to stimulated HCAEC controls. Furthermore, pre-treatment of HCAECs with ETC-642 reduced the TNF-␣-induced monocyte adhesion to HCAECs by 60% (p < 0.01). 3.5. Effect of ETC-642 on adhesion molecule and chemokine gene expression Stimulation with TNF-␣ substantially increased the mRNA levels of VCAM-1, ICAM-1, fractalkine and MCP-1 (5.6-fold, 7.5-fold, 36fold and 10-fold respectively), p < 0.001 (Fig. 3). The TNF-␣-induced increase in VCAM-1 was reduced in cells that were pre-incubated with either rHDL (52%; p < 0.001) or ETC-642 (20%; p < 0.01), when compared to the stimulated control cells. Pre-incubation of cells with rHDL or ETC-642, prior to TNF-␣ stimulation, also reduced fractalkine mRNA levels by 44% (p < 0.001) and 25% (p < 0.01), respectively and MCP-1 levels by 45% (p < 0.001) and 46% (p < 0.001). Finally, we demonstrate that neither rHDL nor ETC-642 were able to reduce the TNF-␣ induced increase in ICAM-1
3.4. Monocyte adhesion 3.6. ETC-642 reduces NF-B gene expression Here we investigated the ability of rHDL and ETC-642 to alter the adherence of monocytes to endothelial cells. When HCAECs were incubated with TNF-␣, adhesion of THP-1 monocytes to the
NF-B regulates a host of inflammatory genes including VCAM1, MCP-1 and fractalkine. To determine if ETC-642 and rHDL
Fig. 1. ETC-642 reduces endothelial adhesion molecule expression. Panel A: Immunohistochemical staining for ICAM-1 and VCAM-1 in representative collared carotid artery sections from NZW rabbits infused with either saline, rHDL or ETC-642 (30 mg/kg). Panel B: Endothelial expression of ICAM-1 and VCAM-1 were quantified as described in Section 2. Results are expressed as mean ± SEM. *p < 0.05 compared to saline; **p < 0.01 compared to saline.
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Fig. 4. ETC-642 reduces NF-B. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC-642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. mRNA levels of the p65 subunit of NF-B were determined by real time PCR. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0001.
Fig. 2. ETC-642 reduces monocyte adhesion to endothelial cells. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC-642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. HCAECs were washed with PBS and incubated with fluorescently labelled THP-1 monocytes (1 × 106 cell/well) for 1 h. Fluorescence was measured using 490 nm (excitation) and 540 nm (emission). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01.
decrease the expression of these genes via NF-B, we measured mRNA levels of the p65 subunit. Results show that stimulation with TNF-␣ increased the level of p65 by 3.4-fold, p < 0.001. Preincubation of HCAECs with rHDL and ETC-642 significantly reduced mRNA levels of the p65 subunit (45% and 52%, respectively; p < 0.001), when compared to stimulated controls (Fig. 4).
4. Discussion Due to the multiple beneficial properties of HDL, there has been considerable interest in developing HDL-related therapeutics. ApoA-I is a large protein, containing 243 amino acids arranged in 10 amphipathic helices, thereby making its synthesis as a therapeutic agent difficult. This has driven the impetus to develop smaller peptides that are easily synthesised but still exhibit the same beneficial properties as apoA-I. A number of investigators have developed short peptides that bear no amino acid homology to apoA-I but retain the ability to efflux cholesterol from cells, one of the main functions of HDL [14]. The fact that short peptide sequences are considerably easier and cheaper to prepare than full length apoA-I make this an attractive option in the development of new therapies for treatment of cardiovascular disease [15].
Fig. 3. Effect of ETC-642 on chemokine and adhesion molecule gene expression. HCAECs were pre-incubated with PBS, rHDL (1 mg/mL, final apoA-I concentration) or ETC642 (1.11 mg/mL, final mimetic peptide concentration) for 24 h and stimulated with TNF-␣ (0.1 ng/mL) for 5 h. VCAM-1, ICAM-1, fractalkine and MCP-1 mRNA levels were quantified by real time PCR. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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In this article, we compare the anti-inflammatory properties of a new apoA-I mimetic peptide, ETC-642, with those of rHDL in order to validate its efficacy as an anti-inflammatory agent. ETC-642 is a complex consisting of an apoA-I mimetic peptide combined with phospholipids. In this study we found that administration of ETC642 significantly decreases adhesion molecule expression (VCAM1, ICAM-1) in collared rabbit carotid arteries and causes transient elevations in plasma lipid levels. Also, in vitro studies in HCAECs show that ETC-642 reduces monocyte adhesion, adhesion molecule expression and chemokine expression, via inhibition of NF-B, a pivotal regulator of inflammation. As reported previously, infusions of rHDL into NZW rabbits did not alter their lipid profiles [16]. In contrast, 1 h post-infusion with 30 mg/kg ETC-642 total cholesterol were significantly elevated. This appeared to be predominantly due to a significant increase in free cholesterol. Interestingly, analysis of the lipoprotein fractions revealed that there was a significant increase in HDL cholesterol at this time point, indicating that the extra free cholesterol was in the HDL fraction. This suggests that ETC-642 is promoting the efflux of free cholesterol from cells to HDL particles. The exact mechanism for these effects is not able to be delineated in the current study but is consistent with previous observations in which ETC-642 was found to mobilize cholesterol in pre-clinical studies and to transiently elevate circulating HDL cholesterol levels in patients [17,18]. Furthermore, another study found that a single injection of ETC-642 transiently elevated plasma cholesterol levels, 45 min after administration in rats [19]. In the current study it was found that the LDL cholesterol fraction was elevated at the time of sacrifice, 48 h after the administration of ETC-642. This implies that cholesterol was transferred from HDL to LDL via cholesterol ester transport protein [20]. An increase in LDL cholesterol could be considered adverse and suggests that a lower dose of ETC-642, such as 10 mg/kg, is potentially more beneficial as it still exhibits the same anti-inflammatory properties without the unwanted rise in LDL cholesterol. However, it is likely that this elevation in LDL cholesterol is transient. Human intervention studies have found that weekly infusions for 5 weeks of mimetic peptide ETC-216, did not change LDL cholesterol levels [10]. Application of peri-arterial collars resulted in a substantial increase in endothelial expression of both VCAM-1 and ICAM1 in the carotid artery. This increased expression was reduced by infusion of apoA-I-containing rHDL, consistent with previous studies [4,16]. The expression of VCAM-1 and ICAM-1 was also significantly and dose-dependently reduced by administration of ETC-642. Another mimetic peptide 5A, that interacts specifically with the ATP binding cassette transporter, ABCA-1, has also recently been found to reduce both ICAM-1 and VCAM-1 in collared rabbit carotid arteries [21], further supporting an anti-inflammatory role for mimetic peptides. Circulating monocytes adhere to endothelial cells after stimulation by proinflammatory cytokines. This process is mediated through a complex co-ordination of numerous proteins, including adhesion molecules and chemokines, on both the endothelial cells and the monocytes. It is likely that the decreased expression of VCAM-1, MCP-1 and fractalkine by rHDL and ETC-642, contributed to the inhibition of monocyte adhesion to endothelial cells. Interestingly, when cells are pre-treated with rHDL or ETC642, it leads to a reduction in endothelial cell VCAM-1 expression in vitro, even when removed from cells prior to cytokine stimulation [23]. This implies that exposure of cells to rHDL and ETC-642 modifies the endothelial cells making them resistant to stimulation for a prolonged period of time [24]. A possible mechanism for the rHDL/ETC-642-mediated suppression of inflammatory markers is via inhibition of the NF-B activation pathway. NF-B regulates VCAM-1, MCP-1 and fractalkine [25,26]. We found that both rHDL and ETC-642 reduced the p65 NF-B subunit. Consistent with this,
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the apoA-I mimetic peptide, 5A, was found to also suppress VCAM1 and ICAM-1 via a reduction in NF-B [21]. Additional studies with the 5A peptide delineated this mechanism further. The 5A peptide was found to also suppress the phosphorylation of IB␣, an upstream mediator of the NF-B pathway. Furthermore, siRNA studies revealed that inhibition of p65 by 5A was mediated through ABCA1 [21], thereby raising the possibility that ETC-642 may reduce p65 via this same pathway. The expression of endothelial cell adhesion proteins is increased in vitro in response to proinflammatory cytokines. HDL has been shown to inhibit cytokine-induced expression of cell surface adhesion molecules VCAM-1 and ICAM-1 in activated endothelial cells both in vitro [27,28] and in vivo [16,21,29]. In the current study we found that the apoA-I mimetic peptide, ETC-642, also reduces VCAM-1 and ICAM-1 expression on the endothelial surface of the arterial wall in vivo. In vitro studies also found that both rHDL and ETC-642 reduce the expression of VCAM-1 and the proinflammatory chemokines MCP-1 and fractalkine. This study provides further support for an anti-inflammatory role of rHDL and now also ETC-642. We did not, however, find that ETC-642 reduced ICAM1 expression in vitro, which is in contrast to our in vivo findings where ICAM-1 was significantly reduced. This maybe explained via post-translational modification of the ICAM-1 protein. Furthermore, ICAM-1 expression is less dependent on NF-B signaling than VCAM-1 or chemokines, which all have NF-B response elements upstream of their promoter regions [26]. ICAM-1 is also regulated by the ERK and JAK/STAT signaling pathways, which are not upstream of NF-B [30]. The lack of effect of rHDL and ETC642 on ICAM-1 maybe due to its regulation via these alternative signaling systems. One of the limitations of this study is that it is impossible to conclude whether the mimetic peptide or the phospholipid complexes are responsible for the anti-inflammatory properties of ETC-642. However, previous investigations, using the same rabbit model of acute inflammation have demonstrated that the phospholipid DPPC, as used for ETC-642, had no effect on adhesion molecule expression [16]. Furthermore, in vitro studies have found that lipidfree apoA1 was not as effective at reducing inflammation as rHDL (apoA1 + phospholipid) and that phospholipid vesicles alone had no effect [22]. Take together, it appears that whilst phospholipids enhance the anti-inflammatory properties of apoA1, they are ineffective on their own. This suggests that the anti-inflammatory properties of ETC-642 are likely to be maximal when the peptide and phospholipids are complexed together and that the peptide or phospholipids alone are likely to have either no effect or modest affects on inflammation. Consistent with this, in the optimisation of ETC-642 by Esperion, DPPC, sphingomyelin and the peptide were tested for their ability to mobilise and esterify cholesterol, two of the main steps in the reverse cholesterol transport pathway. Following injection into rabbits the peptide and the phospholipids were found to individually have no effect on mobilising cholesterol but when tested as a combination, achieved maximal effects on both cholesterol mobilisation and esterification (unpublished data). Despite the immense potential of mimetic peptides to treat a range of cardiovascular disorders, their translation to clinic has not been entirely successful due to findings that they cause adverse effects [10]. For example, patients receiving the mimetic peptide ETC-216 over a 5-week period were reported to have increased gastrointestinal problems, nausea and headaches. One other clinical study however, found that oral administration of D-4F to high-risk patients was tolerated well but only a single dose was given [31]. Overall, further studies on mimetic peptides seem warranted to facilitate their translation into clinic. In conclusion the apoA-I mimetic peptide, ETC-642, exhibits anti-inflammatory properties that are comparable to rHDL in vitro
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and in vivo. These studies highlight the therapeutic potential of mimetic peptides for the prevention of vascular inflammation and cardiovascular disease. Acknowledgements This study was supported by a Heart Research Institute PhD scholarship (Dr Di Bartolo), a Heart Foundation Career Development Fellowship (CR07S3331) and a Bushell Foundation grant (Dr Bursill). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.04.001. References [1] Förstermann U, Pollock JS, Schmidt HHHW, Heller M, Murad F. Calmodulindependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991;88:1788–92. [2] Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Annu Rev Physiol 1991;53:161–77. [3] Plautz G, Nabel EG, Nabel GJ. Introduction of vascular smooth muscle cells expressing recombinant genes in vivo. Circulation 1991;83:578–83. [4] Dinerman JL, Lawson DL, Mehta JL. Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation. Am J Physiol 1991;260:H698–701. [5] Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487–91. [6] Gibbs RA. DNA amplification by the polymerase chain reaction. Anal Chem 1990;62:1202–14. [7] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1989. [8] Ausubel FM, Brent R, Kingston RE, et al. Current protocols in molecular biology; 1991. [9] Luscher TF, Richard V, Tanner FC. Endothelium-derived vasoactive factors and their role in the coronary circulation. Trends Cardiovasc Med 1991;1:179–85. [10] Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J 1990;4:2868–80. [11] Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, Gewert D. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucleic Acids Res 1991;19:184. [12] Cercek B, Fishbein MC, Forrester JS, Helfant RH, Fagin JA. Induction of insulinlike growth factor I messanger RNA in rat aorta after balloon denudation. Circ Res 1990;66:1755–60.
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