The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins
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 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], [7], [8], [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 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peptide based on the sequence of L-amino acid residues: P-V-L-D-L-F-R-E-L-L-N-E-L-L-E-A-L-K-Q-K-L-K (ESP 24218; unpublished, Pfizer).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 ETC-642) 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 α-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-κB p65 subunit as effectively as rHDL. These studies demonstrate the efficacy of ETC-642 as a potential agent for reducing acute vascular inflammation.
Section snippets
Isolation of apolipoprotein A-I
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].
Preparation of rHDLs containing apoA-I and PLPC
Discoidal rHDL containing apoA-I complexed to 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) (Avanti
Plasma lipid concentrations
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 ETC-642 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
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
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).
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