Apolipoprotein E mimetic is more effective than apolipoprotein A-I mimetic in reducing lesion formation in older female apo E null mice

doi:10.1016/j.atherosclerosis.2012.05.040

Atherosclerosis

Volume 224, Issue 2, October 2012, Pages 326-331

https://doi.org/10.1016/j.atherosclerosis.2012.05.040 Get rights and content

Abstract

Objective

The apolipoprotein E mimetic peptide Ac-hE18A-NH₂, capable of reducing plasma cholesterol and possessing anti-inflammatory properties, was compared with the well-studied anti-atherogenic apoA-I mimetic peptide 4F for reducing lesion formation in female apoE null mice with already existing lesions.

Methods and results

In initial experiments, Ac-hE18A-NH₂ was administered retro-orbitally two or three times weekly for 6–8 weeks, while peptide 4F was administered intraperitoneally every day for the same period. Age matched controls were injected with saline every day. At the end of the treatment period, plasma cholesterol levels of Ac-hE18A-NH₂ administered mice were significantly lower than in 4F and control mice. However, both 4F and Ac-hE18A-NH₂ showed reduced lesion areas in en face lesion analysis to a similar extent compared to the control group, while paraoxonase-1 (PON-1) activity was increased only in the Ac-hE18A-NH₂ group. In the third experiment, both peptides were administered at the same dose, frequency, and route of administration. The reduction in en face lesions with Ac-hE18A-NH₂ was significantly greater than the 4F and control groups, although lesions in 4F-treated mice were also significantly reduced compared with controls. Both peptide groups had significantly reduced plasma lipid hydroperoxides, but only the Ac-hE18A-NH₂ group had significantly reduced serum amyloid A levels. HDL and plasma inflammatory indices were significantly reduced in both peptide groups compared with controls.

Conclusions

Although both peptides had similar anti-inflammatory properties, Ac-hE18A-NH₂ was more effective in inhibiting lesions than 4F at the same dose, frequency, and route of administration, perhaps due to its cholesterol reducing properties.

Highlights

► The apoE mimetic Ac-hE18A-NH₂ was more effective than the apoA-I mimetic 4F in reducing aortic lesions in apo E null mice. ► Peptides were administered at a similar dosage, frequency, and route of administration. ► Both peptides exhibited similar anti-inflammatory and anti-oxidative properties. ► The cholesterol-reducing property of Ac-hE18A-NH₂ may be responsible for the greater atheroprotection.

Introduction

Major markers for atherosclerosis are increased plasma cholesterol levels (especially apolipoprotein (apo) B containing lipoproteins), low levels of high density lipoproteins (HDL), and inflammation due to increased levels of oxidized lipids [1], [2], [3]. Human apoA-I, the major protein component of HDL has been shown to inhibit atherosclerosis in several dyslipidemic animal models and in humans [4], [5]. On the other hand, apoE, the protein component of very low density lipoproteins (VLDL), inhibits atherosclerosis by enhancing the uptake of atherogenic lipoproteins by the liver and thus lowering plasma cholesterol levels [6], [7], [8]. ApoE has also been shown to regress lesion formation independently of plasma cholesterol levels [9]. These two proteins have been studied extensively in several laboratories.

Over the years, we and others have designed and studied synthetic peptide analogs that are much shorter than apolipoproteins A-I and E and do not possess sequence homology to either apoA-I or apoE but possess secondary structural motifs similar to these two proteins [10], [11], [12], [13], [14]. The structural motif that is common to both of these proteins is the presence of class A amphipathic helices that are characterized by the presence of a polar and a nonpolar face with positively charged residues at the polar–nonpolar interface and negatively charged residues at the center of the polar face [10]. Based on this motif, the first peptide designed and studied in detail was 18A with the sequence DWLKAFYDKVAEKLKEAF [15]. This peptide, despite being only 18 residues in length, mimicked several lipid-associating properties of apoA-I, which has 243 amino acids. Protection of N-and C-terminal ends with acetyl and NH₂ groups respectively produced Ac-18A-NH₂ (also referred to as 2F because of the presence of 2 phenylalanine (Phe) residues on the nonpolar face) with increased lipid-associating ability [16]. Administration of this peptide in a dyslipidemic mouse model did not inhibit atherosclerosis [17]. However, when the nonpolar face of this class A amphipathic helical peptide was modified by substituting the existing aliphatic hydrophobic amino acids with the aromatic amino acid Phe, the resulting peptides 4F and 5F were able to inhibit atherosclerosis in dyslipidemic mouse models without changing plasma cholesterol levels [17], [18]. Class A amphipathic helical peptides, (especially 4F analogs) have been studied extensively by our and several other laboratories [19], [20], [21], [22].

Addition of apoE to VLDL enhances its uptake via the heparan sulfate proteoglycan pathway in the space of Disse [23] due to the clustering of positively charged arginine (Arg) residues in the N-terminal putative receptor binding region of apoE (residues 141–150). It has been shown that the lipid-associating C-terminal class A amphipathic helix must be present for the binding and uptake of atherogenic lipoproteins by hepatocytes [24]. Based on these observations that the dual-domain nature of apoE is important for its association with apoB-containing lipoproteins, we covalently linked the putative receptor binding domain (residues 141–150) from apoE to 18A. The resulting peptide, Ac-hE18A-NH₂, has been shown to enhance the uptake of atherogenic apoB-containing lipoproteins both in vitro and in vivo via the heparin sulfate proteoglycan (HSPG) pathway in dyslipidemic mouse and rabbit models [25], [26], [27], [28]. Recently we also described the anti-inflammatory and recycling properties of this peptide [29]. A single administration of Ac-hE18A-NH₂ has the ability to dramatically decrease plasma cholesterol in dyslipidemic mouse and rabbit models. However, the cholesterol levels return to the original levels within 24 h. This peptide also inhibits atherosclerosis in apoE null mice [30]. A comparison of the efficacy of this peptide to the extensively studied class A amphipathic helical peptide 4F has not yet been reported. In the present paper, we have compared the ability of the apoE mimetic peptide Ac-hE18A-NH₂ and the apoA-I mimetic peptide 4F in decreasing the progression of atherosclerosis in apoE null mouse model with established aortic lesions. We have compared the effect of prolonged administration of these two peptides on plasma cholesterol levels and the properties of lipoproteins that may be responsible for the beneficial properties of these two peptides.

Section snippets

Synthesis of peptides

Peptides Ac-hE18A-NH₂ with the amino acid sequence LRKLRKRLLR-DWLKAFYDKVAEKLKEAF and 4F with the amino acid sequence DWFKAFYDKVAEKFKEAF were synthesized using solid-phase synthesis as previously described [31].

Effect of intravenous administration of peptides on plasma cholesterol in apoE null mice

Female apoE null mice were purchased from Jackson Laboratories (Bar Harbor, MI). After acclimatization for several weeks they were randomized into three groups, control (receiving vehicle only), 4F, and Ac-hE18A-NH₂. Doses and periods of administration were as described in the results and

Results

Three experiments were performed, with differing frequencies and modes of administration. In the first experiment, peptide treatment was begun in 22 week old female apo E null mice and continued for 8 weeks. The both peptides were administered at a dose of 100 μg; peptide 4F was administered daily intraperitoneally, while peptide Ac-hE18A-NH₂ was administered twice weekly retro-orbitally. 24 h after the final treatment, plasma cholesterol in the Ac-hE18A-NH₂ group was significantly lower and

Discussion

Previously we reported that chronic administration of peptide Ac-hE18A-NH₂ reduced plasma cholesterol and inhibited atherosclerotic lesion formation in apoE null mice [30]. This peptide was also effective in stimulating PON-1 activity, reducing lipid hydroperoxide levels, and exhibited recycling properties similar to apoE [28], [29]. In our earlier paper, we had compared the properties of Ac-hE18A-NH₂ to a peptide derived by attaching the residue 151–160 region of apoE to the class A

Conclusions

At a similar dosage, frequency, and route of administration, Ac-hE18A-NH₂ is more effective than 4F in reducing atherosclerotic aortic lesions in older apo E null mice. Although both peptides exhibited similar anti-inflammatory and anti-oxidative properties, the cholesterol-reducing property of Ac-hE18A-NH₂ may be responsible for the greater atheroprotection.

Disclosures

G. M. Anantharamaiah is a principal in Bruin Pharma, a startup biotechnology company.

Acknowledgments

We thank Dr. Mohammad Navab for his assistance in inflammatory index measurements. This study was partially supported by grants R01 HL090803 and P01 HL34343 from NIH, and by a gift from the Julius H. Caplan Charity Foundation, Inc. in memory of Miles D. Garber Jr., M.D.

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Apolipoprotein E mimetic is more effective than apolipoprotein A-I mimetic in reducing lesion formation in older female apo E null mice

Abstract

Objective

Methods and results

Conclusions

Highlights

Introduction

Section snippets

Synthesis of peptides

Effect of intravenous administration of peptides on plasma cholesterol in apoE null mice

Results

Discussion

Conclusions

Disclosures

Acknowledgments

Methods Enzymol

J Lipid Res

J Biol Chem

J Lipid Res

J Lipid Res

Biochem Biophys Res Commun

J Lipid Res

Atherosclerosis

Atherosclerosis

J Biol Chem

J Lipid Res

Kidney Internat

Interaction of low density lipoproteins with extracellular matrix

Curr Opin Lipidol

Drugs in development: targeting high-density lipoprotein metabolism and reverse cholesterol transport

Curr Opin Cardiol

Emerging role of high density lipoprotein in the prevention of cardiovascular disease

Drug Discov Today

Somatic gene transfer of human apoA-I inhibits atherosclerosis progression in mouse models

Circulation

Apolipoprotein A-I deficiency results in markedly increased atherosclerosis in mice lacking the LDL receptor

Arterioscler Thromb Vasc Biol

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation

J Clin Invest