Interaction of hyaluronic acid-linked phosphatidylethanolamine (HyPE) with LDL and its effect on the susceptibility of LDL lipids to oxidation

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Abstract

The amphiphilic polysaccharide hyaluronic acid-linked phosphatidylethanolamine (HyPE), synthesized by covalently binding dipalmitoyl-phosphatidylethanolamine (DPPE) to short chain hyaluronic acid (mol. wt.≅30 000), interacts with low-density lipoproteins (LDL), to form a ‘sugar-decoration’ of the LDL surface. This results in an increase in the apparent size of the LDL particles, as studied by photon correlation spectroscopy, and in broadening of the 1H NMR signals of the LDL’s phospholipids. Experiments conducted with fluorescently-labeled HyPE indicate that the interaction of HyPE with LDL involves incorporation of the hydrocarbon chains of this amphiphilic polysaccharide into the outer monolayer of the LDL. This interaction also inhibits the copper-induced oxidation of the LDL polyunsaturated fatty acids, avoiding oxidation altogether when the concentration of HyPE is higher than a tenth of the concentration of the LDL’s phospholipids. This can not be attributed to competitive binding of copper by HyPE. We propose that the protection of LDL lipids against copper-induced oxidation is due to formation of a sugar network around the LDL.

Introduction

Hyaluronic acid (sodium hyaluronate; hyaluronan; n-HyA) is a naturally-occurring linear polysaccharide glycosaminoglycan, composed of alternating residues of the monosaccharide d-glucuronic acid and N-acetyl-d-glucosamine linked in repeating units. It is widely distributed in the extracellular matrix of connective tissues, and is present in synovial fluids and in the aqueous and vitreous humour of the eye (Goa and Benfield, 1994). At physiological pH it is polyanionic, and it exists in a random coil configuration, forming entangled networks even at low concentrations (<1 mg/ml) (Goa and Benfield, 1994, Laurent et al., 1996). The consequent unique rheological properties of these networks are responsible for the physiological roles of hyaluronic acid as the prime constituent of the matrix of connective tissues and as a lubricant in the synovial fluid and in the humour of the eye (Scott et al., 1991, Goa and Benfield, 1994, Laurent et al., 1996).

Several studies have shown that n-hyaluronic acid protects tissues and cells from damage caused by reactive oxygen species (ROS) in human arthritic synovial fluid (Sato et al., 1988), chick embryo cartilage (Cortivo et al., 1996) and explanted bovine cartilage (Larsan et al., 1992). This effect may be attributed to the antioxidative effect of n-HyA due to scavenging of free radicals (Sato et al., 1988, Larsan et al., 1992, Cortivo et al., 1996) and/or to the reduction of lipid oxidation (Artola et al., 1993).

Oxidative modification of low density lipoprotein (LDL) lipids plays a major role in the genesis of atherosclerosis (Steinberg et al., 1989, Esterbauer et al., 1992). LDL peroxidation involves free radicals that interact with the polyunsaturated free and esterified fatty acids (PUFAs) contained in the LDL. Antioxidants can protect the LDLs’ PUFAs against oxidation by scavenging free radicals (Esterbauer et al., 1992, Hoffman and Garewal, 1995).

In view of the protective effect of n-HyA against lipid peroxidation (Artola et al., 1993), we found it of interest to study its effect on lipid peroxidation in LDL. In the present study we first investigated the effect of high molecular weight n-HyA on copper-induced LDL oxidation and found that this compound has only a slight antioxidative effect. Then we investigated the effect of low molecular weight HyA, made by cleavage of n-HyA (Saari et al., 1993), and found somewhat larger effects. Finally, in an attempt to enhance the antioxidative capability of hyaluronic acid, we studied the antioxidative efficacy of a novel substance, hyaluronic acid-linked-PE (HyPE), composed of N-derivatized phosphatidylethanolamine (PE) linked to hyaluronic acid of about 30 000 mol. wt. (Yedgar and Dagan, 1991). This compound has recently been shown to incorporate into PC bilayers (Dan et al., 1998) and we therefore expected it to incorporate into the surface monolayer of LDL, thus raising the local concentration of HyA near the LDL’s surface. We found that HyPE indeed interacts with LDL and inhibits copper-induced LDL oxidation much more than HyA.

Section snippets

Materials and methods

CuCl2 was purchased from Sigma (St. Louis, MO). 2,2′-Azo-bis(2-amidinopropane) hydrochloride (AAPH) was purchased from Poly Sciences (Warrington, PA). 13(s)-Hydroperoxyoctadeca-9Z,11E-dienoic acid (hydroperoxy linoleic acid) was purchased from ICN-Biomedicals (Costa Mesa, CA). High molecular weight hyaluronic acid was a gift from Biotechnology General (Rehovot, Israel). Low molecular weight hyaluronic acid (HyA) was produced from n-HyA as previously described (Saari et al., 1993). Hyaluronic

Structural studies

Being amphiphilic, HyPE can be expected to self-assemble in water (Tanford, 1980). Given the low turbidity of sonicated HyPE solutions we think that the aggregates present in these solutions are micelles that scatter only limited fraction of light (Vinson et al., 1989). As an example, the 90° scattering of light by a 50 μM (0.167 g.%) solution of HyPE, as observed by the photon count in a QLS measurement, was only 3 kcounts/s, as compared to 39 kcounts/s in a dispersion of 0.5 μM LDL (0.125

Acknowledgements

This work was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities–Centers of Excellence Program and by the Lady Davis Fund (to ES). We also thank Professor Y. Talmon and S. Carmeli for helpful discussions.

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