Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout—clearance via scavenger receptors

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Abstract

Oxidative modifications of low density lipoprotein (LDL) convert LDL into a ligand recognized by a variety of scavenger receptors (SR) in mammals. This oxidized LDL (oxLDL) activate several cell types, and have been shown to induce expression of a variety of genes in mammals. Lipoproteins of poikilothermic animals like salmonid fishes contain high levels of polyunsaturated fatty acids susceptible to oxidative modifications. We have investigated, and found trout LDL to be susceptible to oxidation in the presence of Cu2+. When oxidized or acetylated trout LDL was injected intravenously, the clearance rate was increased compared to that of native LDL. Modified LDL was taken up almost exclusively in the kidney, whereas native LDL was also taken up in the liver. Uptake of both oxLDL and acetylated LDL in the kidney was significantly inhibited by lipoteichoic acid (LTA) and formaldehyde treated BSA (fBSA), both of which are known ligands of SR.

Introduction

Clearance of substances like modified plasmaproteins, exogenic and endogenic toxins, inflammatory enzymes, invading microorganisms, and other foreign or endogenic substances is of vital importance as part of the first line of defense of the immune system. Modification of plasmaproteins include processes like free radical oxidation of lipoproteins. Upon oxidation of low density lipoproteins (LDL), the LDL is converted into a ligand recognized by several classes of the scavenger receptors (SR). Oxidized LDL (oxLDL) and/or acetylated LDL (acLDL) is recognized by class A SR: SR-A (mainly expressed by macrophages (Mφ) and sinusoidal endothelial cells (SEC)), class B SR: CD36 (expressed by mammary epithelial cells, adipocytes, platelets, erythrocyte precursors, Mφ, endothelial cells (EC) and smooth muscle cells (SMC)) and SR-BI (mainly expressed stereogenetic tissue and hepatocytes, but also other cell types) (reviewed in [1], [2]). Other oxLDL and/or acLDL receptors are class D SR: CD68/macrosialin (mainly expressed by Mφ), class E SR: lectin-like oxLDL receptor (LOX-1) [3] (expressed by cardiovascular endothelium, SMC, Mφ), class F SR: SR expressed by EC or SREC (reviewed in [1], [2]). An additional SR for phosphatidylserine and oxidized lipoprotein named SR-PSOX which is expressed by Mφ in atherosclerotic lesions has also been cloned [4]. The quantitative importance of these receptors in vivo has not been directly assed, but knock out studies of SR-A [5] and CD36 [6] in ApoE deficient mice show their involvement in atherosclerotic lesion development.

Lipoproteins can be oxidized in vitro by cultured arterial cells such as monocyte-macrophages (Mφ), EC and SMC, or by Cu2+ [7]. Mφ, EC and SMC all express SR recognizing oxLDL, and this interaction may stimulate these cells to produce reactive oxygen species (ROS), causing further oxidative modifications of lipids and lipoproteins [8]. The mechanisms of these oxidation processes in vivo are still uncertain, but involvement of transition metals have been proposed [7], [9]. To prevent or delay these processes the lipoproteins and serum contain a wide range of antioxidants [10], [11], protecting the lipids from oxidation. In addition, both cytosol and extracellular fluid contain a range of water-soluble antioxidants and radical scavengers [7].

However, oxidative modification of LDL do occur in the body [12], [13]. One good indication of this is the presence of autoantibodies EO6 and T15 against oxidized phospholipids in mouse [14]. OxLDL has been shown to activate vascular component cells such as EC, Mφ, and SMC, and induces several proatherogenic genes (ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), heparin-binding epidermal growth factor (EGF) like protein (HB-EGF), platelet-derived growth factor (PDGF-A-B), cyclooxygenase-2 (COX-2) and endothelial nitric oxide synthase (eNOS)) [15], [16], [17], matrix metalloproteinases (MMPs), CD40, CD40L and tissue factor (TF) [18], [19]. OxLDL also induces apoptosis in SMC [20], and impairs nitric oxide (NO) production in EC [21].

It is not clear how oxLDL and its receptors interact with each other, although interaction of the charged collagen domain containing a lysine cluster of SR-A with acetylated LDL (acLDL) and oxLDL has been shown [22]. A recent review by Boullier et al. [23] suggest that some SR may have been selected during evolution because of their involvement in recognition and clearance of bacteria and apoptotic cells. This recognition is in part due to the presence of oxidized phospholipids on their surface. Oxidation of LDL generates modified phospholipids of similar structures, and is thus also recognized by these SR [23].

Salmonid tissue and lipoproteins contain high amounts of polyunsaturated fatty acids, and may therefore be susceptible to these potentially fatal oxidative alterations [24]. Oxidative modifications may lead to an altered metabolism of the piscine lipoproteins.

In salmonid fishes, the existence of a SR pathway has been demonstrated in kidney with ligands such as modified albumin, protein coated paramagnetic beads [25], [26], [27] and acetylated LDL [28]. In Atlantic Cod (Gadus morhua), SR activity has been demonstrated in the endocardial EC [29], [30]. In this report, we have investigated the metabolism of both oxidized and acetylated LDL from rainbow trout. The susceptibility of trout LDL to Cu2+ oxidation has been analyzed, and the clearance and tissue distribution of intravenously injected modified LDL has been followed by radiotracer studies. In addition, we have studied the possible inhibition of both oxLDL and acLDL uptake by preinjection with putative receptor competitors.

Section snippets

Chemicals

Carrier free Na125I with a specific activity of 644 MBq/μg I, was obtained from Institutt for Energiteknikk, Kjeller, Norway. Tyramine cellobiose was kindly donated by Dr Helge Tolleshaug, Nycomed AS, Oslo. RPMI-1640, penicillin, streptomycin and fetal calf serum were obtained from Flow Laboratories, Irvine, Scotland. Kit for lipid peroxide determination was obtained from Kamiya Biomedical Company, CA, USA. Lipoteichoic acid (LTA), bovine serum albumin (BSA), formaldehyde, polyinosinic acid

Oxidation of trout LDL with Cu2+

Freshly isolated LDL from trout was incubated in the presence of 5 mM Cu2+, and the formation of conjugated dienes was followed by OD234 nm measurements (Fig. 1). The results shown in Fig. 1 indicate that the formation of conjugated dienes increased both with time and concentration of lipoprotein. We prepared EDTA free LDL samples both by overnight dialysis under N2 or gel filtration on PD10 columns, without any detectable effect on subsequent oxidation (not shown).

In Table 1 the formation of

Discussion

The present study describes the catabolic fate of oxLDL and chemically modified LDL in rainbow trout. As described above, oxLDL is a potent inducer of several proatherogenic genes and other proteins, and of apoptosis in certain cell types (reviewed in Ref. [3]). The SR were initially identified by their recognition and uptake of modified LDL which contribute to accumulation of LDL cholesterol and foam cell formation [13], [44], [45]. The list of SR members is steadily growing, as well as their

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