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Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout—clearance via scavenger receptors
Developmental and Comparative Immunology 26 (2002) 723–733 www.elsevier.com/locate/devcompimm
Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout—clearance via scavenger receptors Marianne K. Frøystada, Vivi Voldenb, Trond Bergc, Tor Gjøena,* a
School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo, Norway Institute of Nutrition Research, University of Oslo, P.O. Box 1046, Blindern, 0316 Oslo, Norway c Department of Molecular Cell Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway b
Received 12 October 2001; revised 22 April 2002; accepted 16 May 2002
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 Cu2þ. 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. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Scavenger receptor; Rainbow trout; Oxidized low density lipoprotein; Acetylated low density lipoprotein; Lipoteichoic acid; Formaldehyde treated BSA
Abbreviations: SR, scavenger receptor; SR-A, class A scavenger receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; oxLDL, oxidized low density lipoprotein; acLDL, acetylated low density lipoprotein; HDL, high density lipoprotein; LTA, lipoteichoic acid; LPS, lipopolysaccarides; Poly I, polyinosinic acid; BSA, bovine serum albumin; fBSA, formaldehyde treated BSA; LPO, lipid peroxides; TBARS, thiobarbituric acid reactive substances; DiI, 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; HBEGF, heparin-binding EGF like protein; EGF, epidermal growth factor; PDGF-A-B, platelet-derived growth factor; COX-2, cyclooxygenase-2; eNOS, endothelial nitric oxide synthase. * Corresponding author. Tel.: þ47-228-44943; fax: þ 47-22844944. E-mail address: [email protected] (T. Gjøen).
1. 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
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macrophages (Mf) and sinusoidal endothelial cells (SEC)), class B SR: CD36 (expressed by mammary epithelial cells, adipocytes, platelets, erythrocyte precursors, Mf, endothelial cells (EC) and smooth muscle cells (SMC)) and SR-BI (mainly expressed stereogenetic tissue and hepatocytes, but also other cell types) (reviewed in Refs. [1,2]). Other oxLDL and/or acLDL receptors are class D SR: CD68/ macrosialin (mainly expressed by Mf), class E SR: lectin-like oxLDL receptor (LOX-1) [3] (expressed by cardiovascular endothelium, SMC, Mf), class F SR: SR expressed by EC or SREC (reviewed in Refs. [1, 2]). An additional SR for phosphatidylserine and oxidized lipoprotein named SR-PSOX which is expressed by Mf 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 (Mf), EC and SMC, or by Cu2þ [7]. Mf, 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, Mf, 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), plateletderived growth factor (PDGF-A-B), cyclooxygenase2 (COX-2) and endothelial nitric oxide synthase (eNOS)) [15 – 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 –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 Cu2þ 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.
2. Materials and methods 2.1. Chemicals Carrier free Na125I with a specific activity of 644 MBq/mg 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
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Company, CA, USA. Lipoteichoic acid (LTA), bovine serum albumin (BSA), formaldehyde, polyinosinic acid (poly I), trichloroactetic acid (TCA) and Iodogen were purchased from Sigma, St Louis, USA. DiI (1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl-indocarbocyanine perchlorate) was obtained from Molecular Probes, Eugene, Oregon, USA. LIPO lipoprotein electrophoresis kit, P/N 655910 was obtained from Beckman Instruments Inc., Diagnostic System Group, Brea, CA, USA. 2.2. Animals Farmed rainbow trout (Oncorhynchus mykiss) weighing 300– 500 g were obtained from a local fish farm. They were kept in tanks containing running fresh water at a temperature of 8 –12 8C, and fed standard pellets for salmon (Ewos EST 93, with energy distribution 40% protein, 49.5% fat and 10.5% carbohydrates). 2.3. Isolation of lipoproteins Ten ml blood samples were pooled from fish anaesthetized with 0.003% benzocaine. The blood was withdrawn with a syringe inserted in the caudal vein and placed in tubes on ice. 0.01% EDTA was present in the syringe and throughout centrifugation and dialysis to prevent oxidation and proteolysis. Plasma was obtained by centrifugation (1000g, 20 min). Four lipoprotein fractions were prepared by sequential density ultracentrifugation [31]. The four fractions; very LDL (VLDL) (d , 1.015; intermediate density lipoprotein (IDL): 1.015 , d , 1.040 g/ ml), LDL (1.040 , d , 1.085 g/ml), HDL (1.085 , d , 1.210 g/ml), were dialyzed against PBS containing 0,01% EDTA (PBS-EDTA)(or against EDTA free PBS when proceeding with oxidation procedures) to remove NaBr, and stored at 4 8C before characterization and labeling. All preparations were used within 3 weeks after isolation. Human LDL was prepared from a normolipemic male donor by sequential ultracentrifugation (1.019 , LDL , 1.063 g/ml). 2.4. Oxidation of lipoproteins Unlabeled and
125
I-TC labeled lipoproteins were
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dialyzed against EDTA free PBS, pH 7.4, overnight under N2 or gelfiltrated on a PD10 column (Pharmacia, Sweden) before oxidation in a cell-free system [32]. Routinely, 100 mg/ml LDL was incubated at 37 8C in the presence of 5 mM CuSO2. Oxidation was terminated by addition of EDTA (10 mM) and butylated hydroxy toluene (BHT) (40 mM) before storage at 4 8C. The formation of conjugated dienes were followed by the change in absorbance at 234 nm (Beckman DU-62 Spectrophotometer, CA, USA). Absorbance was measured every 5 min for 240 min. Lipid peroxides (LPO) were determined by a colorimetric method (LPO kit, Kamiya Biomedical Company, Thousand Oaks, USA). Thiobarbituric acid reactive substances (TBARS) were measured as described [33]. Briefly, 500 ml samples were mixed with 250 ml 20% TCA and centrifuged. Equal amounts of supernatant and 1% thiobarbituric acid were mixed, boiled for 10 min and the absorbance measured at 535 nm. The change in electrophoretic mobility of oxidized lipoproteins was analyzed by agarose gel electrophoresis in 0.05 M barbitalbuffer pH 8.6 stained with Sudan Black (LIPO lipoprotein electrophoresis kit from Beckman) [34]. 2.5. Acetylation of lipoproteins Acetylation of LDL was performed as described by Basu et al. [35]. Briefly, the dialyzed LDL was reacted with acetic anhydride in a saturated sodium acetate buffer, which results in acetylation of the LDL lysine residues. This modification increases the net negative charge of the LDL by substitution of the 1-aminogroups of lysine. The acLDL was subsequently dialyzed against PBS-EDTA at 4 8C. 2.6. Radiolabeling of lipoproteins with 125I-tyraminecellobiose, or direct labeling with 125I For the study of biodistribution of oxLDL, radiolabeling was performed with 125I-tyraminecellobiose (TC), as TC is trapped in the degradative organs with minimal leakage. Direct labeling will give the same biodistribution at early time points after injection (before degradation takes place). Whereas at later time points redistribution will take place because of protein degradation and release of tracer [36].
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Native or oxidized trout LDL was labeled with 125Ityramine-cellobiose according to the method described by Pittman et al. [36]. A specific activity of 200 –800 cpm/ng LDL protein was achieved by this method. All radioactivity analyses were made in a Packard Cobra Autogamma counter with 72% counting efficiency. The biodistribution study of acLDL was performed with directly labeled ligand prepared as described later, to confirm earlier biodistribution results obtained with TC labeled acLDL [28]. For the competition assay, oxidized or acetylated LDL was radiolabeled directly using Iodogenw coated tubes. The distribution was analyzed 1 h after injection, to avoid degradation of the competitors. Approximately 0.5 mg of acLDL or oxLDL was added to a Iodogenw coated tube, and reacted with 40 mBq Na125I for 30 min on ice. The reaction was then terminated by adding 40 ml 0.1 M KI, and 40 ml 0.1 m NaS2O5. The radiolabeled acLDL or oxLDL was then dialyzed against PBS-EDTA to remove unbound radioactivity. A specific activity of 100 cpm/ng oxidized LDL, and 4000 cpm/ng acetylated LDL was obtained with this method. 2.7. Blood clearance The fish were anaesthetized with 0.003% benzocaine before handling. Three hundred microliters (, 0.35 mg/ml) 125I-TC-oxLDL or 125I-TC-LDL in saline were injected into the tail vein through a 23 gauge needle. The animals (three fishes in each group) were then returned to the tanks for sampling of 50 ml blood from the tail vein of each fish at predetermined time points. The samples were diluted to 250 ml with water and precipitated with 10% TCA for a minimum of 30 min. The precipitate was analyzed for radioactivity. 2.8. Uptake of lipoproteins in J774 macrophages The murine macrophage cell line J774 expresses a high number of SR and is routinely used for biological analysis of lipoprotein modification. Cells were seeded in 35 mm Costar wells and maintained in RPMI-1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum. The macrophages were incubated for 5 h at 37 8C in the presence of 10 mg/ml 125I-TC labeled lipoprotein.
The cells were washed with cold PBS and assayed for radioactivity and protein by the method of Bradford [37]. 2.9. Biodistribution Oxidized and native LDL: 25 mg of TC-labeled lipoprotein was intravenously injected as described before and sacrificed 24 h later. Before sacrifice the fishes were anaesthetized with 0.003% benzocaine and whole body perfused with PBS via the heart to remove residual radioactivity. Liver, spleen, heart and kidney were excised, weighed and analyzed for radioactivity. Acetylated LDL: 1 h after intravenous injection of , 30 mg directly labeled lipoprotein the animals were sacrificed, blood was drawn and samples of organs were excised, weighed, and analyzed for radioactivity. 2.10. Competition assay Fish were preinjected intravenously with SR ligands [38]: 2 mg of either LTA, or formaldehyde treated BSA (fBSA) were injected 15 min prior to injection with , 30 mg 125I-oxLDL or 125I-acLDL. The control groups were noninjected fish or fish injected with 2 mg unmodified BSA. After 60 min the fish were sacrificed, and blood samples as well as various organs were collected, weighed and analyzed for radioactivity.
3. Results 3.1. Oxidation of trout LDL with Cu2þ Freshly isolated LDL from trout was incubated in the presence of 5 mM Cu2þ, 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 LPO and tiobarbituric
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Fig. 1. Kinetics of Cu2þ stimulated oxidation of trout LDL analyzed by increase in OD234 (conjugated dienes). Four different LDL concentrations (125, 63, 32 and 16 ug/ml from top to bottom) were incubated with 5 mM CuSO4 in PBS buffer pH 7.4. OD234 was measured every 5 min for 7 h and plotted against time.
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Fig. 2. Uptake of LDL in J774 mouse macrophages. The cells were grown in 35 mm wells and incubated with 0.2 mg 125I-TC-LDL/ml or 125I-TC-oxLDL/ml for 5 h. The cells were then washed three times with PBS and analyzed for radioactivity and protein. Rainbow trout native LDL and oxidized LDL are abbreviated as LDL T, and LDLOX T, respectively. Human native LDL and oxidized LDL are abbreviated as LDL H and LDLOX H, respectively.
acid reactive substances is shown. About 250 nmol LPO/mg protein were present in trout LDL after 24 h of oxidation, compared to a pretreatment level below 60 nmol/mg. TBARS increased from a pretreatment level at 9.2 nmol/mg protein to 63.5 nmol/mg protein. These results show that trout LDL is highly suceptible to oxidation by Cu2þ under these conditions.
with Cu2þ. This clearly shows that the Cu2þ oxidation of trout LDL has transformed the lipoprotein into a ligand that is recognized and effectively endocytosed by these cells.
3.2. Uptake of oxidized LDL in macrophages
Trout LDL was subjected to Cu2þ oxidation for 24 h before 125I-TC labeling and injection into the fishes. Fig. 3 shows the plasma clearance of oxidized 125 I-TC-LDL and native 125I-TC-LDL. The clearance of LDL was enhanced after oxidation, and the modified lipoprotein was rapidly removed from plasma. This removal was mainly effected by a increased uptake by the kidney (Fig. 4(A)). More than 90% of the recovered dose of oxLDL were found in the kidney, while less than 3% were found in the liver 24 h after injection. The corresponding values for unmodified LDL were 50 and 35%, respectively. Whole body perfusion with PBS was performed to remove residual radioactivity from organs (significant for native LDL). Negligible amounts of the tracers were found in other organs than kidney and liver.
A much used functional assay of LDL modification, is the amount of LDL taken up in a macrophage cell line with high expression of SR, the murine J774 cell line [39 – 43]. These cells were used to follow uptake of 125I-TC labeled trout LDL in its native or oxidized form. Fig. 2 shows that uptake of LDL in these cells increased six-fold after oxidation Table 1
LPO nmol/mg protein TBARS nmol/mg protein
Before oxidation
After oxidation
57 9.2
247.7 63.5
3.3. Plasma clearance and biodistribution of native and oxidized LDL
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Fig. 3. Blood clearance of 125I-TC-LDL and 125I-TC-oxLDL after intravenous injection in trout. About 25 mg labeled trout lipoprotein was injected into each fish. Fifty microliter blood samples were drawn into a syringe and analyzed for radioactivity. The values are the mean of three fish ^SD.
3.4. Biodistribution of acLDL Injection of radiolabeled acetylated LDL (125IacLDL) showed that also this ligand was mainly cleared by kidney cells. One hour after injection more than 83% of acLDL were found in the kidney, while about 10% was found to remain in the blood (Fig. 4(B)). Relatively small amounts were found in liver (less than 4%), spleen (less than 2%), and heart (less than 2%). Body perfusion with PBS was omitted as previous experiments have shown that more than 90% is cleared from blood within 1 h after injection [28].
3.5. Competition assay Negatively charged SR ligands inhibited uptake of both radiolabeled oxLDL and acLDL in kidney cells when injected prior to the labeled ligand. A concurrent increase in the amount of oxLDL or acLDL remaining in the blood was observed. Preinjection with both LTA and fBSA significantly inhibited uptake of both oxLDL and acLDL in the kidney compared to the control. Preinjection with unmodified BSA showed tissue distribution of oxLDL and acLDL similar to the control (Fig. 5(A) and (B)).
Fig. 4. Biodistribution. (A) Uptake of 125I-TC-LDL and 125I-TCoxLDL in different organs after intravenous injection of 25 mg TC labeled lipoprotein. 24 h after injection the animals were sacrificed, organs excised, weighed and analyzed for radioactivity. Values are mean of three fishes ^SD and expressed as percent of activity recovered in the analyzed organs. (B) Uptake of 125I-acLDL in different organs after intravenous injection of ,30 mg directly labeled lipoprotein. One hour after injection, the animals were sacrificed, blood drawn and organs excised, weighed, and analyzed for radioactivity. Values are mean of three fishes ^SD and expressed as percent of activity recovered in the analyzed organs.
4. 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,
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Fig. 5. Competition assay. Two milligrams of either LTA, fBSA or BSA was intravenously injected 15 min prior to injection of ,30 mg 125IacLDL (A) or ,30 mg 125I-oxLDL (B). After 60 min the fish were sacrificed, blood was drawn and organs excised, weighed and analyzed for radioactivity. Control fish were not preinjected with competing ligand, and BSA was injected as a negative control. Values are the mean of three different fishes in each experiment ^SD and expressed as percent of activity recovered in the analyzed organs. Asterix denotes significantly different values compared to the control, with p , 0.05.
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 partly overlapping lists of ligands (reviewed in Refs. [1,2, 46]). SR may have been selected during evolution due to recognition and clearance of bacteria, apoptotic cells and possibly oxLDL via interaction with
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oxidized phospholipids [23]. It has been demonstrated that binding and phagocytosis of oxidatively damaged red blood cells or apoptotic thymocytes are inhibited by oxLDL [47,48]. Boullier et al. have shown that binding of oxLDL to CD36 transfected cells are in part mediated by oxidized phospholipids associated with both the protein and the lipid moieties of oxLDL [49]. In addition, a monoclonal antibody, EO6, against oxLDL [50] has been shown to recognize a series of oxidized phospholipids; structural motifs responsible for antigeneticity is outlined in Ref. [14]. This antibody inhibits uptake of oxLDL in Mf and in cells transfected with CD36 or SR-BI, and was shown to have a variable region that is 100% identical to a naturally occurring autoantibody designated T15. EO6 and T15 are structurally and functionally equivalent [51]. The T15 autoantibody has been extensively studied for three decades and has been shown to recognize phosphorylcholine (choline phosphate, the head group of phosphatidylcholine), which is a common constituent of the cell wall of many pathogens. Production of this autoantibody even under germ-free conditions in mice may in part be due to oxidatively modified phospholipids on the surface of apoptotic cells, or in oxLDL, acting as endogenous antigens modulating the level of expression of this antibody [23]. This may indicate that oxidized phospholipids of cells and LDL are common ligands for both SR and natural antibodies. Previous studies in trout have described lipoprotein synthesis, composition, and catabolism [28,52 – 54]. However, few studies have focused on the role of oxidative modifications of lipoproteins and their effect on metabolism [7]. Salmonid lipoproteins are highly enriched in polyunsaturated fatty acids, making them prone to oxidative modifications. Salmonids also develop atherosclerotic plaques [55 – 57] during migration and spawning although the coronary lesions in salmonids are typically lipid-free, despite high plasma levels of both total cholesterol and LDLs [55]. Our results show that rainbow trout LDL is susceptible to oxidation by Cu2þ. The relevance of this mode of modification to the in vivo situation is debated, but transition metal ions may be essential for cell-induced oxidation, and the lipoprotein composition is similar for metal-induced and cell-induced oxidation [7,9,58]. The temporal development of diene formation in trout LDL was different from that observed with human
LDL. In mammalian lipoproteins, a lag phase is normally observed before OD234 starts to rise. This lag phase represents depletion of vitamin E from the lipoproteins [7]. Diene formation in trout LDL increased immediately after Cu2þ addition without any detectable lag phase. This rapid onset of diene formation might be caused by the high PUFA content in trout LDL. The level of LPO after 24 h of oxidation was about five-fold higher than the pretreatment value. The pretreatment value was more than twice the level normally found in human LDL [7]. The increase was probably higher, since LPO may have been formed during the preparation of EDTA-free LDL. In one study, fully oxidized human LDL was obtained by overnight dialysis in PBS under air [59]. In addition, LPO are decomposed as the oxidative changes proceed through later stages. Initially, LPO’s increase concurrently with diene formation, but are subsequently decomposed to aldehydes in the terminal decomposition phase [7]. The level of TBARS reflects this decomposition, as well as the increase in net negative charge of the lipoprotein particle as the aldehydes start to react with amino groups of apoB. The mobility of trout LDLs in agarose gels before and after Cu2þ induced oxidation was compared. An increase in negative charge was observed both with oxidized and acetylated LDL as the relative electrophoretic mobility was 1.26 and 1.24, respectively, compared to native LDL (not shown). The relative increase in electrophoretic mobility was moderate compared to previously reported values for human LDL (up to 3 £ ) [7]. Assessed by a more functional assay such as receptor-mediated endocytosis in macrophages, trout LDL was found to be transformed into a ligand that was readily endocytosed by the SR of J774 cells. Uptake of human oxLDL was higher than uptake of trout oxLDL in J774 cells. This may be explained by the more negative charge in human oxidized LDL, or by the fact that J774 mouse macrophages express several other classes of SR (some are CD36, SR-BI and MARCO) in addition to class A SR (SRA) [40,42, 43], that may have different affinities for the two species of oxLDL. In mammals, the main scavenging site for effete plasma macromolecules resides in the liver EC [60]. In contrast, salmonid fishes express SR activity in kidney cells [25–28]. The accelerated plasma clearance and
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the increased uptake of oxidized LDL compared to native LDL, suggests that it was catabolized via the SR pathway. This is corroborated by the tissue distribution of injected oxLDL and acLDL, and in agreement with previous results [28], and also by work performed by others [26]. Earlier we have shown uptake of 2,8 mm polystyrene beads in rainbow trout head kidneyderived macrophages, demonstrating a role for SR in phagocytosis [27]. Further support for the involvement of SR in oxLDL uptake was the finding that when fish were preinjected with ligands typical of the mammalian SR-A (LTA, fBSA) (reviewed in Ref. [38]), endocytosis of oxLDL and acLDL was significantly reduced. LTA is a component of the surface of gram positive bacteria, which has been shown to be recognized by class A SR [61], in addition to recognition of whole gram positive bacteria by several SR classes [61 – 65]. Polyinosinic acid was also tested as a putative competitor, but no significant reduction was observed (not shown). In conclusion our study show that trout LDL is susceptible to oxidative modifications, leading to increased negative charge and subsequent endocytosis by kidney cells. The same route of metabolism is used by acetylated LDL. The cell types in kidney responsible for this clearance are capillary EC/SEC in the case of acLDL [28]. Both SEC and leucocytes in the rainbow trout kidney has been shown to take up other SR ligands [26]. In addition, our results show that uptake of both oxLDL and acLDL in kidney is significantly inhibited by LTA and fBSA indicating a broad specificity for these receptors in fishes as in mammals.
[5]
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[7]
[8]
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[12]
[13]
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[17] Sano H, Sudo T, Yokode M, Murayama T, Kataoka H, Takakura S, Nishikawa S, Nishikawa SI, Kita T. Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein Edeficient mice. Circulation 2001;103:2955–60. [18] Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995;91:2844– 50. [19] Mach F, Schonbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 1998;394:200– 3. [20] Bjorkerud B, Bjorkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscler Thromb Vasc Biol 1996; 16:416–24. [21] Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature 1990;344:160 –2. [22] Doi T, Higashino K, Kurihara Y, Wada Y, Miyazaki T, Nakamura H, Uesugi S, Imanishi T, Kawabe Y, Itakura H. Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors. J Biol Chem 1993;268:2126–33. [23] Boullier A, Bird DA, Chang MK, Dennis EA, Friedman P, Gillotte-Taylor K, Horkko S, Palinski W, Quehenberger O, Shaw P, Steinberg D, Terpstra V, Witztum JL. Scavenger receptors, oxidized LDL, and atherosclerosis. Ann NY Acad Sci 2001;947:214–22. discussion 222– 3. [24] Bell JG, Cowey CB, Adron JW, Shanks AM. Some effects of vitamin E and selenium deprivation on tissue enzyme levels and indices of tissue peroxidation in rainbow trout (Salmo gairdneri). Br J Nutr 1985;53:149–57. [25] Dannevig BH, Berg T. Localization of the scavenger receptor in salmonid fishes. In Kirn A, Knook DL, Wisse E, editors. Cells of the hepatic sinusoid. Rijswiik (Netherlands): Kupffer Cell Foundation, p. 143– 4. [26] Dannevig BH, Lauve A, Press CM, Landsverk T. Receptormediated endocytosis and phagocytosis by rainbow trout. Fish Shellfish Immunol 1994;4:3–18. [27] Froystad MK, Rode M, Berg T, Gjoen T. A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages. Dev Comp Immunol 1998;22:533– 49. [28] Gjøen T, Berg T. Metabolism of low density lipoproteins in rainbow trout. Fish Physiol Biochem 1992;9:453 –61. [29] Sorensen KK, Melkko J, Smedsrod B. Scavenger-receptormediated endocytosis in endocardial endothelial cells of Atlantic cod Gadus morhua. J Exp Biol 1998;201:1707–18. [30] Seternes T, Oynebraten I, Sorensen K, Smedsrod B. Specific endocytosis and catabolism in the scavenger endothelial cells of cod (Gadus morhua L.) generate high-energy metabolites. J Exp Biol 2001;204:1537–46. [31] Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifually separated lipoproteins in human serum. J Clin Invest 1955;34:1345 –53.
[32] Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism. Arteriosclerosis 1987;7:135–43. [33] Joyeux M, Rolland A, Fleurentin J, Mortier F, Dorfman P. tertButyl hydroperoxide-induced injury in isolated rat hepatocytes: a model for studying anti-hepatotoxic crude drugs. Planta Med 1990;56:171–4. [34] Noble RP. Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid Res 1968;9:693–700. [35] Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci USA 1976;73: 3178–82. [36] Pittman RC, Carew TE, Glass CK, Green SR, Taylor Jr. CA, Attie AD. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 1983;212:791 –800. [37] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. [38] Platt N, Haworth R, Darley L, Gordon S. The many roles of the class A macrophage scavenger receptor. Int Rev Cytol 2002; 212:1–40. [39] Ding Y, Hakamata H, Matsuda H, Kawano T, Kawasaki T, Miyazaki A, Horiuchi S. Reduced expression of the macrophage scavenger receptors in macrophage-like cell mutants resistant to brefeldin A. Biochem Biophys Res Commun 1998; 243:277–83. [40] Han J, Hajjar DP, Febbraio M, Nicholson AC. Native and modified low density lipoproteins increase the functional expression of the macrophage class B scavenger receptor, CD36. J Biol Chem 1997;272:21654–9. [41] Malerod L, Juvet K, Gjoen T, Berg T. The expression of scavenger receptor class B, type I (SR-BI) and caveolin-1 in parenchymal and nonparenchymal liver cells. Cell Tissue Res 2002;307:173–80. [42] Matveev S, van der Westhuyzen DR, Smart EJ. Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages. J Lipid Res 1999;40:1647–54. [43] van der Laan LJ, Dopp EA, Haworth R, Pikkarainen T, Kangas M, Elomaa O, Dijkstra CD, Gordon S, Tryggvason K, Kraal G. Regulation and functional involvement of macrophage scavenger receptor MARCO in clearance of bacteria in vivo. J Immunol 1999;162:939–47. [44] Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci USA 1979;76:333 –7. [45] Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223– 61. [46] Peiser L, Gordon S. The function of scavenger receptors
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[55] Farrell AP, Johansen JA. Reevaluation of regression of coronary arteriosclerotic lesions in repeat-spawning steelhead trout. Arterioscler Thromb 1992;12:1171–5. [56] Farrell AP, Saunders RL, Freeman HC, Mommsen TP. Arteriosclerosis in Atlantic salmon. Effects of dietary cholesterol and maturation. Arteriosclerosis 1986;6:453– 61. [57] Saunders RL, Farrell AP. Coronary arteriosclerosis in Atlantic salmon. No regression of lesions after spawning. Arteriosclerosis 1988;8:378– 84. [58] Bruckdorfer KR. Non-enzymatic oxidation of lipids and lipoproteins: the role of metals and nitric oxide. Curr Opin Lipid 1993;4:238–43. [59] Schuh J, Fairclough Jr. GF, Haschemeyer RH. Oxygenmediated heterogeneity of apo-low-density lipoprotein. Proc Natl Acad Sci USA 1978;75:3173–7. [60] Smedsrød B, Pertoft H. Gustafson Scavenger functions of the liver endothelial cell. Biochem J 1990;266:313– 27. [61] Dunne DW, Resnick D, Greenberg J, Krieger M, Joiner KA. The type I macrophage scavenger receptor binds to grampositive bacteria and recognizes lipoteichoic acid. Proc Natl Acad Sci USA 1994;91:1863– 7. [62] Greenberg JW, Fischer W, Joiner KA. Influence of lipoteichoic acid structure on recognition by the macrophage scavenger receptor. Infect Immun 1996;64:3318–25. [63] Kraal G, van der Laan LJ, Elomaa O, Tryggvason K. The macrophage receptor MARCO. Microbes Infect 2000;2:313– 6. [64] Shimaoka T, Kume N, Minami M, Hayashida K, Sawamura T, Kita T, Yonehara S. LOX-1 supports adhesion of Grampositive and Gram-negative bacteria. J Immunol 2001;166: 5108–14. [65] Nakamura K, Funakoshi H, Miyamoto K, Tokunaga F, Nakamura T. Molecular cloning and functional characterization of a human SR with C-type lectin (SRCL), a novel member of a SR family. Biochem Biophys Res Commun 2001;280:1028–35.
Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout—clearance via scavenger receptors Marianne K. Frøystada, Vivi Voldenb, Trond Bergc, Tor Gjøena,* a
School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo, Norway Institute of Nutrition Research, University of Oslo, P.O. Box 1046, Blindern, 0316 Oslo, Norway c Department of Molecular Cell Biology, University of Oslo, P.O. Box 1066, Blindern, 0316 Oslo, Norway b
Received 12 October 2001; revised 22 April 2002; accepted 16 May 2002
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 Cu2þ. 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. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Scavenger receptor; Rainbow trout; Oxidized low density lipoprotein; Acetylated low density lipoprotein; Lipoteichoic acid; Formaldehyde treated BSA
Abbreviations: SR, scavenger receptor; SR-A, class A scavenger receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; oxLDL, oxidized low density lipoprotein; acLDL, acetylated low density lipoprotein; HDL, high density lipoprotein; LTA, lipoteichoic acid; LPS, lipopolysaccarides; Poly I, polyinosinic acid; BSA, bovine serum albumin; fBSA, formaldehyde treated BSA; LPO, lipid peroxides; TBARS, thiobarbituric acid reactive substances; DiI, 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; HBEGF, heparin-binding EGF like protein; EGF, epidermal growth factor; PDGF-A-B, platelet-derived growth factor; COX-2, cyclooxygenase-2; eNOS, endothelial nitric oxide synthase. * Corresponding author. Tel.: þ47-228-44943; fax: þ 47-22844944. E-mail address: [email protected] (T. Gjøen).
1. 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
0145-305X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 2 ) 0 0 0 3 5 - 6
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macrophages (Mf) and sinusoidal endothelial cells (SEC)), class B SR: CD36 (expressed by mammary epithelial cells, adipocytes, platelets, erythrocyte precursors, Mf, endothelial cells (EC) and smooth muscle cells (SMC)) and SR-BI (mainly expressed stereogenetic tissue and hepatocytes, but also other cell types) (reviewed in Refs. [1,2]). Other oxLDL and/or acLDL receptors are class D SR: CD68/ macrosialin (mainly expressed by Mf), class E SR: lectin-like oxLDL receptor (LOX-1) [3] (expressed by cardiovascular endothelium, SMC, Mf), class F SR: SR expressed by EC or SREC (reviewed in Refs. [1, 2]). An additional SR for phosphatidylserine and oxidized lipoprotein named SR-PSOX which is expressed by Mf 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 (Mf), EC and SMC, or by Cu2þ [7]. Mf, 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, Mf, 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), plateletderived growth factor (PDGF-A-B), cyclooxygenase2 (COX-2) and endothelial nitric oxide synthase (eNOS)) [15 – 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 –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 Cu2þ 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.
2. Materials and methods 2.1. Chemicals Carrier free Na125I with a specific activity of 644 MBq/mg 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
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Company, CA, USA. Lipoteichoic acid (LTA), bovine serum albumin (BSA), formaldehyde, polyinosinic acid (poly I), trichloroactetic acid (TCA) and Iodogen were purchased from Sigma, St Louis, USA. DiI (1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl-indocarbocyanine perchlorate) was obtained from Molecular Probes, Eugene, Oregon, USA. LIPO lipoprotein electrophoresis kit, P/N 655910 was obtained from Beckman Instruments Inc., Diagnostic System Group, Brea, CA, USA. 2.2. Animals Farmed rainbow trout (Oncorhynchus mykiss) weighing 300– 500 g were obtained from a local fish farm. They were kept in tanks containing running fresh water at a temperature of 8 –12 8C, and fed standard pellets for salmon (Ewos EST 93, with energy distribution 40% protein, 49.5% fat and 10.5% carbohydrates). 2.3. Isolation of lipoproteins Ten ml blood samples were pooled from fish anaesthetized with 0.003% benzocaine. The blood was withdrawn with a syringe inserted in the caudal vein and placed in tubes on ice. 0.01% EDTA was present in the syringe and throughout centrifugation and dialysis to prevent oxidation and proteolysis. Plasma was obtained by centrifugation (1000g, 20 min). Four lipoprotein fractions were prepared by sequential density ultracentrifugation [31]. The four fractions; very LDL (VLDL) (d , 1.015; intermediate density lipoprotein (IDL): 1.015 , d , 1.040 g/ ml), LDL (1.040 , d , 1.085 g/ml), HDL (1.085 , d , 1.210 g/ml), were dialyzed against PBS containing 0,01% EDTA (PBS-EDTA)(or against EDTA free PBS when proceeding with oxidation procedures) to remove NaBr, and stored at 4 8C before characterization and labeling. All preparations were used within 3 weeks after isolation. Human LDL was prepared from a normolipemic male donor by sequential ultracentrifugation (1.019 , LDL , 1.063 g/ml). 2.4. Oxidation of lipoproteins Unlabeled and
125
I-TC labeled lipoproteins were
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dialyzed against EDTA free PBS, pH 7.4, overnight under N2 or gelfiltrated on a PD10 column (Pharmacia, Sweden) before oxidation in a cell-free system [32]. Routinely, 100 mg/ml LDL was incubated at 37 8C in the presence of 5 mM CuSO2. Oxidation was terminated by addition of EDTA (10 mM) and butylated hydroxy toluene (BHT) (40 mM) before storage at 4 8C. The formation of conjugated dienes were followed by the change in absorbance at 234 nm (Beckman DU-62 Spectrophotometer, CA, USA). Absorbance was measured every 5 min for 240 min. Lipid peroxides (LPO) were determined by a colorimetric method (LPO kit, Kamiya Biomedical Company, Thousand Oaks, USA). Thiobarbituric acid reactive substances (TBARS) were measured as described [33]. Briefly, 500 ml samples were mixed with 250 ml 20% TCA and centrifuged. Equal amounts of supernatant and 1% thiobarbituric acid were mixed, boiled for 10 min and the absorbance measured at 535 nm. The change in electrophoretic mobility of oxidized lipoproteins was analyzed by agarose gel electrophoresis in 0.05 M barbitalbuffer pH 8.6 stained with Sudan Black (LIPO lipoprotein electrophoresis kit from Beckman) [34]. 2.5. Acetylation of lipoproteins Acetylation of LDL was performed as described by Basu et al. [35]. Briefly, the dialyzed LDL was reacted with acetic anhydride in a saturated sodium acetate buffer, which results in acetylation of the LDL lysine residues. This modification increases the net negative charge of the LDL by substitution of the 1-aminogroups of lysine. The acLDL was subsequently dialyzed against PBS-EDTA at 4 8C. 2.6. Radiolabeling of lipoproteins with 125I-tyraminecellobiose, or direct labeling with 125I For the study of biodistribution of oxLDL, radiolabeling was performed with 125I-tyraminecellobiose (TC), as TC is trapped in the degradative organs with minimal leakage. Direct labeling will give the same biodistribution at early time points after injection (before degradation takes place). Whereas at later time points redistribution will take place because of protein degradation and release of tracer [36].
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Native or oxidized trout LDL was labeled with 125Ityramine-cellobiose according to the method described by Pittman et al. [36]. A specific activity of 200 –800 cpm/ng LDL protein was achieved by this method. All radioactivity analyses were made in a Packard Cobra Autogamma counter with 72% counting efficiency. The biodistribution study of acLDL was performed with directly labeled ligand prepared as described later, to confirm earlier biodistribution results obtained with TC labeled acLDL [28]. For the competition assay, oxidized or acetylated LDL was radiolabeled directly using Iodogenw coated tubes. The distribution was analyzed 1 h after injection, to avoid degradation of the competitors. Approximately 0.5 mg of acLDL or oxLDL was added to a Iodogenw coated tube, and reacted with 40 mBq Na125I for 30 min on ice. The reaction was then terminated by adding 40 ml 0.1 M KI, and 40 ml 0.1 m NaS2O5. The radiolabeled acLDL or oxLDL was then dialyzed against PBS-EDTA to remove unbound radioactivity. A specific activity of 100 cpm/ng oxidized LDL, and 4000 cpm/ng acetylated LDL was obtained with this method. 2.7. Blood clearance The fish were anaesthetized with 0.003% benzocaine before handling. Three hundred microliters (, 0.35 mg/ml) 125I-TC-oxLDL or 125I-TC-LDL in saline were injected into the tail vein through a 23 gauge needle. The animals (three fishes in each group) were then returned to the tanks for sampling of 50 ml blood from the tail vein of each fish at predetermined time points. The samples were diluted to 250 ml with water and precipitated with 10% TCA for a minimum of 30 min. The precipitate was analyzed for radioactivity. 2.8. Uptake of lipoproteins in J774 macrophages The murine macrophage cell line J774 expresses a high number of SR and is routinely used for biological analysis of lipoprotein modification. Cells were seeded in 35 mm Costar wells and maintained in RPMI-1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum. The macrophages were incubated for 5 h at 37 8C in the presence of 10 mg/ml 125I-TC labeled lipoprotein.
The cells were washed with cold PBS and assayed for radioactivity and protein by the method of Bradford [37]. 2.9. Biodistribution Oxidized and native LDL: 25 mg of TC-labeled lipoprotein was intravenously injected as described before and sacrificed 24 h later. Before sacrifice the fishes were anaesthetized with 0.003% benzocaine and whole body perfused with PBS via the heart to remove residual radioactivity. Liver, spleen, heart and kidney were excised, weighed and analyzed for radioactivity. Acetylated LDL: 1 h after intravenous injection of , 30 mg directly labeled lipoprotein the animals were sacrificed, blood was drawn and samples of organs were excised, weighed, and analyzed for radioactivity. 2.10. Competition assay Fish were preinjected intravenously with SR ligands [38]: 2 mg of either LTA, or formaldehyde treated BSA (fBSA) were injected 15 min prior to injection with , 30 mg 125I-oxLDL or 125I-acLDL. The control groups were noninjected fish or fish injected with 2 mg unmodified BSA. After 60 min the fish were sacrificed, and blood samples as well as various organs were collected, weighed and analyzed for radioactivity.
3. Results 3.1. Oxidation of trout LDL with Cu2þ Freshly isolated LDL from trout was incubated in the presence of 5 mM Cu2þ, 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 LPO and tiobarbituric
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Fig. 1. Kinetics of Cu2þ stimulated oxidation of trout LDL analyzed by increase in OD234 (conjugated dienes). Four different LDL concentrations (125, 63, 32 and 16 ug/ml from top to bottom) were incubated with 5 mM CuSO4 in PBS buffer pH 7.4. OD234 was measured every 5 min for 7 h and plotted against time.
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Fig. 2. Uptake of LDL in J774 mouse macrophages. The cells were grown in 35 mm wells and incubated with 0.2 mg 125I-TC-LDL/ml or 125I-TC-oxLDL/ml for 5 h. The cells were then washed three times with PBS and analyzed for radioactivity and protein. Rainbow trout native LDL and oxidized LDL are abbreviated as LDL T, and LDLOX T, respectively. Human native LDL and oxidized LDL are abbreviated as LDL H and LDLOX H, respectively.
acid reactive substances is shown. About 250 nmol LPO/mg protein were present in trout LDL after 24 h of oxidation, compared to a pretreatment level below 60 nmol/mg. TBARS increased from a pretreatment level at 9.2 nmol/mg protein to 63.5 nmol/mg protein. These results show that trout LDL is highly suceptible to oxidation by Cu2þ under these conditions.
with Cu2þ. This clearly shows that the Cu2þ oxidation of trout LDL has transformed the lipoprotein into a ligand that is recognized and effectively endocytosed by these cells.
3.2. Uptake of oxidized LDL in macrophages
Trout LDL was subjected to Cu2þ oxidation for 24 h before 125I-TC labeling and injection into the fishes. Fig. 3 shows the plasma clearance of oxidized 125 I-TC-LDL and native 125I-TC-LDL. The clearance of LDL was enhanced after oxidation, and the modified lipoprotein was rapidly removed from plasma. This removal was mainly effected by a increased uptake by the kidney (Fig. 4(A)). More than 90% of the recovered dose of oxLDL were found in the kidney, while less than 3% were found in the liver 24 h after injection. The corresponding values for unmodified LDL were 50 and 35%, respectively. Whole body perfusion with PBS was performed to remove residual radioactivity from organs (significant for native LDL). Negligible amounts of the tracers were found in other organs than kidney and liver.
A much used functional assay of LDL modification, is the amount of LDL taken up in a macrophage cell line with high expression of SR, the murine J774 cell line [39 – 43]. These cells were used to follow uptake of 125I-TC labeled trout LDL in its native or oxidized form. Fig. 2 shows that uptake of LDL in these cells increased six-fold after oxidation Table 1
LPO nmol/mg protein TBARS nmol/mg protein
Before oxidation
After oxidation
57 9.2
247.7 63.5
3.3. Plasma clearance and biodistribution of native and oxidized LDL
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Fig. 3. Blood clearance of 125I-TC-LDL and 125I-TC-oxLDL after intravenous injection in trout. About 25 mg labeled trout lipoprotein was injected into each fish. Fifty microliter blood samples were drawn into a syringe and analyzed for radioactivity. The values are the mean of three fish ^SD.
3.4. Biodistribution of acLDL Injection of radiolabeled acetylated LDL (125IacLDL) showed that also this ligand was mainly cleared by kidney cells. One hour after injection more than 83% of acLDL were found in the kidney, while about 10% was found to remain in the blood (Fig. 4(B)). Relatively small amounts were found in liver (less than 4%), spleen (less than 2%), and heart (less than 2%). Body perfusion with PBS was omitted as previous experiments have shown that more than 90% is cleared from blood within 1 h after injection [28].
3.5. Competition assay Negatively charged SR ligands inhibited uptake of both radiolabeled oxLDL and acLDL in kidney cells when injected prior to the labeled ligand. A concurrent increase in the amount of oxLDL or acLDL remaining in the blood was observed. Preinjection with both LTA and fBSA significantly inhibited uptake of both oxLDL and acLDL in the kidney compared to the control. Preinjection with unmodified BSA showed tissue distribution of oxLDL and acLDL similar to the control (Fig. 5(A) and (B)).
Fig. 4. Biodistribution. (A) Uptake of 125I-TC-LDL and 125I-TCoxLDL in different organs after intravenous injection of 25 mg TC labeled lipoprotein. 24 h after injection the animals were sacrificed, organs excised, weighed and analyzed for radioactivity. Values are mean of three fishes ^SD and expressed as percent of activity recovered in the analyzed organs. (B) Uptake of 125I-acLDL in different organs after intravenous injection of ,30 mg directly labeled lipoprotein. One hour after injection, the animals were sacrificed, blood drawn and organs excised, weighed, and analyzed for radioactivity. Values are mean of three fishes ^SD and expressed as percent of activity recovered in the analyzed organs.
4. 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,
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Fig. 5. Competition assay. Two milligrams of either LTA, fBSA or BSA was intravenously injected 15 min prior to injection of ,30 mg 125IacLDL (A) or ,30 mg 125I-oxLDL (B). After 60 min the fish were sacrificed, blood was drawn and organs excised, weighed and analyzed for radioactivity. Control fish were not preinjected with competing ligand, and BSA was injected as a negative control. Values are the mean of three different fishes in each experiment ^SD and expressed as percent of activity recovered in the analyzed organs. Asterix denotes significantly different values compared to the control, with p , 0.05.
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 partly overlapping lists of ligands (reviewed in Refs. [1,2, 46]). SR may have been selected during evolution due to recognition and clearance of bacteria, apoptotic cells and possibly oxLDL via interaction with
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oxidized phospholipids [23]. It has been demonstrated that binding and phagocytosis of oxidatively damaged red blood cells or apoptotic thymocytes are inhibited by oxLDL [47,48]. Boullier et al. have shown that binding of oxLDL to CD36 transfected cells are in part mediated by oxidized phospholipids associated with both the protein and the lipid moieties of oxLDL [49]. In addition, a monoclonal antibody, EO6, against oxLDL [50] has been shown to recognize a series of oxidized phospholipids; structural motifs responsible for antigeneticity is outlined in Ref. [14]. This antibody inhibits uptake of oxLDL in Mf and in cells transfected with CD36 or SR-BI, and was shown to have a variable region that is 100% identical to a naturally occurring autoantibody designated T15. EO6 and T15 are structurally and functionally equivalent [51]. The T15 autoantibody has been extensively studied for three decades and has been shown to recognize phosphorylcholine (choline phosphate, the head group of phosphatidylcholine), which is a common constituent of the cell wall of many pathogens. Production of this autoantibody even under germ-free conditions in mice may in part be due to oxidatively modified phospholipids on the surface of apoptotic cells, or in oxLDL, acting as endogenous antigens modulating the level of expression of this antibody [23]. This may indicate that oxidized phospholipids of cells and LDL are common ligands for both SR and natural antibodies. Previous studies in trout have described lipoprotein synthesis, composition, and catabolism [28,52 – 54]. However, few studies have focused on the role of oxidative modifications of lipoproteins and their effect on metabolism [7]. Salmonid lipoproteins are highly enriched in polyunsaturated fatty acids, making them prone to oxidative modifications. Salmonids also develop atherosclerotic plaques [55 – 57] during migration and spawning although the coronary lesions in salmonids are typically lipid-free, despite high plasma levels of both total cholesterol and LDLs [55]. Our results show that rainbow trout LDL is susceptible to oxidation by Cu2þ. The relevance of this mode of modification to the in vivo situation is debated, but transition metal ions may be essential for cell-induced oxidation, and the lipoprotein composition is similar for metal-induced and cell-induced oxidation [7,9,58]. The temporal development of diene formation in trout LDL was different from that observed with human
LDL. In mammalian lipoproteins, a lag phase is normally observed before OD234 starts to rise. This lag phase represents depletion of vitamin E from the lipoproteins [7]. Diene formation in trout LDL increased immediately after Cu2þ addition without any detectable lag phase. This rapid onset of diene formation might be caused by the high PUFA content in trout LDL. The level of LPO after 24 h of oxidation was about five-fold higher than the pretreatment value. The pretreatment value was more than twice the level normally found in human LDL [7]. The increase was probably higher, since LPO may have been formed during the preparation of EDTA-free LDL. In one study, fully oxidized human LDL was obtained by overnight dialysis in PBS under air [59]. In addition, LPO are decomposed as the oxidative changes proceed through later stages. Initially, LPO’s increase concurrently with diene formation, but are subsequently decomposed to aldehydes in the terminal decomposition phase [7]. The level of TBARS reflects this decomposition, as well as the increase in net negative charge of the lipoprotein particle as the aldehydes start to react with amino groups of apoB. The mobility of trout LDLs in agarose gels before and after Cu2þ induced oxidation was compared. An increase in negative charge was observed both with oxidized and acetylated LDL as the relative electrophoretic mobility was 1.26 and 1.24, respectively, compared to native LDL (not shown). The relative increase in electrophoretic mobility was moderate compared to previously reported values for human LDL (up to 3 £ ) [7]. Assessed by a more functional assay such as receptor-mediated endocytosis in macrophages, trout LDL was found to be transformed into a ligand that was readily endocytosed by the SR of J774 cells. Uptake of human oxLDL was higher than uptake of trout oxLDL in J774 cells. This may be explained by the more negative charge in human oxidized LDL, or by the fact that J774 mouse macrophages express several other classes of SR (some are CD36, SR-BI and MARCO) in addition to class A SR (SRA) [40,42, 43], that may have different affinities for the two species of oxLDL. In mammals, the main scavenging site for effete plasma macromolecules resides in the liver EC [60]. In contrast, salmonid fishes express SR activity in kidney cells [25–28]. The accelerated plasma clearance and
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the increased uptake of oxidized LDL compared to native LDL, suggests that it was catabolized via the SR pathway. This is corroborated by the tissue distribution of injected oxLDL and acLDL, and in agreement with previous results [28], and also by work performed by others [26]. Earlier we have shown uptake of 2,8 mm polystyrene beads in rainbow trout head kidneyderived macrophages, demonstrating a role for SR in phagocytosis [27]. Further support for the involvement of SR in oxLDL uptake was the finding that when fish were preinjected with ligands typical of the mammalian SR-A (LTA, fBSA) (reviewed in Ref. [38]), endocytosis of oxLDL and acLDL was significantly reduced. LTA is a component of the surface of gram positive bacteria, which has been shown to be recognized by class A SR [61], in addition to recognition of whole gram positive bacteria by several SR classes [61 – 65]. Polyinosinic acid was also tested as a putative competitor, but no significant reduction was observed (not shown). In conclusion our study show that trout LDL is susceptible to oxidative modifications, leading to increased negative charge and subsequent endocytosis by kidney cells. The same route of metabolism is used by acetylated LDL. The cell types in kidney responsible for this clearance are capillary EC/SEC in the case of acLDL [28]. Both SEC and leucocytes in the rainbow trout kidney has been shown to take up other SR ligands [26]. In addition, our results show that uptake of both oxLDL and acLDL in kidney is significantly inhibited by LTA and fBSA indicating a broad specificity for these receptors in fishes as in mammals.
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