Published online before print August 30, 2007, doi: 10.1161/CIRCRESAHA.107.149666
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Retention of Low-Density Lipoprotein in Atherosclerotic Lesions of the Mouse
Evidence for a Role of Lipoprotein Lipase
From the Sahlgrenska Center for Cardiovascular and Metabolic Research/Wallenberg Laboratory, Department of Molecular and Clinical Medicine (M.G., M.L., K.S., J.P., V.F., P.J., S.-O.O., J.B.), Göteborg University, Sweden; Division of Cardiovascular Medicine (S.F., M.F.L.), Vanderbilt University Medical Center, Nashville, Tenn; Department of Medicine (C.F.S.), Washington University, St Louis, Mo; and Department of Medical Biosciences (G.O.), Umeå University, Sweden.
Correspondence to Jan Borén, MD, PhD, Wallenberg Laboratory, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail Jan.Boren{at}wlab.gu.se
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Abstract |
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Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event in atherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB–proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131I-tyramine cellobiose–labeled control LDL (131I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL were retained in atherosclerotic aortae from ldlr–/– mice transplanted with lpl–/– compared with lpl+/+ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. Furthermore, transgenic mice expressing catalytically active or inactive LPL developed the same extent of atherosclerosis. Thus, retention of LDL in the artery wall is initiated by direct LDL–proteoglycan binding but shifts to indirect binding with bridging molecules such as LPL.
Key Words: apolipoprotein B • low-density lipoproteins • retention • atherosclerosis • lipoprotein lipase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The processes involved in the initiation of atherosclerosis have been under debate for many years, and several hypotheses have been postulated.1 We tested the response-to-retention hypothesis2 in an earlier study using genetically modified mice that expressed human recombinant low-density lipoproteins (LDLs) with reduced proteoglycan-binding activity and provided direct evidence showing that subendothelial retention of apolipoprotein (apo)B100-containing lipoproteins is the initiating event in atherogenesis.3 Furthermore, we demonstrated that the atherogenicity of LDL is linked to their proteoglycan-binding activity.3
Lipoproteins associate with artery wall proteoglycans via both direct and indirect interactions. Direct binding between LDL and proteoglycans involves an ionic interaction between basic amino acids in apoB100 (Site B; residues 3359 to 3369) and negatively charged sulfate groups on the glycosaminoglycan (GAG) chains of proteoglycans.4,5 Indirect binding between LDL and proteoglycans is facilitated by apoE,3 which is found in human atherosclerotic plaques together with apoB.6 ApoE binds vascular proteoglycans, and apoE-enrichment of proteoglycan-binding-defective LDL (in which Site B has been inactivated by mutagenesis) partly restores proteoglycan binding.3 Mouse LDL contains significant amounts of apoE, and consequently proteoglycan-binding-defective LDL isolated from mouse plasma displays 
30% of normal proteoglycan binding.3
Subendothelial retention of LDL via indirect binding to GAGs can also be facilitated by lipoprotein lipase (LPL).7 We have shown previously that the binding between LPL and LDL is mediated through an interaction between LDL lipids and LPL.8 LPL facilitates the interaction between GAG chains and extensively oxidized LDL (which cannot bind directly to GAG because of the reduced number of positive charges).9,10 LPL produced by macrophages in the vessel wall has been shown to promote foam cell formation and atherosclerosis in vivo.11,12 It is still unclear whether the proatherogenic role of LPL is solely attributable to bridging functions13 or, at least in part, to the localized generation of smaller particles through the enzymatic activity of LPL.
In this study, we investigated the importance of the direct binding of LDL-apoB to proteoglycans in the development of atherosclerosis over time and assessed the role of LPL to facilitate LDL retention at later stages of development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
For details regarding materials, refer to the expanded Materials and Methods section in the online data supplement at http://circres. ahajounrnals.org.
Human ApoB Transgenic Mice
Two different lines of human apoB transgenic mice (C57BL/6) were used in this study.14 The first line was human recombinant control apoB100 without any mutation in the apoB gene, except a CAA to CTA mutation in codon 2153, which was introduced into both apoB constructs used in the study.14 The mutation does not interfere with the receptor-binding activity of the recombinant LDL or its interaction with proteoglycans, but eliminates the formation of apoB48.14 A second line of transgenic mice expressed human apoB100, in which the arginine residues were converted to serines and the lysine residues were changed to alanines in site B of apoB100 (RK3359-3369SA).14 All animal studies received prior approval by the local Animal Ethics Committee.
Lipid Analysis of Plasma Lipoproteins
Total cholesterol and triglyceride levels were measured in fresh plasma samples obtained after a 4-hour fast. Cholesterol and triglycerides were measured using the Konelab multianalyzer (Kone, Espoo, Finland). The distribution of lipids within the plasma lipoprotein fractions was assessed by FPLC gel filtration using a Superose 6 HR 10/30 column.15
Quantification of Atherosclerosis
The aortic roots were embedded in OCT Tissue-Tec medium, frozen in dry ice and isopentane, cut into 10-µm-thick cross-sections, and stained in 0.5% oil red O.16 Distal aortae were pinned out by en face technique and fixed in 70% ethanol for 5 minutes, stained with 0.5% Sudan IV for 6 minutes, and differentiated for 3 minutes in 80% ethanol.
Retention of LDL In Vivo
Recombinant control LDL and RK3359-3369SA LDL were isolated by sequential ultracentrifugation (d=1.02 to 1.05 g/mL).14 The recombinant LDL were radiolabeled with 125I using Iodogen (Pierce),17 125I-tyramine cellobiose (125I-TC),18 or 131I-TC18 after immunoaffinity chromatography to remove mouse apoE and apoB.14 Retention of a mixture of 125I-LDL (3x107 counts per minute [cpm]) and 131I-TC-LDL (3x107 cpm) or 125I-TC-LDL (3x107 cpm) was analyzed as described previously.17 125I-LDL is mainly an indicator of retained extracellular LDL, as 125I-LDL is rapidly degraded after cellular uptake and its degradation products are immediately released from the cells. In contrast, 131I-TC-LDL and 125I-TC-LDL are not degraded but remain cell associated and therefore are indicators of both extra- and intracellular LDL.18 The specific activity of the recombinant control 125I-LDL, 131I-TC-LDL, or 125I-TC-LDL was 300 cpm/ng. Blood samples were collected at 2 minutes, 8 minutes, 30 minutes, 6 hours, 12 hours, and 24 hours after injection. Retention of 125I-LDL, 131I-TC-LDL, or 125I-TC-LDL was measured in aortae after 72 hours.
Radiolabeling and Secretion of GAGs
See the online data supplement.
Gel Mobility Shift Assay
Recombinant control LDL and RK3359-3369SA LDL were isolated by sequential ultracentrifugation (d=1.02 to 1.05 g/mL),14 and endogenous mouse apoE and apoB were removed by immunoaffinity chromatography.14 Analysis of the binding of recombinant control LDL and RK3359-3369SA LDL to GAGs isolated from atherosclerotic or nonatherosclerotic aortae was performed as described previously.19 To analyze the impact of LPL on the binding of LDL to GAGs, the gel mobility shift assay was also performed in the presence of 10 µg/mL LPL (purified from bovine milk as previously described20).
Bone Marrow Transplantation Using Fetal Liver lpl–/– Cells
Fetal liver cells with either genotype lpl+/+ or lpl–/– were generated by heterozygous breeding of lpl+/– mice.21 Fetal liver cells were isolated at day 1511 and injected into ldlr–/– mice that had been radiated with 9 Gy.11 Three weeks after the bone marrow transplantation, mice were fed an atherogenic diet.
Generation of Transgenic Mice With Macrophage-Specific Expression of Human Active or Inactive LPL
See the online data supplement.
Characterization of Macrophages From Transgenic Mice
Peritoneal macrophages were isolated as described previously.22 Isolated peritoneal macrophages were cultured in RPMI medium 1640 containing 10% FCS overnight at 37°C and 5% CO2, and labeled with 35S-methionine as described previously.23 Recombinant hLPLwt and hLPL156 were recovered from the media by immunoprecipitation using a monoclonal anti-LPL antibody. To analyze the lipase activity, isolated peritoneal macrophages were cultured for 24 hours before heparin 5 U/mL was added to the cells. LPL activity was measured under the same conditions as detailed previously,24 using the commercial phospholipid stabilized emulsion Intralipid (10%) into which 3H-labeled triolein had been incorporated (courtesy of Pharmacia-Upjohn, Stockholm, Sweden). The values were then corrected with the total cellular protein using Bradford.25
To obtain minimally modified recombinant control LDL and RK3359-3369SA LDL, recombinant control LDL and RK3359-3369SA LDL were isolated by sequential ultracentrifugation (d=1.02 to 1.05 g/mL)14 and stored at 4°C for 4 months.26 The uptake of minimally modified recombinant control LDL or RK3359-3369SA LDL into macrophages was analyzed by incubating peritoneal macrophages for 24 hours with 50 µg/mL minimally modified recombinant control LDL or RK3359-3369SA LDL under normal cell culture conditions, as described previously.27 Quantification of the total oil red O surface area was performed using the BioPix software as described previously.28
Western Blotting
Total cellular extracts of mouse aortae (5 mm in length) were prepared as described previously.29 Human recombinant LPL was immunoprecipitated from the total cellular extract with the anti-human LPL monoclonal antibody LPL.A4 (Abcam, Cambridge, UK), separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane. Blocking was performed in Tris-buffered saline with 5% nonfat milk, followed by incubation with antibody LPL.A4 in Tris-buffered saline with 5% nonfat milk. Proteins were visualized using enhanced chemiluminescence (GE Healthcare, Uppsala, Sweden) according to the recommendations of the manufacturer. Densitometric readings of bands were performed using ImageQuant software (Bio-Rad, Sundbyberg, Sweden).
Data Analysis
Curve-fit analysis was performed in GraphPad Prism version 3.00 for Windows using a nonlinear curve fit assuming 1-site binding (GraphPad Software, San Diego, Calif). Unless otherwise stated, results are given as means±SD. Statistical significance was tested using 1-way ANOVA, followed by Bonferroni (homogenous variance) or Dunett T3 (nonhomogenous variance). If the samples were not parametric, Kruskal–Wallis was performed, followed by the Mann–Whitney test.
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Results |
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Atherogenesis Is Delayed in Mice Expressing RK3359-3369SA LDL
To investigate the importance of the direct interaction between LDL-apoB and proteoglycans on the development of atherosclerosis over time, human apoB transgenic mice expressing recombinant proteoglycan-binding-defective LDL (RK3359-3369SA) or control LDL were fed an atherogenic diet for 0 to 40 weeks, followed by en face measurement of lesions in the aortae. The distribution of cholesterol into atherogenic lipoproteins (Figure 1) and the total cholesterol plasma levels in mice expressing recombinant RK3359-3369SA LDL or control LDL were similar (7.8±0.8 and 8.2±0.63 mmol/L, respectively). Initiation of atherogenesis was delayed by
7 weeks in mice expressing RK3359-3369SA LDL compared with mice expressing control LDL (Figure 2). However, mice expressing RK3359-3369SA LDL developed the same degree of atherosclerosis as mice expressing control LDL after 30 to 40 weeks (Figure 2).
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) or RK3359-3369SA LDL (
) after 20 weeks on an atherogenic diet. The fractions corresponding to very-low-density lipoproteins (VLDL), LDL, and high-density lipoproteins (HDL) are indicated.
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RK3359-3369SA LDL Is Retained As Efficiently As Control LDL in Atherosclerotic Aortae
We analyzed the retention in vivo of LDL with normal and reduced proteoglycan-binding activity in atherosclerotic and nonatherosclerotic aortae. Control LDL-expressing transgenic mice were fed an atherogenic diet (atherosclerotic aortae) or chow diet (nonatherosclerotic aortae) for 40 weeks and injected with a mixture of either control 125I-LDL and 131I-TC-LDL or RK3359-3369SA 125I-LDL and 131I-TC-LDL to allow simultaneous analysis of both 125I-LDL and 131I-TC-LDL. The decay of labeled recombinant control LDL and RK3359-3369SA LDL was identical in both atherosclerotic and nonatherosclerotic mice (data not shown).
Analysis of nonatherosclerotic aortae 72 hours after injection showed that higher levels of 125I-LDL and 131I-TC-LDL were retained in mice injected with control 125I-LDL and 131I-TC-LDL than in mice injected with RK3359-3369SA 125I-LDL and 131I-TC-LDL (P<0.01; Figure 3A). The retention of 125I-LDL and 131I-TC-LDL in atherosclerotic aortae was several fold higher than the retention observed in nonatherosclerotic aortae (Figure 3A and 3B). Furthermore, there was no significant difference between the mice injected with control LDL or RK3359-3369SA LDL (Figure 3B). These findings indicate that although proteoglycan-binding-defective LDL is retained at significantly lower levels than control LDL in nonatherosclerotic lesions, it is retained to the same extent as control LDL in atherosclerotic aortae.
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In nonatherosclerotic aortae, there was no significant difference between the levels of retained 125I-LDL or 131I-TC-LDL, either for control or RK3359-3369SA LDL (Figure 3A). In contrast, both control 131I-TC-LDL and RK3359-3369SA 131I-TC-LDL were retained at higher levels than control 125I-LDL and RK3359-3369SA 125I-LDL in atherosclerotic aortae (P<0.05; Figure 3B). This indicates that although retention of both extracellular and intracellular LDL is increased in atherosclerotic aortae compared with nonatherosclerotic aortae, the increase in intracellular LDL is more pronounced.
Direct Binding of RK3359-3369SA LDL to GAGs Isolated From Atherosclerotic and Nonatherosclerotic Aortae Is Defective
Analysis of GAG chains in aortae isolated from mice fed an atherogenic diet (atherosclerotic aortae) or chow diet (nonatherosclerotic aortae) for 40 weeks showed accumulation of longer GAG chains in atherosclerotic aortae (Figure I in the online data supplement).
We used a gel mobility shift assay to investigate the direct binding of recombinant RK3359-3369SA LDL and control LDL to the total GAG fraction isolated from these atherosclerotic and nonatherosclerotic aortae. Control LDL bound to GAGs from atherosclerotic aortae with a higher affinity than to GAGs from nonatherosclerotic aortae (dissociation constants [Kd] 8.3 versus 15.2 nmol/L; n=3 per group). In contrast, we did not observe binding of RK3359-3369SA LDL to GAGs from either atherosclerotic or nonatherosclerotic aortae (Kd>120 nmol/L; n=3 per group).
These findings suggest that proteoglycan-binding-defective LDL is retained in atherosclerotic lesions by interactions other than direct binding of LDL-apoB to proteoglycans.
LPL Is Important for Retention of LDL in Atherosclerotic Lesions
To analyze the effect of LPL on the binding of LDL to the total GAG fraction isolated from atherosclerotic aortae, we incubated RK3359–33609SA LDL and control LDL with LPL (10 µg/mL) before repeating the gel mobility shift assay. Control LDL bound to GAGs with a higher affinity in the presence of LPL than in the absence of LPL (Kd, 4.2 versus 8.8 nmol/L; n=3 per group). Furthermore, in the presence of LPL, RK3359-3369SA LDL bound to GAGs almost as efficiently as control LDL (Kd, 5.4 nmol/L; n=3 per group).
We next transplanted ldlr–/– mice with fetal liver cells from LPL-deficient (lpl–/–) and control (lpl+/+) mice to test the importance of macrophage-specific expression of LPL in the artery wall for retention of LDL. As the development of atherosclerosis is delayed in mice with transplanted lpl–/– cells, they were fed an atherogenic diet for longer than the control mice (18 versus 14 weeks) to ensure similar levels of atherosclerosis in both groups (8.6±1.9% and 7.9±2.4% sudanophilic lesions in pinned-out aortae from mice with transplanted lpl–/– and lpl+/+ cells, respectively, means±SD; n=6 per group).
Analysis of the aortae 72 hours after injection with either recombinant control 125I-TC-LDL or RK3359-3369SA 125I-TC-LDL showed that significantly more labeled LDL was retained in atherosclerotic aortae from mice transplanted with lpl+/+ cells than from mice transplanted with lpl–/– cells (P<0.01; Figure 4). We did not observe any difference in retained recombinant control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL in mice transplanted with lpl+/+ cells (Figure 4). There was a trend toward more retained recombinant control 125I-TC-LDL than RK3359-3369SA 125I-TC-LDL in mice transplanted with lpl–/– cells, but the difference was not significant (Figure 4). The decay of 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL was identical in both groups (data not shown). Analysis of the lipoprotein profiles in mice transplanted with lpl–/– or lpl+/+ cells showed identical profiles (data not shown).
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) and RK3359-3369SA LDL (
) were isolated by sequential ultracentrifugation and radiolabeled with 125I-TC after immunoaffinity chromatography to remove mouse apoE and apoB. 125I-TC-LDL (3x107 cpm) or RK3359-3369SA 125I-TC-LDL (3x107 cpm) was injected into the lpl–/– and lpl+/+ mice, and retention of 125I-TC-LDL or RK3359-3369SA 125I-TC-LDL was measured after 72 hours. The data are shown as means±SD (n=6 for 125I-TC-LDL and n=3 for RK3359-3369SA 125I-TC-LDL).
Both Active and Inactive LPL Facilitate LDL Uptake into Macrophages
To elucidate if the proatherogenic role of LPL is due solely to bridging functions or at least in part to its catalytic activity, we generated transgenic mice with macrophage-specific expression of human catalytically active (hLPLwt) or inactive (hLPL156) LPL. ApoE facilitates an indirect interaction between LDL and artery wall proteoglycans,3 and to avoid this confounding factor, the hLPLwt and hLPL156 mice were bred on an apoe–/– background. Peritoneal macrophages were isolated from the mice and labeled with 35S-methionine for 2 hours, followed by immunoprecipitation of hLPL from the media of the macrophages. Similar hLPL expression was observed in the media of macrophages from both hLPLwt and mutant hLPL156-expressing macrophages (Table 1). The lipase activity in media isolated from mutant hLPL156-expressing macrophages was similar to the activity in media isolated from nontransgenic peritoneal macrophages but significantly increased in media isolated from hLPLwt-expressing macrophages (Table 1).
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To study the effect of LPL on the uptake of minimally modified LDL into macrophages in vitro, peritoneal macrophages were isolated from hLPLwtxapoe–/–, hLPL156xapoe–/–, and apoe–/– mice and incubated with 50 µg/mL minimally modified recombinant control LDL or RK3359-3369SA LDL for 24 hours. Quantification of the total oil red O surface area of the cells showed significantly higher uptake of LDL in macrophages isolated from hLPLwtxapoe–/– and hLPL156xapoe–/– mice than from apoe–/– mice (Figure 5). There was no difference in LDL uptake between macrophages overexpressing hLPLwt or mutant hLPL156 (Figure 5) or between uptake of recombinant control LDL or RK3359-3369SA LDL in the 3 groups (Figure 5). These results were confirmed in studies where peritoneal macrophages isolated from hLPLwtxapoe–/–, hLPL156xapoe–/–, and apoe–/– mice were incubated with 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL (data not shown).
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Taken together, these results show that macrophage-specific expression of LPL induces lipid uptake and that this is independent of lipase activity.
Both Active and Inactive LPL Facilitate Progression of Atherosclerosis
To further investigate the proatherogenic role of both active and inactive LPL, we performed an atherosclerosis study. To minimize cholesterol diet-induced macrophage expression of endogenous LPL,30 the mice were fed an atherogenic diet for 6 weeks, followed by a chow diet for an additional 6 or 12 weeks. There were no significant differences in lipid profiles (data not shown) or plasma cholesterol in apoe–/–, hLPLwtxapoe–/–, or hLPL156xapoe–/– mice at the 2 different time points (9.3±1.0, 9.3±0.6, and 8.8±2.2 mmol/L, respectively, after 6+6 weeks and 9.4±1.1, 8.7±2.1, and 9.2±1.6 mmol/L, respectively, after 6+12 weeks). Western blots of aortic extracts from hLPLwtxapoe–/– or hLPL156xapoe–/– mice analyzed after 6+6 and 6+12 weeks with an antibody against human LPL showed accumulation of equal amounts of hLPLwt and hLPL156 (Figure 6).
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At 6+6 weeks, hLPLwtxapoe–/– and hLPL156xapoe–/– mice showed increased atherosclerosis in the proximal aorta compared with apoe–/– mice (P<0.01; Table 2). At 6+12 weeks, both hLPLwtxapoe–/– and hLPL156xapoe–/– mice displayed significantly more atherosclerosis than apoe–/– mice in the distal aorta (P<0.01; Table 2). These results show that the noncatalytic bridging function of LPL is sufficient for its proatherogenic role.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we observed that although the initiation of atherosclerosis is delayed in mice expressing RK3359-3369SA LDL with reduced proteoglycan-binding activity, they eventually develop the same level of atherosclerosis as mice expressing control LDL. Proteoglycan-binding-defective LDL was not retained in nonatherogenic aortae, but similar levels of retention were observed for proteoglycan-binding-defective LDL and control LDL in atherosclerotic lesions. We further showed an involvement of LPL in retention of LDL in later stages of atherogenesis, and the enzyme did not need to be active to mediate its proatherogenic role. Our findings indicate that direct LDL–proteoglycan binding is necessary to initiate atherogenesis, but at later stages of development, facilitated binding leads to accelerated retention of LDL in lesional aortae.
Our finding that the initiation of atherogenesis was delayed in mice expressing proteoglycan-binding-defective LDL is in agreement with our earlier results showing that subendothelial retention of apoB100-containing lipoproteins is the initiating event in atherogenesis.3 In the present study, however, we showed that the growth and plateau phases of lesion development were almost identical in mice expressing either control or proteoglycan-binding-defective LDL, demonstrating the importance of analyzing several time points in atherosclerosis studies. As we did not observe binding of proteoglycan-binding-defective LDL to GAGs isolated from either atherosclerotic or nonatherosclerotic aortae, processes other than apoB–proteoglycan interaction must promote LDL retention at later stages of atherogenesis.
We showed that cellular degradation of both control LDL and RK3359-3369SA LDL was markedly increased in atherosclerotic compared with nonatherosclerotic aortae, which is consistent with accumulation of lipids in macrophages in atherosclerotic aortae. Indeed, a recent autopsy study of children and young adults who died of noncardiac causes indicate that extracellular subendothelial retention precedes macrophage infiltration and foam cell formation.31
LDL and other apoB-containing lipoproteins bind weakly to vascular proteoglycans in vitro in physiological ionic-strength environments.32 In contrast, molecules such as LPL bind tightly to the LDL lipids8 and facilitate a much stronger LDL retention in the vessel wall by bridging LDL to either macrophages or the extracellular matrix.33 Here, we showed that LPL facilitated the binding of recombinant control LDL to GAGs in vitro and that RK3359-3369SA LDL bound almost as efficiently as control LDL in the presence of LPL. Furthermore, the retention of both recombinant control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL was increased in atherosclerotic aortae from mice transplanted with lpl+/+ cells compared with mice transplanted with lpl–/– cells, demonstrating the importance of macrophage expression of LPL for retention of LDL in lesional aorta. As mice transplanted with lpl–/– bone marrow develop less atherosclerosis,11 they were fed an atherogenic diet for longer than the control mice to induce similar amounts of atherosclerosis in pinned-out aortae.
The importance of macrophage-specific expression of LPL in the development of atherogenesis has been shown previously by several groups,11,34–36 but these studies did not elucidate whether LPL acts as a noncatalytic bridge or whether its lipase activity is at least partly involved. Here, we generated transgenic mice with macrophage-specific expression of active and inactive (D156N) LPL and showed that both forms of the protein were expressed at similar levels in isolated, activated macrophages. Furthermore, both active and inactive LPL accumulated to the same extent in the aortic wall, indicating that the 2 forms have similar stability in the vasculature. The expression of the transgene was low, resulting in only a modest increase in the lipase activity in cultured peritoneal macrophages but with the advantage that the lipoprotein profiles were not affected. Despite the low expression of the transgenes, the uptake of minimally modified recombinant control LDL and RK3359-3369SA LDL was increased in macrophages from mice expressing either catalytically active or inactive LPL compared with macrophages from control mice. Furthermore, mice expressing either active or inactive LPL had significantly more atherosclerotic lesions than control mice. These results indicate that macrophage-specific expression of LPL has a key role in atherogenesis and that the noncatalytic bridging function of LPL is sufficient for its proatherogenic role.
Extracellular matrix changes can also promote LDL retention at later stages of atherosclerosis development. We observed proteoglycans with longer GAG chains in atherosclerotic aortae compared with nonatherosclerotic aortae, in agreement with Kunjathoor et al.37 Enhanced binding of LDL to proteoglycans can also be caused by the modification and aggregation of LDL by sphingomyelinases or phospholipases, which are expressed in atherosclerotic lesions.38 These modifications activate GAG-binding sites on apoB100 to work synergistically with the principal proteoglycan-binding site (Site B), leading to increased interaction with proteoglycans.39
In summary, retention of LDL in the artery wall is initially governed by direct binding of LDL to proteoglycan GAG chains, but then there is a shift to indirect binding when macrophages infiltrate the intima and secrete bridging molecules such as LPL. These bridging molecules act in concert or in parallel with proatherogenic modifications of the extracellular matrix and subendothelial modification of LDL, leading to accelerated retention of atherogenic lipoproteins.
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Acknowledgments |
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We thank Dr Rosie Perkins for excellent scientific editing.
Sources of Funding
This work was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Göran Gustafsson Foundation, the Swedish Foundation for Strategic Research, the Torsten and Ragnar Söderberg Foundation, and NIH grants HL65709 and HL57986 (to S.F.) and HL65405 (to M.F.L.).
Disclosures
Dr. Borén has received honoraria from Sanofi-Aventis, and Dr. Semenkovich has received support from Speakers’ Bureau.
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Footnotes |
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Original received February 2, 2007; revision received August 13, 2007; accepted August 20, 2007.
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References |
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