Accumulation of Biglycan and Perlecan, but Not Versican, in Lesions of Murine Models of Atherosclerosis
Proteoglycan accumulation within the arterial intima has been implicated in lipoprotein retention and in atherosclerosis progression in humans. Two commonly studied murine models of atherosclerosis, the apolipoprotein E (apoE)-deficient (apoE−/−) mouse and the low density lipoprotein receptor–deficient (LDLR−/−) mouse, develop arterial lesions similar to those of human atherosclerosis. However, specific proteoglycan classes that accumulate in lesions of these mice and their relation to the retention of specific apolipoproteins have not been previously determined. In this report, we characterized the distribution of proteoglycans (versican, biglycan, and perlecan) and apolipoproteins (apoB, apoA-I, and apoE) in proximal aortic lesions of chow-fed apoE−/− and LDLR−/− mice at 10, 52, and 73 weeks of age. We observed that similar to the apoE−/− mice, the LDLR−/− mice develop intermediate and advanced plaques within 52 weeks of age. Perlecan and biglycan (both are proteoglycans) appeared early in lesion development with distinct expression patterns as the plaques advanced. Versican, a major proteoglycan detected in human plaques, was mostly absent in both strains. ApoA-I and apoB were detected in early through advanced lesions in regions of proteoglycan accumulation in both strains. Our results indicate that proteoglycans may contribute to the retention of lipoproteins at the earliest stage of atherosclerosis in murine models of atherosclerosis.
A prominent feature of atherosclerotic lesions is the accumulation of extracellular matrix proteoglycans and lipids. Results from multiple studies support the concept that proteoglycans are responsible for trapping lipoproteins in the arterial wall.1 Prolonged retention of lipoproteins may render them susceptible to chemical modifications, such as oxidation, lipase modification, or glycation, leading to their aggregation and/or cellular uptake and lipid accumulation in macrophages and vascular smooth muscle cells.2 This response-to-retention hypothesis of atherosclerosis is considered to be a “central paradigm” contributing to the etiology of atherosclerosis.
A consistently reported change in arterial proteoglycans during the progression of human atherosclerosis is an increase in chondroitin sulfate proteoglycans (CSPGs) and dermatan sulfate proteoglycans and a decrease in heparan sulfate proteoglycans.3–5⇓⇓ CSPG-LDL complexes have been detected in injured rabbit intima, and they have also been isolated from human atherosclerotic lesions, confirming a role for CSPGs in atherosclerosis.3,6⇓ Although dermatan sulfate proteoglycans (eg, biglycan) and heparan sulfate proteoglycans (eg, perlecan) have been detected in the intimal lesions of nonhuman primates and humans, few studies have investigated the role of these proteoglycan classes in atherosclerosis.7–9⇓⇓ Given the diversity in structure and function of the proteoglycan family, it is critical to delineate the presence and role of proteoglycans in atherosclerosis.10
Two murine models of atherosclerosis used widely for studies of atherosclerosis are the apoE-deficient (apoE−/−) and the LDL receptor–deficient (LDLR−/−) mice.11,12⇓ Plasma total cholesterol levels in apoE−/− and LDLR−/− mice are elevated ≈4-fold and 2-fold, respectively, over the levels in wild-type mice when the mice are fed rodent chow, and the levels are 7-fold higher when the mice are fed diets containing cholesterol. Although apoE−/− mice develop lesions when they are fed either diet, the LDLR−/− mice are reported to develop lesions only when they are fed diets rich in fat and cholesterol.12–14⇓⇓ Unfortunately, even in apoE−/− mice, many atherosclerotic lesion characterizations have been performed after the feeding of high cholesterol diets, which induce aberrant lipid perturbations and rapid (in weeks) plaque development. Thus, these studies may not reflect the aortic remodeling seen in the long-term progression of atherosclerotic plaques. Furthermore, no studies have investigated either the proteoglycan composition of lesions or the relation of proteoglycans to apolipoproteins in lesions of either strain.
The purpose of the present study was to characterize proteoglycans and their interactions with apolipoproteins during the long-term development of lesions in apoE−/− and LDLR−/− mice fed rodent chow. Our results demonstrate that perlecan and biglycan appear early in lesion development in both strains. Versican, the major CSPG detected in human atherosclerosis, was mostly absent in both strains. ApoA-I and apoB were detected in early through advanced lesions in regions of proteoglycan accumulation in both strains, suggesting that proteoglycans may contribute to the retention of lipoproteins at the earliest stages of murine atherosclerosis.
Animals and Diets
Male and female apoE−/− and LDLR−/− mice on a primarily C57BL/6J genetic background were obtained from the Jackson Laboratory (Bar Harbor, Me). Mice were maintained on a 12-hour light/dark cycle and given free access to food and water. Mice were fed pelleted rodent chow (Wayne Rodent BLOX 8604, Teklad), which is low in fat and cholesterol content (4% [wt/wt] fat and 0.04% [wt/wt] cholesterol by manufacturer’s estimate) for the entire period of the present study. At the time of death, blood was collected, and plasma was stored at −70°C before analysis.15 Mice were killed with pentobarbital (80 mg/kg IP) and were perfusion-fixed with 10% neutral buffered formalin as described previously.15 Mice were killed at 10 to 11 weeks, 50 to 54 weeks, and 70 to 73 weeks of age. Mouse protocols were approved by the Animal Care and Use Committee of the University of Washington.
Aortic Sinus Lesion Area
Quantification of atherosclerotic fatty streak lesions was performed by evaluation of lesion size in the aortic sinus as described previously.15
Rabbit antisera directed against core proteins of human biglycan (LF-51, dilution 1:500; a kind gift of Dr Larry Fisher, Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Md) and versican (dilution 1:800; kindly provided by Drs Richard LeBaron, University of Texas at San Antonio, and Erkki Ruoslahti, La Jolla Cancer Research Center, La Jolla, Calif) were used in sections pretreated with chondroitinase ABC lyase (Sigma Chemical Co) to remove chondroitin sulfate and dermatan sulfate glycosaminoglycan chains.9 Rabbit antisera against human perlecan (diluted 1:200) was a kind gift of Dr Alan Snow, University of Washington, Seattle, and is monospecific and reactive to mouse perlecan.16
Specificity of antisera for detecting murine biglycan and versican was confirmed by Western blot analysis of the murine heart and liver (Figure 1). Approximately 0.7 g of mouse heart or liver tissue was homogenized in buffer (4 mol/L guanidine hydrochloride and 50 mmol/L sodium acetate, pH 6.0, with 5 mmol/L benzamidine, 100 mmol/L 6-aminohexanoic acid, 10 mmol/L N-ethylmaleimide, and 1 mmol/L phenylmethylsulfonyl fluoride) and extracted overnight at 4°C. The homogenate was dialyzed (against 8 mol/L urea, 0.1 mol/L Tris, pH 7.4, 0.2 mol/L NaCl, and 0.5% Triton X-100), concentrated over DEAE-Sephacel, and precipitated with ethanol. Proteoglycans were digested with 0.5 U chondroitinase ABS lyase (Sigma), applied to SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell). Specific core proteins were visualized after incubation with antibodies to biglycan (1:3000 dilution) and versican (1:1500 dilution), followed by goat anti-rabbit IgG linked to horseradish peroxidase. Blots were developed by enhanced chemiluminescence (ECL, Amersham) and exposed to film.
Goat polyclonal antiserum raised against human apoE (dilution 1:7500, a kind gift of Dr John Albers, University of Washington, Seattle) and apoA-I (dilution 1:10 000, a kind gift of Dr John F. Oram, University of Washington, Seattle) were described previously.17,18⇓ Specificity of these antisera for murine apoA-I and apoE was confirmed by Western blot of murine plasma (data not shown). A rabbit polyclonal antibody specific for murine apoB (diluted 1:1000) was a kind gift of Dr Thomas Innerarity, Gladstone Research Laboratories, San Francisco, Calif.
Serial sections were used to perform immunohistochemisty.19 Diaminobenzidine with nickel chloride was used as the peroxidase substrate, yielding a black reaction product. Slides were counterstained with methyl green. Negative controls included substitution of primary antiserum with PBS or normal rabbit or goat serum.
Quantification of Areas Immunostained for Proteoglycans and Apolipoproteins
Immunostained areas of proteoglycans and apolipoproteins in the aortic sinus were quantified by using Optimas Image Analysis. The extent of immunostained areas was determined by using a selection of threshold for gray scale, brightness, and contrast. The immunostained pixels and total pixels for the lesion were converted to units of squared micrometer.
Measurement of Plasma Cholesterol Levels
Plasma levels of total cholesterol were measured enzymatically (Liberty Scientific, No. 225-26) as described previously.20
Measurement of Medial Thickness
Medial thickness was measured by using Optimas Image Software from Movat-stained sections of aortic sinuses of apoE−/− and LDLR−/− mice. Three values were averaged for each section. Three sections for each mouse were evaluated and averaged to provide 1 value of medial thickness per mouse. Data are expressed as mean±SEM for 5 mice.
All experimental data are expressed as mean±SEM. Mean values were compared by the Student t test, the Tukey highly significant difference multiple comparisons test, or ANOVA. The Pearson coefficient of correlation was used. A value of P<0.05 was accepted as statistically significant.
Extent of Aortic Sinus Atherosclerosis
The mice appeared healthy and gained weight throughout the study. Body weights were not significantly different between strains at 11, 52, and 73 weeks of age. No xanthomatous tissues were observed in LDLR−/− mice, whereas mild to moderate xanthomas (skin, infiltration of the ears, and foot thickening) were observed in apoE−/− mice by 52 weeks.
Cholesterol levels did not differ between sexes or change significantly between 11 and 52 weeks of age for LDLR−/− mice (253±12 and 300±10 mg/dL, respectively) and apoE−/− mice (405±45 and 360±10 mg/dL, respectively). By 73 weeks, cholesterol levels increased significantly for LDLR−/− mice (440±60 mg/dL, P<0.001) and apoE−/− mice (654±20 mg/dL, P<0.001).
Within each strain and time point, the lesion areas tended to be greater for female mice than for male mice, but the differences were not significantly different. Therefore, we have collapsed the data with respect to sex of the animal for the remaining analyses. Aortic sinus lesion areas increased with age for LDLR−/− and apoE−/− mice, and apoE−/− mice had larger lesions than LDLR−/− mice at each time point (P<0.006 to <0.0001). Mean lesion sizes for LDLR−/− mice were 1599±986 μm2 (n=10), 111 934±35 836 μm2 (n=10), and 432 755±59 650 μm2 (n=10) at 11, 52, and 73 weeks, respectively. For apoE−/− mice, values were 10 106±2523 μm2 (n=10), 657 402±141 303 μm2 (n=10), and 1 170 975±105 718 μm2 (n=8) at 11, 52, and 73 weeks, respectively.
Morphology of Aortic Sinus Atherosclerosis
At 11 weeks of age, foam cell deposits were present in 8 of 9 apoE−/− mice (Figure 2B). These early foam cell lesions progressed to intermediate fibrous plaques at 52 weeks (Figure 2D) and advanced necrotic plaques at 73 weeks (Figure 2F). Lesions at 52 weeks were characterized by deposits of proteoglycans (blue, by Movat’s stain), collagen (yellow [does not show well on photographs]), and smooth muscle cells (red, by Movat’s stain) as well as several lipid cores. These lesions resembled type IV intermediate fibrous plaques in humans.21 At 73 weeks, most apoE−/− lesions were lipid-laden and mostly devoid of nuclei. Marked decreases in overall proteoglycan and collagen contents were seen in conjunction with an increase in necrotic core formation. Fibrous caps were thin to nonexistent. Coronaries separating ventricles were largely occluded with foam cells and proteoglycans (Figure 2F, arrow). In some cases, several lipid cores separated by thin layers of extracellular proteoglycan and elastin layers (Figure 2F) were stacked irregularly, one above the other, resembling type Va advanced human lesions.21 Thus, lesions in apoE−/− mice fed rodent chow began as fibrofatty plaques and progressed to necrotic plaques.
In contrast to apoE−/− mice, LDLR−/− mice at 11 weeks of age were mostly free of lesions (8 of 10 mice had no aortic sinus lesions; Figure 2A). Lesions of LDLR−/− mice also progressed to intermediate (52 weeks, Figure 2C), and advanced (73 weeks, Figure 2E) lesions. Intermediate lesions and advanced lesions were largely cellular with accumulations of smooth muscle cells, proteoglycans, and collagen and were covered by a fibrous cap of smooth muscle cells. Foam cells were present near the shoulders of the lesions. At 52 weeks, lesions from LDLR−/− mice shared several features of type III preatheromas in human atherosclerosis.21 Lesions at 73 weeks had increased cholesterol clefts and extracellular lipid cores (Figure 2C). In the intima, smooth muscle cells colocalized with proteoglycans and collagen. Fragmentation of elastin (black, by Movat’s stain) in the media was present in most lesions. Figure 3C highlights the severe intimal disorganization accompanying several lipid cores (near arrows), resulting in disrupted extracellular matrix structure at 73 weeks. Proteoglycans and collagen were distributed around the extracellular lipids. Overall, lesions in the LDLR−/− mice fed rodent chow progressed from fatty streaks at 11 weeks to advanced type V fibrofatty plaques containing an abundance of proteoglycans.21
In intermediate and advanced lesions of apoE−/− mice, medial layers were collapsed, with little or no smooth muscle cytoplasm between elastin layers, indicating smooth muscle cell death (Figure 3B and 3D). Significant medial thinning (P<0.05) was observed for apoE−/− mice between 11 weeks (71±7 μm, n=5) and later time points (25±3 μm, n=5), confirming a previous report.22 The thinnest areas of internal elastic laminae were adjacent to cholesterol clefts.
In contrast, for LDLR−/− mice, the average width of matrix did not change appreciably between 11 weeks (41±1 μm, n=5) and later time points (50±6 μm, n=5). However, the internal elastic laminae of LDLR−/− mice (Figure 3A and 3C) exhibited elastin fragmentation at 52 and 73 weeks (Figure 3C, arrows). A notable feature was the accumulation of proteoglycan (blue, by Movat’s stain) within layers of elastic laminae (Figure 3C, arrows). The blue proteoglycan stain between elastin layers is shown at a higher magnification for another LDLR−/− animal aged 52 weeks in Figure 3E (arrow). In an adjacent section treated with Gomori’s histochemical stain (Figure 3F), this entire matrix region is blue-green in color, indicative of collagen and/or elastin. Contractile elements, which stain red, are absent. Yet, contractile protein staining is evident for the fibrous “cap” located luminally in this section (Figure 3F, asterisk). These observations suggest that a phenotypic shift from contractile to synthetic is occurring among matrix smooth muscle cells in regions of elastin dissolution.
The extent of medial thinning was not correlated with intimal thickness or lesion sizes for either strain (data not shown). Overall, medial thinning was more severe in apoE−/− mice than in LDLR−/− mice.
Perlecan was the most abundant proteoglycan detected in both strains of mice. In intermediate and advanced lesions of LDLR−/− mice, perlecan immunostaining was prominent throughout the intima in regions rich in smooth muscle cells and lipid cores (Figure 4E). In addition, perlecan was detected throughout the media. In intermediate and advanced lesions of apoE−/− mice, perlecan protein expression was very prominent throughout the intima (Figure 5B). Perlecan was present in layers around the regions of extracellular lipid accumulations. In contrast to LDLR−/− mice, perlecan expression was low in regions rich in smooth muscle cells near the lumen and in the media in apoE−/− mice.
Biglycan had an overlapping but distinct distribution compared with that of perlecan. In intermediate and advanced lesions of LDLR−/− mice, biglycan expression was localized to regions containing proteoglycans and not to the collagen-rich regions (compare Figure 4A with Figure 4C). These biglycan-containing areas included regions surrounding foam cells and lipid cores. Low levels of biglycan were detected in the media, particularly in regions of elastin fragmentation, and were correlated with areas of proteoglycan accumulation, as identified by Movat’s stain (not shown). In contrast, in apoE−/− mice, biglycan typically was distributed in the deep intimal layer surrounding the cholesterol clefts (Figure 5D) and was less evident near the lumen.
Versican immunostaining was mostly absent or detected at low levels in the lesions of either mouse strain at all time points studied. For LDLR−/− mice, versican immunostaining was absent in wall (Figure 4G) and valve lesions but was detected in valve cusps (not shown). Advanced lesions of apoE−/− mice showed weak versican immunostaining, except at sites in the deep intima adjacent to the cholesterol clefts (Figure 5C). Overall, the distributions of versican, biglycan, and perlecan were distinct within each strain.
Quantitative assessment of proteoglycan accumulation revealed interesting patterns between LDLR−/− and apoE−/− mice (Figure 6). Perlecan and biglycan showed similar patterns of accumulation with age in apoE−/− mice. At 11 weeks, extensive deposits of perlecan and biglycan were observed throughout the fatty streak lesions of apoE−/− mice (Figure 6B). These levels increased significantly by 52 weeks (for biglycan, P<0.01; for versican, P<0.0001) and then decreased to initial levels at 73 weeks, likely reflecting necrosis of the proteoglycan matrix with time. In contrast, LDLR−/− mouse lesions showed a steady increase in perlecan with time (between 11 and 52 weeks, P<0.00001). Biglycan increased and leveled off between 52 and 73 weeks (between 11 and 52 weeks, P<0.0001; Figure 6A). Versican was not observed in lesions from LDLR−/− mice. Overall, proteoglycan accumulation reflected increased lesion development until such time as necrotic events deteriorated lesion architecture.
For LDLR−/− mice at 11 weeks, apolipoproteins were observed coincident with tiny fatty streak lesions but were also detected in adjacent subendothelial regions where foam cells had not yet accumulated (Figure 7D and 7F). Perlecan and biglycan (Figure 7B and 7C, respectively) were also present in these regions. These data suggest strongly that proteoglycan accumulation and subsequent lipoprotein deposition may represent the first morphologically identifiable step in atherogenesis.
In intermediate and advanced lesions of LDLR −/− mice, apoA-I, apoB, and apoE had overlapping but distinct accumulation profiles (eg, see Figure 4B, 4D, and 4F). ApoE and apoB accumulated primarily in the deep intima around foam cells and in smooth muscle cell–rich regions. ApoA-I and apoB were prevalent in apoE−/− aortic sinus lesions at all time points and had overlapping distribution patterns (not shown).
ApoA-I content was quantified in LDLR−/− and apoE−/− lesions (Figure 6). ApoA-I accumulation increased steadily in LDLR−/− mice with age, mirroring that of perlecan. Similar results were observed for apoE −/− lesions, in which apoA-I levels also mirrored the distribution and content of perlecan. Although we observed apolipoproteins in regions of perlecan and biglycan accumulation, we could not determine specific associations of proteoglycans with apolipoproteins in the present study.
In the present study, we have demonstrated that the major proteoglycans detected in murine atherosclerosis are perlecan and biglycan. Perlecan was present extensively in the lesions of apoE−/− and LDLR−/− mice. Perlecan was detected in intermediate and advanced lesions of hypercholesterolemic nonhuman primates and in cultures of medial smooth muscle cells from human atherosclerotic tissue, but its expression in human lesions has not yet been reported.7–9⇓⇓ Several functions have been attributed to perlecan, but its role at the arterial wall is unclear.10 The LDL receptor class A module domain in perlecan shares homology with the binding domain of the LDL receptor and could potentially bind lipoproteins.10 Perlecan can bind growth factors that promote angiogenic and tumor growth functions.23 At the arterial wall, perlecan may inhibit the proliferation of smooth muscle cells as it does in cell culture.24,25⇓ Our results support a role for perlecan in murine atherosclerosis. However, further studies are needed to determine the role of perlecan at the arterial wall.
Biglycan was detected in apoE−/− and LDLR−/− strains below the fibrous cap and adjacent to matrix and was absent in the endothelium in both strains. In vitro studies have demonstrated that biglycan can bind apoB- and apoE-containing particles.9,26⇓ Biglycan deposits colocalize with apoB and apoE in human atherosclerotic plaques.9 Biglycan interacts with extracellular matrix components, such as collagen types I and V, and can bind to phospholipase A2, which may act on arterial wall LDL, thus contributing to the migration of endothelial cells.27,28⇓ Biglycan expression in both strains of mice supports a role for biglycan in binding lipoproteins. In intermediate and advanced lesions, the general pattern of immunostaining for apoE (in LDLR−/− mice) and apoB was more closely matched to that of biglycan than of perlecan. Taken together, these results suggest that biglycan may be proatherogenic at the arterial wall. Our results support a role for biglycan and perlecan in lipoprotein binding and/or retention at the arterial wall in apoE−/− and LDLR−/− mice.
In contrast, versican, an abundant proteoglycan in human lesions, was essentially absent. Versican was detected in humans and in nonhuman primates as well as in spontaneous and experimentally induced atherosclerosis in rabbits, swine, and pigeons.29–31⇓⇓ The absence or low levels of versican immunostaining in the LDLR−/− mice and apoE−/− mouse lesions may be due to the macrophage-rich nature of mouse lesions. Macrophages can induce versican degradation and modulate its synthesis.32 Overall, the paucity of versican in mouse lesions precludes this as an important contributor to lipoprotein retention in these strains.
The abundance of apoA-I in lesions of both strains (and also demonstrated in humans) has important implications.9 It demonstrates the retention not only of atherogenic lipoproteins (such as LDL and VLDL remnants) but also of HDL. It also raises the question of how apoA-I retention is mediated. So far, in vitro studies have failed to demonstrate an interaction of apoA-I with biglycan, arguing against a direct interaction with biglycan.9 Instead, interactions between biglycan and the apoE component of HDL have been suggested.26 Because apoA-I also accumulated in the lesions of apoE−/− mice and because apoA-I immunostaining was most consistently coincident with that of perlecan, we suggest that perlecan is a major proteoglycan responsible for binding apoA-I.
Deposits of biglycan, perlecan, apoA-I, and apoB were observed in the earliest detectable subendothelial foam cells in mouse aortic lesions. ApoA-I and apoB immunostaining were seen coincident with foam cells and in adjacent regions lacking foam cells. This is consistent with immunocytochemical studies in humans and rabbit arteries for which apolipoprotein deposition seems to precede fatty streak development.33,34⇓ We also show that perlecan and biglycan, but not versican, were present at and adjacent to the mouse type I lesions. Our data are consistent with the idea that lipoprotein retention occurs and is one of the earliest events in atherogenesis.
Finally, we observed several differences between the lesions of the 2 strains. Lesions in LDLR−/− mice progressed slower than did lesions in apoE−/− mice. LDLR−/− lesions (intermediate and advanced) were composed of proteoglycans and collagen deposits around lipid cores with fibrous caps. However, lesions in apoE−/− mice became progressively lipid-laden with age, finally becoming necrotic, as evidenced by decreases in proteoglycan and apolipoprotein content. Also, there were differences in medial thickness, suggesting that apoE (or the lack thereof) at the arterial wall influenced the architectural development of these lesions. These observations and the fact that macrophage-specific expression of human apoE reduced atherosclerosis in apoE −/− mice independent of systemic cholesterol levels35 suggest that functions for apoE have broad implications for arterial wall biology in general. In contrast, the absence or presence of LDL receptors in the macrophage did not influence the atherosclerotic process.36 In addition, local apoE production has been implicated in lipid efflux,37 modulating immune responses,38 lipoprotein retention,26 and macrophage function.39,40⇓ These functions of apoE have potentially important consequences for understanding the pathogenesis of atherosclerosis. We believe that the use of apoE−/− to model aspects of human atheroma may warrant caution. We recommend the use of LDLR−/− mice that are fed diets containing modest amounts of cholesterol and fat. Initiation and progression can easily be studied in this strain, lesion architecture is less necrotic in nature, matrix structure is retained during much of lesion expansion, and the mice show lipoprotein profiles more akin to those of humans with respect to LDL levels.
In conclusion, our data show that proteoglycans are abundant in mouse lesions and that perlecan and biglycan are possible substrates for lipoprotein entrapment. This process may occur during lesion initiation. The identification and characterization of proteoglycans is of importance because of the plethora of functions attributed to each type of matrix molecule. These functions can be tested by using mice genetically engineered to express altered levels of proteoglycans, cytokines, metalloproteinases, and other factors affecting proteoglycan expression.
This work was supported in part by American Heart Association Fellowship Grant 94-WA-109; by Grant 95-WA-113R from the American Heart Association, Washington Affiliate; and by grants HL-02788, HL-52848, and DK-02345 from the National Institutes of Health, Bethesda, Md.
Received December 13, 2001; revision accepted January 7, 2002.
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