Published online before print May 1, 2008, doi: 10.1161/CIRCRESAHA.107.170662
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© 2008 American Heart Association, Inc.
Molecular Medicine |
Discoidin Domain Receptor 1 (Ddr1) Deletion Decreases Atherosclerosis by Accelerating Matrix Accumulation and Reducing Inflammation in Low-Density Lipoprotein Receptor–Deficient Mice
From the Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
Correspondence to Dr Michelle Bendeck, PhD, Department of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Building, Room 6213, 1 King’s College Circle, Toronto, ON M5S 1A8. E-mail michelle.bendeck{at}utoronto.ca
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Abstract |
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Collagens are abundant within the atherosclerotic plaque, where they contribute to lesion volume and mechanical stability and influence cell signaling. The discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds to collagen, is expressed in blood vessels, but evidence for a functional role during atherogenesis is incomplete. In the present study, we generated Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice and fed them an atherogenic diet for 12 or 24 weeks. Targeted deletion of Ddr1 resulted in a 50% to 60% reduction in atherosclerotic lesion area in the descending aorta at both 12 and 24 weeks. Ddr1–/–;Ldlr–/– plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1+/+;Ldlr–/– plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1–/– smooth muscle cells in vitro, revealed increased mRNA levels for procollagen
1(I) and 1(III) and tropoelastin, suggesting an enhancement of matrix synthesis in the absence of DDR1. Furthermore, whereas plaque smooth muscle cell content was unchanged, Ddr1–/–;Ldlr–/– plaques had a 49% decrease in macrophage content at 12 weeks, with a concomitant reduction of in situ gelatinolytic activity. Moreover, mRNA expression of both monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 was reduced in vivo, and Ddr1–/–;Ldlr–/– macrophages demonstrated impaired matrix metalloproteinase expression in vitro. These data suggest novel roles for DDR1 in macrophage recruitment and invasion during atherogenesis. In conclusion, our data support a role for DDR1 in the regulation of both inflammation and fibrosis early in plaque development. Deletion of DDR1 attenuated atherogenesis and resulted in the formation of matrix-rich plaques.
Key Words: atherosclerosis • discoidin domain receptor 1 • collagen • inflammation • macrophage
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Introduction |
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Atherosclerosis is a fibroinflammatory disease of the arterial wall. The atherosclerotic plaque is home to multiple cell types, including endothelial cells, smooth muscle cells (SMCs), and bone marrow–derived monocyte/macrophages, all interacting within a chronically inflamed, lipid-rich, and highly dynamic extracellular matrix microenvironment. Collagens are critical components of the extracellular matrix present within atherosclerotic plaques, where they contribute to lesion volume and can constitute up to 60% of total plaque protein.1 Collagens also provide mechanical stability to the fibrous cap and protect against plaque rupture, a major cause of the clinical complications associated with atherosclerosis.2 Furthermore, collagens stimulate diverse cellular responses that are central to plaque development. For example, collagen synthesis and degradation are important for smooth muscle cell migration,3,4 and degraded type I collagen fragments stimulate the disassembly of focal adhesion complexes in SMCs.5 By contrast, intact type I collagen inhibits SMC proliferation.6 Additionally, type I collagen promotes monocyte differentiation into macrophages and stimulates the production of inflammatory cytokines.7,8
The discoidin domain receptors (DDRs) are a family of receptor tyrosine kinases that bind triple helical collagens and transduce signals controlling the proliferation, migration, and differentiation of multiple cell types.9 There are 2 Ddr genes in the human and mouse genome, Ddr1 and Ddr2, and 6 differentially spliced isoforms of DDR1 have been identified.10,11 Both DDR1 and DDR2 bind to several collagen subtypes and require an intact triple helical domain for signaling. DDR1 has been shown to bind to types I to VI and VIII collagens, and DDR2 binds to fibrillar collagens, particularly types I to III and V but also to type X collagen.12–14 Denatured collagen, or gelatin, does not induce signaling through DDRs.14 Emerging evidence suggests important roles for the DDRs in the pathogenesis of atherosclerosis. DDR1 and DDR2 are both present in the atherosclerotic plaques of nonhuman primates.15 To date, the most research has centered on DDR1, and, in previous studies using Ddr1–/– mice, we determined that DDR1 played a critical role mediating neointimal hyperplasia after arterial injury.13 Furthermore, Ddr1–/– SMCs exhibited reduced migration, proliferation and matrix metalloproteinase (MMP) activity in vitro,13 and overexpression of DDR1 rescued these deficits.16 In agreement with this, overexpression of DDR1 or DDR2 in human SMCs increased MMP-1 expression and decreased procollagen 1(I) mRNA expression.15 Taken together, this evidence positions DDR1 at an important crossroad mediating the detection, expression, and turnover of collagen. However, these experiments examining SMCs in vitro and after a simple mechanical injury of the arterial wall do not accurately reflect the complex, multicellular, and inflammatory environment of the atherosclerotic plaque, and evidence for a functional role of DDR1 during atherogenesis remains incomplete.
In the present study, we have investigated the role of DDR1 in atherogenesis using the Ldlr–/– mouse model of atherosclerosis. Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice were fed an atherogenic diet for 12 or 24 weeks. Our data support a role for DDR1 as a positive regulator of atherosclerosis; capable of influencing both inflammation and fibrosis early in plaque development.
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Materials and Methods |
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Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal Care. At 8 to 12 weeks of age, male and female Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice were placed on an atherogenic diet containing 40% kilocalories of fat and 1.25% cholesterol by weight (Research Diets, D12108) for 12 or 24 weeks.17 Atherosclerotic plaque burden in the descending aorta, lipid composition of atherosclerotic plaques, immunocytochemistry, and picrosirius red (PSR) and Verhoeff–van Gieson (VVG) staining were performed as described in the expanded Materials and Methods section, available in the online data supplement at http://circres.ahajournals.org. Fibrillar collagen content and organization in PSR-stained longitudinal sections of aortic arch were assessed by quantitative polarization microscopy using the LC-PolScope (CRI Systems), as we have described previously.18 Details of the methods used for fluorescence in situ zymography, laser capture microdissection of mouse atheromata, quantitative real-time PCR, and culture of mouse vascular SMCs and bone marrow–derived macrophages can be found in the online data supplement.
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Results |
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DDR1 Was Expressed in Ldlr–/– Mice Fed an Atherogenic Diet
To confirm the expression of DDRs in murine atherosclerotic plaques, DDR1 and DDR2 mRNA levels were measured in laser microdissected plaques from Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice after 12 or 24 weeks on the atherogenic diet (Figure I in the online data supplement). DDR1 mRNA was expressed in Ddr1+/+;Ldlr–/– plaques and was absent in Ddr1–/–;Ldlr–/– plaques. DDR2 mRNA was expressed equally in plaques from mice of both genotypes. Immunostaining with an antibody against DDR1 revealed that the protein was expressed in atherosclerotic plaques of Ldlr–/– mice fed an atherogenic diet for 12 or 24 weeks (supplemental Figure I). Negative control sections stained using an equivalent concentration of normal rabbit IgG demonstrated minimal staining. There was, however, a slight signal in sections from Ddr1–/–;Ldlr–/– mice stained with the DDR1 primary antibody resulting from cross-reactivity of the antibody with other proteins.
Generation of Ddr1–/–;Ldlr–/– Mice, Physiology, and Plasma Lipids
Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice exhibited no significant differences in mean arterial pressure measured after 24 weeks on the atherogenic diet, nor were there any differences in body weight after 12 or 24 weeks on the atherogenic diet (supplemental Figure II). Two-way ANOVA of the data split by gender revealed a significant difference in body weight between females and males (P=0.045) but no significant difference in body weight between genotypes (P=0.477) (supplemental Figure IIIB). The sample size is greater in the body weight measurement data because we have included data from all mice used in the study, irrespective of whether the mice were used for measurements of atherosclerotic plaque burden, lesion composition, mRNA analysis, or measurement of blood pressure. Total plasma cholesterol and triglycerides were not significantly different between genotypes at either 12 or 24 weeks (supplemental Figure II).
Atherogenesis Was Attenuated in Ddr1–/–;Ldlr–/– Mice
There was a pronounced reduction in atherosclerotic plaque burden in Ddr1–/–;Ldlr–/– mice compared with Ddr1+/+; Ldlr–/– mice at 12 and 24 weeks (Figure 1A). Measurement of the percentage of aortic surface area occupied by plaque revealed a 50% reduction in atherosclerotic plaque area in the descending aortae of Ddr1–/–;Ldlr–/– mice at 12 weeks and a persistent 60% reduction in area after 24 weeks on atherogenic diet (Figure 1B). Two-way ANOVA analyzing the data split by sex revealed a significant decrease in plaque burden in both males and females of the Ddr1–/–;Ldlr–/– group (P=0.002) but no sex-dependent difference in plaque burden (P=0.07) (supplemental Figure IIIA). Lipid content of atherosclerotic lesions (measured by staining longitudinal sections of the aortic arch with oil red O and calculating the percentage positive lesion area) was comparable between genotypes (supplemental Figure IV). This suggests that the reduction in oil red O–positive area in the en face preparations reflects a change in lesion size rather than a decrease in lipid accumulation in the lesions.
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Matrix Accumulation Was Accelerated in Ddr1–/–;Ldlr–/– Mice
Fibrillar collagen content and organization in atherosclerotic plaques was assessed using PSR staining and the LC-PolScope, a quantitative polarization microscopy imaging system. PSR-stained sections of the aortic arch viewed by light microscopy demonstrated increased total collagen in the plaques of Ddr1–/–;Ldlr–/– mice compared with Ddr1+/+; Ldlr–/– mice at 12 weeks (Figure 2A and 2B). However, there was no difference in PSR stain between genotypes at 24 weeks, indicating that total collagen content was comparable between genotypes (Figure 2C and 2D). In PolScope images, the brightness of each pixel is proportional to the amount of birefringent fibrillar collagen present in the atherosclerotic plaque. PolScope images showed the presence of collagen fibers in the adventitia and sparsely distributed in the plaques of Ddr1+/+;Ldlr–/– mice at 12 weeks (Figure 2E). The absence of birefringent signal in the media was consistent with the circumferential orientation of collagen fibrils in this layer of the vessel wall, which would not be visible in these longitudinal sections. Fibrillar collagen birefringence was increased in the Ddr1–/–;Ldlr–/– plaques compared with Ddr1+/+;Ldlr–/– plaques at 12 weeks (Figure 2E and 2F). At 24 weeks, there was a dramatic increase in the birefringent signal in the Ddr1+/+;Ldlr–/– mice, resulting in comparable birefringence between genotypes, therefore indicating equivalent fibrillar collagen content (Figure 2G and 2H). Quantification of the mean birefringence retardance, determined by calculating the average retardance per pixel throughout the whole plaque, showed that fibrillar collagen content was significantly greater in the Ddr1–/–;Ldlr–/– mice compared with the Ddr1+/+;Ldlr–/– mice at 12 weeks, but there was no difference between genotypes at 24 weeks (Figure 2I). Plaques in the abdominal aorta at 12 weeks exhibited similar genotype-dependent differences in collagen content to those in the aortic arch (supplemental Figure V).
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Elastin accumulation in lesions was assessed by staining sections with VVG. Thick black elastic fibers were more prominent in the fibrous caps of the plaques from Ddr1–/–; Ldlr–/– mice compared with Ddr1+/+;Ldlr–/– mice at 12 weeks (Figure 3A and 3B). However, by 24 weeks, VVG staining increased in Ddr1+/+;Ldlr–/– plaques and was comparable to Ddr1–/–;Ldlr–/– mice (Figure 3C and 3D). Measurement of the percentage of plaque area stained for elastin revealed that elastin content was significantly greater in Ddr1–/–;Ldlr–/– mice at 12 weeks but was not different between genotypes at 24 weeks (Figure 3E).
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Matrix accumulation in atherosclerotic plaques is the net result of synthesis and degradation. To evaluate matrix synthesis, the mRNA levels for procollagen 1(I), procollagen 1(III), and tropoelastin were measured in laser microdissected plaques using quantitative real-time PCR (Figure 4). The mRNA levels for all 3 matrix molecules were significantly greater in the Ddr1–/–;Ldlr–/– mice compared with Ddr1+/+;Ldlr–/– mice at 12 weeks (Figure 4A). mRNA for procollagen 1(I) was also elevated at 24 weeks in the Ddr1–/–;Ldlr–/– mice (Figure 4B).
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SMCs are the major collagen-producing cell type in the atherosclerotic plaque; to determine whether DDR1-deficiency directly influenced collagen expression, we measured the mRNA expression levels of procollagen 1(I) and procollagen 1(III) in SMCs in vitro using quantitative real-time PCR. Ddr1–/– SMCs expressed 134±13-fold more procollagen 1(I) and 3.3±0.36-fold more procollagen 1(III) mRNA compared with Ddr1+/+ smooth muscle cells (P<0.05).
Matrix degradation in the plaque is accomplished through the action of proteinases including the MMPs. In situ zymography with DQ gelatin was performed to localize and assess MMP activity in longitudinal cryosections of the aortic arch. Areas of specific proteolytic cleavage were identified by comparing the level of bright green fluorescence in serial tissue sections treated with DQ gelatin and incubated in the absence or presence of the protease inhibitors EDTA or GM6001. The decrease in fluorescent signal intensity on treatment with these protease inhibitors confirms that the signal is indeed attributable to proteolytic activity. Gelatinase activity was localized in the plaque core and the fibrous cap and was minimal in the media (Figure 5). At 12 weeks, there was less gelatinase activity in sections from Ddr1–/–;Ldlr–/– mice compared with Ddr1+/+;Ldlr–/– mice (Figure 5A and 5B). By contrast gelatinase activity was comparable between genotypes at 24 weeks (Figure 5C and 5D). Gelatinase activity was inhibited by incubation with 10 mmol/L EDTA or 100 µmol/L GM6001, implicating MMPs in substrate digestion (Figure 5E and data not shown), although incomplete inhibition suggests that cysteine or serine proteases may also contribute. Sections incubated in the absence of DQ gelatin demonstrated the minimal contribution of tissue autofluorescence to the signal (Figure 5F).
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To further examine the MMPs contributing to matrix degradation, we used laser capture microdissection of the plaques and quantitative real-time PCR to measure mRNA levels of several MMPs, which were selected based on their specificity as collagenases (MMP-8 and -13) or gelatinases (MMP-2, -9, and -14). At 12 weeks, expression of MMP-2 was increased, whereas MMP-13 was decreased in Ddr1–/–; Ldlr–/– plaques (Figure 6A). At 24 weeks, MMP-2 and MMP-13 were elevated, and MMP-8 mRNA was decreased in Ddr1–/–;Ldlr–/– plaques (Figure 6B). MMP-14 expression was not significantly different between genotypes at either time point, and MMP-9 mRNA was not detectable in the microdissected samples.
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Macrophages are a major source of MMPs within the atherosclerotic plaque; to determine whether DDR1 influenced macrophage MMP expression, we examined MMP expression in vitro using bone marrow–derived macrophages cultured from Ddr1+/+;Ldlr–/– and Ddr1–/–;Ldlr–/– mice. Compared with Ddr1+/+ macrophages, the mRNA level of MMP-2, -9, and -14 was reduced in Ddr1–/– macrophages by 82%, 77% and 55%, respectively. Expression of MMP-8 and MMP-13 was not significantly altered (Figure 6C).
Macrophage but Not SMC Accumulation Was Reduced in Plaques From Ddr1–/–;Ldlr–/– Mice
To evaluate changes in the cellular composition of the atherosclerotic plaques, serial longitudinal sections of the aortic arch were stained with antibodies against smooth muscle -actin (SM -actin) or Mac-2 to assess SMC and macrophage content, respectively (Figure 7). Sections incubated in the absence of primary antibody did not show positive staining (supplemental Figure VI). SM -actin staining was evident in the media, plaque core, and fibrous cap at both 12 and 24 weeks (Figure 7A through 7D). Measurement of the percentage of plaque area stained positive for SM -actin showed no significant difference in SMC content in the Ddr1–/–;Ldlr–/– mice when compared with Ddr1+/+; Ldlr–/– mice, at either the 12 or the 24 week time point (Figure 7E).
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Macrophages were distributed throughout the plaque at 12 weeks in the Ddr1+/+;Ldlr–/– mice (Figure 7F), in marked contrast to the Ddr1–/–;Ldlr–/– mice, where there were very few macrophages in the plaque, and those present were confined to the superficial layers of the lesion (Figure 7G). Quantification of the percentage of plaque area stained positive for Mac-2 revealed that macrophage content was significantly lower in the Ddr1–/–;Ldlr–/– mice compared with the Ddr1+/+;Ldlr–/– mice at 12 weeks (Figure 7J). By 24 weeks, there were no significant differences in macrophage distribution or content between the 2 genotypes (Figure 6H through 6J). We observed substantial decreases in the area occupied by cells within the plaque at 24 weeks. This was likely attributable to the robust accumulation of matrix in the lesion at this late time point.
To examine the presence of other types of leukocytes in the lesions, longitudinal sections of the aortic arch were stained with antibodies to CD3 and Ly6G, markers of T cells and neutrophils, respectively. T lymphocytes were found very infrequently in the plaque, whereas neutrophils were observed in the adventitial layer. There were no differences between genotypes (supplementary Figure VII).
Adhesion molecules and chemokines such as vascular cell adhesion molecule (VCAM)-1 and monocyte chemoattractant protein (MCP)-1 are key mediators of macrophage recruitment into the atherosclerotic plaque.19–21 The mRNA expression of MCP-1 and VCAM-1 was evaluated in laser-microdissected plaques using real time PCR. The mRNA levels for both MCP-1 and VCAM-1 were significantly reduced in the Ddr1–/–;Ldlr–/– mice compared with Ddr1+/+; Ldlr–/– mice at 12 weeks (Figure 8A). Furthermore, MCP-1 expression was persistently reduced in the Ddr1–/–;Ldlr–/– mice even after 24 weeks (Figure 8B), suggesting that there was a lasting impairment of the inflammatory response in these mice.
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Discussion |
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Here, we show that DDR1 is a critical regulator of atherogenesis. Deletion of DDR1 attenuates plaque development and results in a substantial change in plaque composition, including an early increase in extracellular matrix content and decreased macrophage accumulation. Ultimately, the plaques in DDR1-deficient mice are smaller and exhibit features consistent with greater stability.
We observed marked acceleration of fibrillar collagen and elastin accumulation in the plaques from the Ddr1–/–;Ldlr–/– mice. The observation of increased matrix accumulation with decreased lesion size may seem counterintuitive. However, it is important to understand that the matrix does not simply add bulk to the plaque; matrix molecules can influence cell behavior. For example, fibrillar collagens and intact elastin have been shown to inhibit SMC proliferation and migration.6,22–25 Furthermore, fibrillar collagen inhibits, whereas monomeric collagen potentiates macrophage MMP production,26 thereby affecting the potential to invade the plaque. Therefore, in our model, it is possible that enhanced matrix accumulation contributed to reduced lesion size by regulating SMC proliferation and migration and macrophage invasion. Consistent with our findings, previous studies have reported that matrix accumulation does not necessarily correlate with increased lesion size: deletion of MMP-13 in the apolipoprotein (Apo)E–/– mouse resulted in increased fibrillar collagen content without an increase in lesion size,27 and similar findings were noted in ApoE–/– mice expressing collagenase-resistant collagen.28 However, this paradigm does not always hold true because deletion of TIMP-129 or overexpression of MMP-1 in macrophages of ApoE–/– mice30 results in decreased plaque size caused by excessive proteolytic activity and clearance of matrix.
The net content of matrix in the atherosclerotic plaque is the result of a balance between matrix synthesis and degradation. We observed an increase in the mRNA levels for procollagen 1(I), procollagen 1(III), and tropoelastin in Ddr1–/–;Ldlr–/– plaques at 12 weeks, suggestive of increased synthesis of these molecules. Moreover Ddr1–/– SMCs cultured in vitro overexpressed procollagen 1(I) and procollagen 1(III). These results are consistent with previous in vitro studies in which overexpression of DDR1 in SMCs decreased type I collagen mRNA.15 Taken together, these data suggest that DDR1-deficient SMCs adopt a matrix-synthetic phenotype and promote accelerated matrix accumulation during atherogenesis.
Coincident with increased matrix accumulation at 12 weeks, we observed reduced in situ gelatinolytic activity in the plaques of Ddr1–/–;Ldlr–/– mice. This decrease in proteolytic activity in the lesions was likely influenced by the reduced macrophage content of the Ddr1–/–;Ldlr–/– plaques, because macrophages are a major source of MMPs and cysteine and serine proteases in the atherosclerotic plaque.31–33 To determine which enzymes were altered in DDR1-deficiency, we measured the mRNA levels of several MMPs known to be involved in atherosclerosis. In addition to its interstitial collagenase activity, MMP-13 is also a gelatinase.34 The decrease in MMP-13 mRNA in the 12-week lesions of DDR1-deficient mice may have contributed to the difference between genotypes evident on in situ zymography. Because MMP-2 is also an important gelatinase, we expected that the expression of this enzyme would vary in parallel with in situ gelatinase activity. However, we observed an increase in MMP-2 mRNA in DDR1-deficient mice, which indicates that DDR1 negatively regulates MMP-2 mRNA expression. However, we cannot exclude the possibility that DDR1 may positively regulate MMP-2 protein expression or activation. The absence of MMP-9 mRNA in the microdissected plaque samples is likely because the steady-state levels were too low to be detected.
The decrease in MMP-13 expression in the 12-week lesion may have contributed to the marked acceleration of fibrillar collagen deposition in the Ddr1–/–;Ldlr–/– plaques, because collagen clearance is similarly reduced in the plaques of MMP-13 deficient mice.27 Furthermore, our in vitro experiments using bone marrow–derived macrophages showed no differences in MMP-13 expression between cells of the 2 genotypes. This suggests that the reduction in MMP-13 mRNA in the microdissected lesions is a result of reduced macrophage content and not a direct effect of DDR1 deficiency on MMP-13 expression in macrophages. Taken together, our data suggest that there are decreases in the proteolytic activity in the plaques of Ddr1–/–;Ldlr–/– mice that result in decreased matrix degradation early in atherogenesis.
By contrast, the changes in MMP expression that we observed at 24 weeks occur in the absence of any measurable difference in the matrix content of the lesions. The increase in MMP-13 expression at 24 weeks may be predicted to reduce collagen content in the lesions. However, the persistent increases in procollagen I expression, along with a decrease in MMP-8 expression, could facilitate collagen accumulation in the Ddr1–/– mice, resulting in no net difference in collagen content between genotypes at this time point. We suggest that the continued expression of collagen and alteration in MMP expression at this later time point may reflect enhanced remodeling of the plaque matrix leading to the formation of smaller, matrix-rich plaques with limited growth potential.
The reduction in macrophage accumulation in Ddr1–/–; Ldlr–/– plaques at 12 weeks is consistent with a role for DDR1 in mediating macrophage recruitment, invasion, or persistence in the atherosclerotic plaque. Decreased mRNA levels of MCP-1 and VCAM-1 (both key mediators of monocyte/macrophage recruitment from the circulation19–21) were correlated with decreased macrophage content in the Ddr1–/–; Ldlr–/– plaque. Consistent with our results, recent studies have demonstrated direct regulation of chemokine expression by macrophage DDR18 and provide evidence of a proinflammatory role for DDR1 in several pulmonary and renal fibroinflammatory pathologies.35–39 Macrophage-invasive ability may also have been altered in Ddr1–/–;Ldlr–/– mice as result of decreased expression of several MMPs by these cells including MMP-2, -9, and -14. Consistent with this observation, DDR1 has been shown to facilitate macrophage invasion into 3D collagen gels,40 and we and others have demonstrated that DDR1 can regulate MMP expression in several cell types.9
The reduction in atherosclerotic plaque size in the Ddr1–/–; Ldlr–/– mouse is in agreement with our previous findings demonstrating reduced neointimal thickening in Ddr1–/– mice after wire injury of the carotid artery.13 However, the reasons for the decrease in lesion size differ greatly between the 2 studies. In the wire injury model, we observed decreased SMC infiltration and collagen accumulation in the neointima of the DDR1-deficient mouse.13 However, in the Ddr1–/–; Ldlr–/– mouse, we see decreased macrophage accumulation and increased matrix content. This is likely attributable to differences between the 2 models; atherosclerotic plaques in the Ldlr–/– mouse are rich in leukocytes, lipids, and oxidation products,17,41 whereas the neointimal lesions formed after wire injury are populated almost exclusively by SMCs, with little inflammatory cell involvement.13 The distinct findings underline the importance of studying DDR1 in different models of arterial injury and allude to an important role for DDR1 in vascular inflammation.
Furthermore, there are similarities in the phenotypes of 1 integrin–deficient and DDR1-deficient mice on the atherosclerotic background. Deletion of the 1 integrin gene in the ApoE–/– mouse resulted in reduced plaque size and the development of a plaque that was rich in collagen and poor in macrophages.42 The similarity in phenotypes suggests that DDR1 may share some functions with the 1β1 integrin receptor in atherosclerosis and underscores the importance of collagen-dependant signaling in plaque development. By contrast, deletion of the 2 integrin collagen receptor did not affect plaque development in the ApoE–/– mouse.43 The role of DDR2 in atherogenesis has not been investigated.
In conclusion, deletion of the gene for DDR1 resulted in the attenuation of atherosclerotic plaque development in the Ldlr–/– mouse. Elimination of DDR1 shifted plaque composition toward increased matrix and decreased macrophage content early in plaque development, with the net result of a decrease in lesion size that persisted even in late lesions. Thus, we have identified DDR1 as an early positive regulator of plaque development that when inhibited, can limit disease progression.
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Acknowledgments |
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We thank Dr Phillip Connelly and Graham Maguire for assistance with measurements of plasma lipoproteins and triglycerides, Dr Scott Heximer for assistance measuring mean arterial pressure, Dr David Courtman for assistance with the LC-PolScope analysis, and Dr Phillip Marsden and Brent Steer for assistance with laser capture microdissection and real-time PCR. We dedicate this article to the memory of Dr Wolfgang F. Vogel.
Sources of Funding
This study was funded by Canadian Institutes of Health Research grant MOP37847 and Heart and Stroke Foundation of Ontario grant NA6069 (to M.B.). M.B. is a Career Investigator of the Heart and Stroke Foundation of Ontario. W.F.V. held a Canada Research Chair (tier II). C.F. was supported by a doctoral research award from the Heart and Stroke Foundation of Canada, a Canada Graduate Scholarship Doctoral Research Award from the Canadian Institutes of Health Research, and the Meredith and Malcolm Silver Scholarship in Cardiovascular Studies. P.A. was supported by a Canada Graduate Scholarship Doctoral Research Award from the Canadian Institutes of Health Research and an Ontario Graduate Scholarship.
Disclosures
None.
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Footnotes |
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Deceased. 
Original received August 15, 2007; resubmission received January 2, 2008; revised resubmission received April 14, 2008; accepted April 17, 2008.
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References |
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