Increased Cell and Matrix Accumulation During Atherogenesis in Mice With Vessel Wall–Specific Deletion of Discoidin Domain Receptor 1
Rationale: Discoidin domain receptor (DDR)1 is a collagen receptor expressed on both smooth muscle cells (SMCs) and macrophages, where it plays important roles regulating cell and matrix accumulation during atherogenesis. Systemic deletion of DDR1 resulted in attenuated plaque growth but accelerated matrix accumulation in LDLR-deficient mice. Deletion of DDR1 solely on bone marrow–derived cells resulted in decreased macrophage accumulation and plaque growth but no change in matrix accumulation.
Objective: These findings led us to hypothesize that accelerated matrix accumulation was attributable to the increased synthetic ability of Ddr1−/− resident vascular wall SMCs.
Methods and Results: We used bone marrow transplantation to generate chimeric mice and investigate the role of SMC DDR1 during atherogenesis. Mice with deficiency of DDR1 in vessel wall–derived cells (Ddr1+/+→−/−) or control mice (Ddr1+/+→+/+) were fed an atherogenic diet for 12 weeks. We observed a 3.8-fold increase in the size of aortic sinus plaques in Ddr1+/+→−/− compared to Ddr1+/+→+/+ mice. This was attributed to pronounced accumulation of collagen, elastin, proteoglycans, and fibronectin and resulted in a thickened fibrous cap. The enhanced matrix accumulation decreased the proportion of plaque area occupied by cells but was associated with a shift in the cellular composition of the lesions toward increased numbers of vessel wall–derived SMCs compared to bone marrow–derived macrophages. In vitro studies confirmed that Ddr1−/− SMCs expressed more matrix, proliferated more, and migrated farther than Ddr1+/+ SMCs.
Conclusions: DDR1 expression on resident vessel wall SMCs limits proliferation, migration and matrix accumulation during atherogenesis.
The accumulation of extracellular matrix regulates both growth and stability of the atherosclerotic plaque. Vascular smooth muscle cells (SMCs) undergo a phenotypic switch from contractile to synthetic as they proliferate and migrate into the lesion, elaborating a collagen-rich matrix.1 Collagen accumulation has a multifaceted role in the etiology of atherosclerosis, whereas excess collagen can contribute to the expansion of lesion volume and vascular stenosis, maintenance of a thick collagen-rich fibrous cap is important for the prevention of plaque rupture. Inflammatory processes influence the turnover of collagens in the plaque. Plaque macrophages are a potent source of matrix metalloproteinases (MMPs), which destabilize the lesion by digesting collagen at the rupture prone plaque shoulders.2 Thus, the balance between synthesis, remodeling, and degradation of collagens determines the content and organization of the atherosclerotic plaque matrix, influencing disease progression and clinical outcomes.
The discoidin domain receptor (DDR)1 is a collagen receptor tyrosine kinase expressed on both SMCs and macrophages3–7 that initiates signaling when bound by triple helical collagens.8,9 Several studies have revealed that DDR1 plays important roles in controlling cell proliferation, migration, and matrix remodeling.10 We have previously determined functional roles for DDR1 in atherogenesis using the hypercholesterolemic Ldlr−/− mouse model. Atherosclerotic plaques that formed in mice doubly deficient in DDR1 and LDLR (Ddr1−/−;Ldlr−/−) were smaller in size and exhibited accelerated matrix deposition, decreased in situ MMP activity, and decreased macrophage content compared to mice deficient in LDLR only (Ddr1+/+;Ldlr−/−).4 DDR1 also mediates atherosclerotic plaque calcification by SMCs, a long-term complication of atherosclerosis.11 We performed studies using bone marrow transplantation to delete DDR1 exclusively in bone marrow–derived cells of Ldlr−/− mice.12 This resulted in decreased plaque macrophage infiltration, because of the inability of macrophages to penetrate the endothelial cell basement membrane, and resulted in a significant decrease in lesion size. However, distinct from the phenotype that was observed in the systemic Ddr1−/−; Ldlr−/− knockout, there was no change in matrix accumulation in the plaques of mice after DDR1-deletion in bone marrow–derived cells only. This finding led us to hypothesize that accelerated and enhanced matrix accumulation was primarily attributable to the increased synthetic ability of resident Ddr1−/− vessel wall SMCs and prompted us to investigate the contribution of DDR1 expressed solely on these cells.
In the study reported herein, we transplanted bone marrow from Ddr1+/+;Ldlr−/− mice into Ddr1−/−;Ldlr−/− hosts, generating chimeric mice with a deficiency of DDR1 specific to resident vessel wall cells (Ddr1+/+→−/−, vessel wall deletion). For the first time, we report that atherosclerotic plaques from Ddr1+/+→−/− mice are significantly larger compared to the control chimeric mice (Ddr1+/+→+/+), because of robust increases in collagen, elastin, proteoglycans, and fibronectin in the extracellular matrix and a thickened fibrous cap. Moreover, the enhanced matrix accumulation reduced the proportion of plaque area occupied by cells, but the cellular composition of the lesions was shifted toward increased numbers of vessel wall–derived cells compared to bone marrow–derived macrophages. Taken together, these data support a distinct and independent role for DDR1 expressed on resident vessel wall SMCs in attenuating proliferation, migration, and matrix accumulation during atherogenesis.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Animal experiments were performed in accordance with the guidelines of the Canada Council on Animal Care. Sex-mismatched bone marrow transplantation was performed using Ddr1−/−;Ldlr−/− mice and their Ddr1+/+;Ldlr−/− littermates.4 The experimental groups included female Ddr1+/+;Ldlr−/− or Ddr1−/−;Ldlr−/− hosts receiving male Ddr1+/+;Ldlr−/− bone marrow to generate control (Ddr1+/+→+/+) or vessel wall deletion (Ddr1+/+→−/−) chimeric mice. Three weeks after transplantation, mice were placed on an atherogenic diet containing 40% kilocalories of fat and 1.25% cholesterol by weight (Research Diets, D12108). After 12 weeks on diet, mice were euthanized by anesthetic overdose with 67 mg/kg xylazine and 333 mg/kg ketamine IP, and the heart and proximal aorta were fixed in 4% paraformaldehyde. Sections of the aortic sinus were used to measure plaque size and composition. Plaques present on the aortic valve leaflets, when present, were excluded from this analysis. SMCs were isolated from the carotid arteries of Ddr1+/+ and Ddr1−/− mice by enzymatic dispersion,9 and experiments were performed to measure matrix protein expression, MMP/TIMP expression, and cell proliferation and migration. Primers used to measure mRNA expression were as previously published.13,14
Bone Marrow Transplantation and Plasma Lipids
Body weight and fasting plasma triglycerides were similar between Ddr1+/+→+/+ and Ddr1+/+→−/− chimeric mice; however, total cholesterol was increased in Ddr1+/+→−/− mice (Online Table II). Male:female chimerism was assessed by genotyping samples of leukocyte DNA with primers against Ddr1 and Sry, a marker of the Y chromosome. The DDR1 wild type allele was present in leukocyte DNA from Ddr1+/+→+/+ and Ddr1+/+→−/− mice, and the abundance of Sry was comparable between groups (Online Figure I, A). Measurement of the Sry/Gapdh ratio using quantitative real-time PCR confirmed similar chimerism between groups (Online Figure I, B).
Deletion of DDR1 in Vessel Wall Cells Increased Atherosclerotic Plaque Size and Resulted in the Formation of Lipid-Poor Plaques
Deletion of DDR1 in resident vessel wall cells (Ddr1+/+→−/−) resulted in a dramatic increase in plaque size in the aortic sinus (compare Figure 1A and 1B). Moreover, the lesions from Ddr1+/+→−/− mice were histologically distinct from the controls. Plaques from Ddr1+/+→+/+ mice were rich in lipid and foam cells (Figure 1C), whereas lesions from Ddr1+/+→−/− mice had less evidence of foam cell accumulation (Figure 1D) and were instead rich in extracellular matrix. Measurement of lesion area in Verhoeff–van Gieson (VVG)-stained cross-sections of the aortic sinus of Ddr1+/+→−/− mice revealed a 3.8-fold increase compared to control Ddr1+/+→+/+ mice (Figure 1E). Taken together, these data suggest that deletion of DDR1 in vessel wall cells resulted in the formation of large fibrous plaques.
To assess lipid content of the lesions, we measured the white area on monochrome images of VVG stained tissue. These measurements correlate well with the percentage of lesion area occupied by oil red O staining, and this is an established method to estimate plaque lipid content.15 Using this method, we found the lipid content of the Ddr1+/+→−/− lesions was significantly lower than lesions from Ddr1+/+→+/+ mice (42±3% versus 60±4% respectively, P<0.05). We also observed similar results after staining frozen sections of the aortic arch with oil red O; specifically, there was a decrease in the percentage of plaque area occupied by lipid in the Ddr1+/+→−/− mice (Online Figure II). This demonstrates that the increase in lesion size in the Ddr1+/+→−/− mice was not attributable to increased lipid accumulation and rules out the possibility that the increased plasma cholesterol content in the Ddr1+/+→−/− mice directly influenced lesion hypertrophy.
Robust Accumulation of Extracellular Matrix in the Plaques of Ddr1+/+→−/− Mice and an Increase in Fibrous Cap Thickness
We examined matrix composition of the lesions by staining serial sections of the aortic sinus with picrosirius red, VVG, and Movat’s pentachrome to label collagen, elastin, and proteoglycans, respectively (Figure 2). Collagen staining was often present throughout the entire thickness in the lesions from Ddr1+/+→−/− mice (Figure 2B). The percentage of lesion area stained positive for collagen was significantly increased in Ddr1+/+→−/− mice compared to Ddr1+/+→+/+ mice (Figure 2C). Analysis of fibrillar collagen content and organization in the lesions was carried out using the LC-PolScope. Images demonstrate organized fibrillar collagen in lesions of Ddr1+/+→−/− mice (Figure 2E), compared to the sparse distribution of collagen fibers in the plaques from control Ddr1+/+→+/+ mice (Figure 2D). Quantification of the average birefringence retardance per pixel confirmed a significant increase in fibrillar collagen content in Ddr1+/+→−/− plaques (Figure 2F). Elastin was also abundant in the fibrous caps of atherosclerotic plaques from Ddr1+/+→−/− mice (Figure 2H) and was sparsely distributed in the lesions from Ddr1+/+→+/+ mice (Figure 2G). Measurement of the percentage of lesion area stained positive for VVG revealed a significant accumulation of elastin in plaques from Ddr1+/+→−/− mice (Figure 2I). Proteoglycans were localized in the core of the plaque and within the fibrous cap (Figure 2J and 2K). Measurement of the percentage of lesion area stained positive for proteoglycan revealed a significant increase in proteoglycan content of Ddr1+/+→−/− plaques (Figure 2L). There was also more fibronectin staining in plaques from Ddr1+/+→−/−mice compared to Ddr1+/+→+/+ mice (Online Figure III).
Measurement of fibrous cap thickness was carried out using picrosirius red–stained sections to identify collagen fibers, and smooth muscle (SM) α-actin stained sections to delineate the cap. Relative fibrous cap thickness was significantly increased in Ddr1+/+→−/− mice (Figure 3). These data suggest that deletion of DDR1 in resident vessel wall cells increased lesion size by enhancing matrix accumulation and favoring the assembly or remodeling of that matrix into a highly organized, thickened fibrous cap.
Increased Matrix and MMP mRNA Levels in Ddr1−/− SMCs In Vitro
To investigate the mechanisms underlying the increased matrix accumulation in the Ddr1+/+→−/− mice, we measured mRNA expression of key matrix molecules and matrix-degrading enzymes in primary culture SMCs isolated from Ddr1+/+ and Ddr1−/− mice. Expression of mRNA for types I and III collagen was significantly increased in Ddr1−/− cells compared to Ddr1+/+ cells (Figure 4A and 4B). This suggested that synthesis of the interstitial collagen matrix was dramatically increased in DDR1-deficient SMCs. By contrast, the mRNA for basement membrane type IV collagen was significantly decreased (Figure 4C). To assess the capacity of Ddr1−/− SMCs to degrade matrix, we measured mRNA for 22 members of the MMP superfamily and 4 tissue inhibitors of metalloproteinases (TIMPs). Eighteen MMPs were expressed in SMCs, and comparing Ddr1−/− to Ddr1+/+ SMCs, mRNA levels were significantly increased for 9 MMPs, significantly decreased for 3 MMPs, and not significantly changed for 6 MMPs in the Ddr1−/− SMCs (Table). TIMPs 1 to 3 were also expressed, and the mRNAs for TIMPs 1 and 2 were decreased, whereas TIMP-3 was not changed in Ddr1−/− compared to Ddr1+/+ SMCs (Table). These data suggest that Ddr1−/− SMCs have the potential for increased matrix proteolysis attributable to a net increase in MMP expression and a net decrease in the expression of TIMPs.
Increased Expression of mRNA for Type I Collagen, Elastin, and MMP-3, -13, and -10 in the Plaques of Ddr1+/+→−/− Mice
To validate the changes in matrix and MMP mRNA that we observed in SMCs in vitro, laser capture microdissection was used to collect tissue from Ddr1+/+→−/− and Ddr1+/+→+/+ mice and measure mRNA in the plaque. mRNA expression was increased by 4-fold for type I collagen, by 17-fold for MMP-13, by 4-fold for MMP-3, and by 5-fold for MMP-10 in the plaques of Ddr1+/+→−/− mice compared to Ddr1+/+→+/+ mice. By contrast, the mRNA for tropoelastin was not increased. These results confirm our findings of increased matrix and MMP expression by Ddr1−/− SMCs in culture (above). Taken together, these data demonstrate that DDR1 on SMCs modulates the expression of matrix molecules and MMPs in the atherogenic microenvironment, altering the balance between matrix synthesis and degradation and resulting in a net increase in matrix accumulation with DDR1 deficiency.
Increases in Type I Collagen and MMP-13 mRNA Expression in Ddr1−/− SMCs Were Mediated via Mitogen-Activated Protein Kinase Signaling
To investigate the mechanisms by which DDR1 deficiency led to increases in mRNA expression for type I collagen and MMP-13, we performed experiments incubating Ddr1−/− SMCs with inhibitors of the mitogen-activated protein kinase family of signaling molecules, as these pathways have been implicated in downstream DDR1-signaling in other cells.16,17 There was a 37% decrease in expression of type I collagen mRNA after incubation of the cells with the p38K inhibitor SB203580, but minimal change after incubation with PD98059 or SP600125, inhibitors of the MEK and c-Jun N-terminal kinase (JNK) pathways respectively (Online Figure IV, A). MMP-13 mRNA expression was decreased by 30% to 40% after incubation with SP600125, PD98059 or SB203580 (Online Figure IV, B). This suggests that type I collagen expression in Ddr−/− SMCs was mediated partially by p38K, whereas the increase in MMP-13 expression was mediated by extracellular signal-regulated kinase (ERK)1/2, JNK, and p38K.
A Reduced Proportion of Plaque Area Was Occupied by Cells in Ddr1+/+→−/− Mice, but the Number of Vessel Wall–Derived Cells Was Increased
Sections of the aortic sinus were immunostained with antibodies against SM α-actin or Mac-2, markers of SMCs and macrophages, respectively (Figure 5). In the Ddr1+/+→+/+ control mice, SM α-actin–positive cells were localized in a thin layer at the luminal aspect of the plaque (Figure 5B). By contrast, SMCs were localized throughout the thickened fibrous cap in Ddr1+/+→−/− mice (Figure 5C). In plaques from Ddr1+/+→+/+ mice, many Mac-2–positive cells had the appearance of foam cells (Figure 5E). By contrast, few Mac-2–positive foam cells were observed in the plaques from Ddr1+/+→−/− mice, instead macrophages were small and punctate (Figure 5F). As a result of the robust accumulation of matrix, the percentage of lesion area positive for either SM α-actin (Figure 5G), or Mac-2 (Figure 5H) was significantly decreased in Ddr1+/+→−/− mice compared to Ddr1+/+→+/+ mice. However, the reduction in the proportion of plaque area occupied by SMCs was not as great as the reduction in the proportion of area occupied by macrophages. Therefore, as an additional measurement of the cellular composition of the lesions, we counted the cells and performed fluorescence in situ hybridization with a Y chromosome probe to identify bone marrow–derived cells. The total number of Y chromosome positive cells in the plaque was comparable between Ddr1+/+→+/+ and Ddr1+/+→−/− mice (Figure 6). The number of vessel wall–derived cells was calculated by subtracting the number of bone marrow–derived cells from total cell number in the lesion. There was a significant increase in the number of vessel wall–derived cells in the plaques of Ddr1+/+→−/− mice, which resulted in a significant increase in total cell number in these plaques (Figure 6). Thus, vessel wall–specific deletion of DDR1 resulted in an increase in the number of vessel-wall derived cells in the plaque, but did not alter the number of bone marrow–derived cells in the plaque.
Increased Proliferation and Migration of Ddr1−/− SMCs Plated on a Complex Extracellular Matrix In Vitro
The increase in the number of DDR1-deficient vessel wall–derived cells in the plaque could be attributable to increases in cell proliferation, migration or both. We performed in vitro experiments to determine whether proliferation or migration was altered in DDR1-deficient SMCs. To mimic the matrix present in the vessels of the Ddr1−/− host mice, we biosynthesized a complex endogenous extracellular matrix substrate by plating Ddr1−/− SMCs for 5 days. These SMCs were lifted from the matrix using EDTA and EGTA, and naïve SMCs of either genotype (Ddr1+/+ or Ddr1−/−) were plated on the biosynthesized matrix. We found that Ddr1−/− SMCs exhibited significantly greater rates of proliferation (Figure 7A) and migration (Figure 7B) compared to Ddr1+/+ SMCs. Furthermore, treatment with the MMP inhibitor doxycycline significantly reduced the distance migrated by Ddr1−/− SMCs to a level which was not significantly different from the distance migrated by Ddr1+/+ SMCs (Figure 7B). This suggests that greater migration of the Ddr1−/− cells was directly related to their expression of MMPs.
In the present study, we identify distinct roles for DDR1 expressed in vessel wall–derived cells in the regulation of matrix and cell accumulation during atherogenesis. Deletion of DDR1 in vessel wall–derived cells dramatically accelerated matrix accumulation, resulting in the formation of large fibrotic lesions with thickened fibrous caps. Our data suggest that by modulating the expression of matrix proteins and MMPs, DDR1 negatively regulates matrix turnover. Furthermore, there was an increase in the number of vessel wall–derived cells in the plaques that was associated with increased proliferation and MMP-dependant migration of Ddr1−/− SMCs on a complex biosynthesized matrix.
Our results strongly suggest that the enhanced matrix accumulation in the Ddr1+/+→−/− mice was a consequence of DDR1-deletion specifically on host vascular wall cells, as DDR1 was intact in the bone marrow–derived cells. We previously reported increased plaque matrix accumulation after the systemic deletion of DDR1 in the Ddr1−/−;Ldlr−/− mouse.4 However, in the former studies we attributed matrix accumulation to the combination of increased collagen expression by Ddr1−/− SMCs and reduced MMP-dependent collagen proteolysis by Ddr1−/− macrophages. Because the deletion of DDR1 solely in bone marrow–derived cells had no influence on plaque matrix accumulation,12 our present studies support the hypothesis that enhanced matrix production is a specific consequence of DDR1-deletion in vascular SMCs. Taken together, our in vivo and in vitro studies demonstrate that DDR1 acts as a sensor for the collagen matrix, and a negative feedback regulator of interstitial collagen expression in SMCs. This is consistent with reports from Ferri et al, showing that overexpression of DDR1 in SMCs inhibited collagen synthesis.3 However, we are the first to prove that endogenous levels of DDR1 impact on the expression of collagen and other matrix molecules, and that DDR1 is an important negative regulator of collagens type I and III, which are the most abundant collagens in the vessel wall.
Another important finding is that fibrous caps were thicker and more prevalent in lesions from Ddr1+/+→−/− mice compared to Ddr1+/+→+/+ mice. Moreover, LC-PolScope analysis revealed more fibrillar collagen in the caps of Ddr1+/+→−/− mice, demonstrating in vivo that DDR1 plays a critical role in inhibiting fibril organization. Previous in vitro studies have suggested that collagen fibril formation and organization are dependent on collagen receptors. The collagen-binding α2β1 integrin promoted collagen fibril formation in SMCs,18 whereas fibril formation was attenuated in osteoblasts overexpressing DDR1.19 In the latter study, the authors concluded that DDR1 locked the collagen molecules into an incomplete fibrillar state.19 Therefore accumulation of fibrillar collagens and the organization of thicker fibrous caps in lesions from Ddr1+/+→−/− mice in vivo is consistent with the hypothesis that DDR1 normally functions to attenuate collagen fibril formation by SMCs. This may have important implications for the treatment of atherosclerotic vascular disease, where a thick organized plaque fibrous cap protects against plaque rupture, and thus studies examining selective inhibition of DDR1 for lesion stabilization may be warranted.
The preferential accumulation of DDR1-deficient vessel wall–derived cells in the lesions of Ddr1+/+→−/− mice prompted us to measure the effects of DDR1 deletion on SMC proliferation and migration. To mimic the microenvironment of the Ddr1−/− vessel wall, we assayed SMCs on a biosynthesized matrix generated by Ddr1−/− SMCs, and found that migration and proliferation of the Ddr1−/− cells was greater than their Ddr1+/+ counterparts. This is consistent with previous studies which showed increased proliferation of DDR1-null mesangial cells,16 and decreased migration in DDR1 overexpressing kidney epithelial cells.20,21 Possible mechanisms include repression of ERK1/2 and p38K by DDR1 to limit proliferation, and/or repression of α2β1 integrin–stimulated cell migration by DDR1. Whereas we have previously shown that the migration of SMCs on purified type I or type VIII collagen is DDR1-dependent,7,9 our present results emphasize the importance of the extracellular microenvironment and suggest that the function of DDR1 is different when the cell is exposed to a complex multicomponent matrix. Taken together, these studies underscore the limitations of studying how cells respond to individual matrix ligands, and highlight the importance of understanding how cells use DDR1 to integrate multiple signals from the matrix.
We found that Ddr1−/− SMCs grown in culture exhibited increased expression of both matrix and MMP genes; however, there was a dramatic net increase in matrix accumulation in the DDR1-deficient host mice. This leads to 2 important conclusions: first, that matrix synthesis and accumulation exceeds degradation; and second, that the major function of the increased MMPs produced by SMCs is not to clear excess matrix but instead to facilitate matrix remodeling and SMC migration into the plaque. Importantly, our finding that plaques from Ddr1+/+→−/− mice contain more vessel wall derived cells than plaques from Ddr1+/+→+/+ mice may be explained by the elevation of MMP expression in Ddr1−/− SMCs, a possibility that is supported by our in vitro data demonstrating that Ddr1−/− SMC migration was attenuated after treatment with the MMP inhibitor doxycycline. Moreover, we propose that as Ddr1−/− SMCs invade the plaque, their enhanced expression of interstitial matrix genes results in extracellular matrix synthesis and assembly which contributes directly to plaque expansion. The mechanisms by which matrix and MMP expression were increased in the Ddr1−/− SMCs are not completely understood. However, our studies show that the increase in type I collagen expression was mediated at least in part by p38K activation, and the increase in MMP-13 expression was mediated through a combination of ERK1/2, JNK and p38K activation.
A striking finding of the present study was the increase in lesion size in the Ddr1+/+→−/− mice. After systemic deletion of DDR1 in Ldlr−/− mice we observed an increase in plaque matrix content, but in that case lesion size was reduced.4 Moreover, bone marrow–specific deletion of DDR1 resulted in the attenuation of lesion size by limiting macrophage accumulation in the developing plaque, without affecting matrix accumulation.12 Thus, we conclude that the expression of DDR1 on different cell types mediates distinct effects on plaque size, inflammation and fibrosis during atherogenesis. We propose the following model: DDR1 expression on macrophages mediates their infiltration into the developing plaque, whereas DDR1 expression in resident vessel wall SMCs limits SMC infiltration and lesion fibrosis. In the context of systemic DDR1 deletion (Ddr1−/−;Ldlr−/−), lesion initiation and growth is slowed by reduced macrophage accumulation, and combined with increased matrix expression by SMCs, this results in the formation of smaller, matrix rich plaques compared to plaques from Ddr1+/+;Ldlr−/− mice. In the context of DDR1 deletion exclusively in bone marrow cells (Ddr1−/−→+/+), impaired monocyte recruitment results in the formation of smaller lesions without affecting matrix accumulation. By contrast, when DDR1 is deleted exclusively in vessel wall cells (Ddr1+/+→−/−), Ddr1+/+ macrophages can colonize and initiate an inflammatory response in the arterial intima of both Ddr1+/+ and Ddr1−/− hosts. Given a comparable platform of lesion development to begin with, enhanced matrix synthesis by DDR1-deficient host SMCs rapidly overtakes the developing lesion and results in unrestricted matrix accumulation and larger plaques.
In conclusion, we have demonstrated novel roles for DDR1 expressed on vessel wall–derived cells in the regulation of matrix accumulation, fibrous cap formation, and SMC migration and proliferation during atherogenesis. Deletion of DDR1 in vessel wall cells accelerated matrix accumulation and resulted in the formation of large matrix-rich lesions with thick, organized fibrous caps. These studies reinforce the importance of studying collagen signaling in lesion development and suggest that inhibiting SMC DDR1 may be a novel therapeutic target to promote the formation of matrix-rich stable plaques.
Sources of Funding
This study was funded by Heart and Stroke Foundation of Ontario grants NA6069 and T6734 (to M.P.B.), who is a Career Investigator of the Heart and Stroke Foundation of Ontario. C.F. was supported by a Doctoral Research Award from the Heart and Stroke Foundation of Canada, a Canada Graduate Scholarship Doctoral Award from the Canadian Institutes of Health Research, and the Meredith and Malcom Silver Scholarship in Cardiovascular Studies. P.J.A. was supported by a Canada Graduate Scholarship Doctoral Award from the Canadian Institutes of Health Research. E.W. was supported by a John D. Schultz Scholarship from the Heart and Stroke Foundation of Ontario and a Charles Hollenberg Summer Studentship from the Banting and Best Diabetes Centre.
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Novelty and Significance
What Is Known?
Accumulation of extracellular matrix (particularly collagens) in atherosclerotic plaque can have both negative (increased plaque size) and positive (decreased plaque rupture) effects on disease progression.
Our understanding of the receptors and the mechanisms that control matrix accumulation is incomplete.
Discoidin domain receptor (DDR)1 is a collagen receptor expressed on both smooth muscle cells (SMCs) and macrophages in the plaque, but the function of this receptor on these cells is not known.
What New Information Does This Article Contribute?
We show for the first time that in vascular SMCs, DDR1 represses matrix synthesis and, at the same time, limits the expression of many MMPs.
The net effect of DDR1-deletion in vascular smooth muscle cells is the accumulation of extracellular matrix during atherogenesis.
DDR1 is also a negative regulator of cell proliferation and migration.
Previous studies from our laboratory have shown that DDR1 is expressed on SMCs and macrophages in atherosclerotic plaques and that systemic deletion of DDR1 results in a reduction in plaque growth and macrophage accumulation but an increase in matrix accumulation and fibrous cap thickening. To identify the distinct contribution of DDR1 expressed on SMCs, we used bone marrow transplantation to generate host chimeric mice with DDR1 deficiency in cells of the vessel wall. We show, for the first time, that DDR1 deletion in SMCs of the vessel wall results in robust matrix accumulation in the plaque and the formation of a large fibrous cap with well-organized collagen fibers, suggesting that these lesions might be better protected against plaque rupture. These studies suggest that, in SMCs, DDR1 acts as a sensor of the collagen matrix. Thus, inhibiting SMC DDR1 may be a useful therapeutic approach for promoting the formation of stable, matrix-rich plaques.
↵*Both authors contributed equally to this work.
Original received November 23, 2009; revision received April 7, 2010; accepted April 22, 2010.