Discoidin Domain Receptor 1 on Bone Marrow–Derived Cells Promotes Macrophage Accumulation During Atherogenesis
Rationale: We described a critical role for the discoidin domain receptor (DDR)1 collagen receptor tyrosine kinase during atherosclerotic plaque development. Systemic deletion of Ddr1 in Ldlr−/− mice accelerated matrix accumulation and reduced plaque size and macrophage content. However, whether these effects reflected an independent role for macrophage DDR1 during atherogenesis remained unresolved.
Methods: In the present study, we performed sex-mismatched bone marrow transplantation using Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice to investigate the role of macrophage DDR1 during atherogenesis. Chimeric mice with deficiency of DDR1 in bone marrow–derived cells (Ddr1−/−→+/+) or control chimeric mice that received Ddr1+/+;Ldlr−/− marrow (Ddr1+/+→+/+) were fed an atherogenic diet for 12 weeks.
Results: We observed a 66% reduction in atherosclerosis in the descending aorta and a 44% reduction in plaque area in the aortic sinus in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice. Furthermore, we observed a specific reduction in the number of donor-derived macrophages in Ddr1−/−→+/+ plaques, suggesting that bone marrow deficiency of DDR1 attenuated atherogenesis by limiting macrophage accumulation in the plaque. We have also demonstrated that the effects of DDR1 on macrophage infiltration and accumulation can occur at the earliest stage of atherogenesis, the formation of the fatty streak. Deficiency of DDR1 limited the appearance of 5-bromodeoxyuridine–labeled monocytes/macrophages in the fatty streak and resulted in reduced lesion size in Ldlr−/− mice fed a high fat diet for 2 weeks. In vitro studies to investigate the mechanisms involved revealed that macrophages from Ddr1−/− mice had decreased adhesion to type IV collagen and decreased chemotactic invasion of type IV collagen in response to monocyte chemoattractant protein-1.
Conclusions: Taken together, our data support an independent and critical role for DDR1 in macrophage accumulation at early and late stages of atherogenesis.
The accumulation of macrophages in the arterial intima is a critical event in atherosclerotic plaque development. Macrophage accumulation depends on a series of well-defined interactions with the endothelium, culminating in transmigration and invasion of the subendothelial extracellular matrix.1 In the atherosclerotic intima, monocytes/macrophages interact with an extracellular matrix rich in several types of collagen. Collagens are important components of the extracellular matrix present within atherosclerotic plaques: contributing to lesion volume, enhancing the mechanical stability of the fibrous cap, and providing key signals that regulate monocyte differentiation, protease expression, and the production of inflammatory mediators.2
The discoidin domain receptors (DDRs) are a subfamily of receptor tyrosine kinases that transduce signals when bound to collagens. There are 2 Ddr genes in the human and mouse genomes, Ddr1 and Ddr2,3 and 6 differentially spliced isoforms of DDR1 have been identified (termed Ddr1a-e, and an isoform only expressed in rat testes).4,5 Both DDR1 and DDR2 bind to several collagen subtypes, but the receptors require an intact triple helical domain for signaling; denatured collagen, or gelatin, does not induce signaling through DDRs.6–8 Importantly, DDR1 has been shown to signal when bound to collagen types I to V and VIII, a ligand repertoire that includes fibril forming interstitial collagens (types I to III), as well as network-forming type IV collagen, a principal component of the endothelial basement membrane.
Ferri et al have reported the expression of DDR1 in the atherosclerotic plaques of nonhuman primates.9 We have recently identified a functional role for DDR1 in the regulation of atherosclerotic plaque inflammation and fibrosis using Ldlr−/− mice fed a high-fat diet.10 Deletion of DDR1 resulted in smaller atherosclerotic plaques that were matrix rich and macrophage poor, both key features of a stable plaque. Accelerated matrix accumulation in Ddr1−/−;Ldlr−/− plaques resulted from enhanced expression of collagens and elastin by Ddr1−/− smooth muscle cells (SMCs) and was associated with decreased in situ matrix metalloproteinase (MMP) activity, as well as reduced lesional macrophage content. However, because DDR1 is expressed both on vessel wall–derived cells such as SMCs and on bone marrow–derived cells such as macrophages, the relative contribution of DDR1 expressed solely by macrophages to plaque development remained unresolved.
In the present study, we have used sex-mismatched bone marrow transplantation to study the role of DDR1 expressed on bone marrow–derived cells such as monocytes/macrophages during atherogenesis. Atherosclerotic plaques from chimeric mice with DDR1-deficient bone marrow (Ddr1−/−→+/+) were smaller in size and exhibited reduced accumulation of bone marrow–derived macrophages. Bromodeoxyuridine (BrdUrd) pulse-labeling experiments performed in Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice fed an atherogenic diet for 2 weeks revealed a novel role for DDR1 in macrophage accumulation in the early fatty streak. The mechanisms behind this were revealed by in vitro experiments showing that macrophages from Ddr1−/−;Ldlr−/− mice exhibited decreased attachment to and invasion of type IV collagen matrix. Our data support an independent role for macrophage DDR1 in atherogenesis and suggest that DDR1 promotes macrophage accumulation and lesion growth by regulating invasion of the intimal basement membrane.
An expanded Methods section can be found in the Online Data Supplement at http://circres.ahajournals.org.
Reciprocal Bone Marrow Transplantation, Analysis of Atherosclerosis, and Histomorphometry
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.10 The experimental groups included female Ddr1+/+;Ldlr−/− hosts receiving male Ddr1+/+;Ldlr−/− bone marrow (Ddr1+/+→+/+: control), and female Ddr1+/+;Ldlr−/− hosts receiving male Ddr1−/−; Ldlr−/− bone marrow (Ddr1−/−→+/+: bone marrow deletion). Three weeks after transplantation, mice were placed on an atherogenic diet containing 40% kcal of fat and 1.25% cholesterol by weight (Research Diets, D12108) for 12 weeks. After 12 weeks, mice were euthanized by anesthetic overdose and the heart, kidneys, and descending aorta (downstream of the left subclavian artery to the iliac bifurcation) were fixed in 4% paraformaldehyde. The aortic sinus was embedded in paraffin and sectioned into 5-μm-thick cross-sections. Details of the methods used for analysis of male:female chimerism can be found in the Online Data Supplement. Plasma lipid analysis, oil red O staining of the descending aorta, Verhoeff’s van Gieson, picosirius red and LC-PolScope analysis of fibrillar collagen were performed as described previously, and additional details can be found in the online supplement.10
Y Chromosome Fluorescence In Situ Hybridization and Analysis of Plaque Cellular Content
After deparaffinization in xylenes, air-dried cross-sections of the aortic sinus were denatured in 10 mmol/L sodium citrate (pH 6.0) for 2 hours at 80°C, rinsed in 2× sodium chloride/sodium citrate buffer (SSC), and digested in Pepsin (0.22 mg/mL) in 0.1 mol/L HCl for 2 hours at 37°C. Dehydrated sections were then incubated with 3.3 μL of CY3-labeled mouse Y chromosome Paint probe (Cambio), cover-slipped, and sealed with rubber cement before overnight hybridization at 37°C. After posthybridization washes in 0.1× SSC/0.3% NP-40 at 75°C for 3 minutes and 4× SSC/0.1% NP-40 for 10 minutes, slides were counterstained with Hoechst 33528 (1:10 000 in distilled water). Y chromosome–positive cells were counted using a Zeiss Axioplan epifluorescence microscope with a ×100 oil immersion lens.
To determine the relative contribution of vessel wall (ie, host) and bone marrow (ie, donor)–derived cells to lesion, the number of Y chromosome–positive cells was subtracted from the total number of nuclei in the plaque. The difference reflects the number of resident vessel wall–derived cells, for example SMCs, in the lesion.
BrdUrd Pulse Labeling and Measurement of Fatty Streak Lesion Size
Macrophage accumulation into the fatty streak was assessed by BrdUrd pulse labeling.11,12 Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice were fed the atherogenic diet for 2 weeks, then given a single IV injection of BrdUrd (10 mg/mL, 0.2 mL volume) to label a cohort of proliferating cells in both the bone marrow and aortic intima at the time of the injection. Mice were euthanized 2 or 24 hours later, and the ascending aorta (aortic sinus to brachiocephalic artery) was whole mount immunostained for BrdUrd using fluorescein isothiocyanate (FITC)-tyramide amplification (Tyramide signal amplification; Perkin Elmer) and mounted en face. Costaining of nuclei and lipids was performed using Hoescht 33528 (1:10 000 in distilled H2O) and oil red O, respectively.
The total number of BrdUrd-positive cells in the ascending aorta was counted using an Olympus Fluoview confocal microscope with a ×60 oil immersion lens. As previously reported, BrdUrd-positive cells cannot be detected in the circulation at 2 hours11; therefore, the number of BrdUrd-positive cells in the aortic intima at 2 hours provides a baseline measurement of intimal macrophage proliferation. In contrast, between 6 and 24 hours, there is a detectable cohort of BrdUrd-positive monocytes present in the circulation.11 Therefore, an increase in the BrdUrd-positive cell number in the aortic intima between 2 and 24 hours reflects the accumulation of newly labeled monocytes/macrophages in the developing fatty streak by a combination of local proliferation and recruitment from the circulation over a 22 hour time period. The number of BrdUrd-positive cells per ascending aorta at each time point is expressed per unit of fatty streak lesion surface area to account for differences in lesion size between groups.
Measurement of fatty streak lesion size was carried out using oil red O fluorescence and confocal microscopy as detailed in the online supplement. Low magnification images of the en face preparations of ascending aorta were captured using a Bio-Rad confocal microscope with a ×4 objective. Measurement of the percentage of aortic surface area occupied by plaque was carried out using Simple PCI quantitative imaging software.
Ddr1+/+;Ldlr−/− or Ddr1−/−;Ldlr−/− mice fed the atherogenic diet for 2 weeks were pulse labeled with BrdUrd for 24 hours, and samples of peripheral blood (100 μL) or bone marrow (106 cells) were taken to measure intracellular staining of BrdUrd using flow cytometry (FITC-BrdUrd flow kit, Becton Dickinson) according to the instructions of the manufacturer. Costaining for monocytes in peripheral blood was performed using the following markers: CD115-phycoerythrin (1:100, eBioscience), Ly-6C-biotin (1:200, BMA), and streptavidin-allophycocyanin (SA-APC, 1:800, Becton-Dickinson). Samples were analyzed on a Beckman Coulter FC500 flow cytometer at the following wavelengths: FITC: 510 nm/540 nm, phycoerythrin: 560 nm/590 nm; APC: 660 nm/690 nm.
Data are presented as means±SEM. All statistical analysis was carried out using Sigma Stat (SyStat Software Inc). Pairwise comparisons between transplant groups or genotypes were performed using Student’s t test. Data that did not fit a normal distribution were analyzed by Mann–Whitney U test for nonparametric comparisons. BrdUrd pulse data were analyzed using a 1-way ANOVA with a Tukey post hoc analysis for pairwise comparisons. Statistical significance was determined at P<0.05.
Bone Marrow Transplantation, Assessment of Sry Chimerism, and Plasma Lipids
Body weight and fasting plasma cholesterol and triglycerides measured after 12 weeks on the atherogenic diet (15 weeks after transplant) were comparable between transplant groups (Online Table I). Male:female chimerism measured after 12 weeks on the atherogenic diet was assessed by analyzing genomic DNA isolated from peripheral blood leukocytes for Ddr1 and Sry, a marker of the Y chromosome (Online Figure I, A). Leukocyte DNA from Ddr1−/−→+/+ mice demonstrated the presence of the donor genotype (Ddr1−/−), with negligible host DNA remaining. (Online Figure I, A, top). The abundance of leukocyte Sry was comparable in all chimeric mice (Online Figure I, A, bottom), and this was confirmed by measuring the ratio of Sry/Gapdh by quantitative real-time PCR (Online Figure I, B).
DDR1 Expressed on Bone Marrow–Derived Cells Promotes Atherogenesis
Deletion of Ddr1 in bone marrow–derived cells (Ddr1−/−→+/+) resulted in a marked reduction in atherosclerotic plaque burden in the descending aorta compared to Ddr1+/+→+/+ mice (Figure 1A). Quantification of the percentage aortic surface area occupied by oil red O–positive plaque revealed a significant 66% decrease in atherosclerotic plaque burden in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice (Figure 1B). Furthermore, atherosclerotic plaque area measured in cross-sections of the aortic sinus was reduced by 44% in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice (Figure 1C). Taken together, these data confirm a critical role for DDR1 expressed on bone marrow–derived cells in atherosclerotic plaque growth and development.
Bone Marrow–Derived Macrophage Accumulation Was Reduced in Ddr1−/−→+/+ Plaques
Because male bone marrow donors were used to generate the chimeric mice, we were able to detect bone marrow–derived cells in the lesions using fluorescence in situ hybridization for the Y chromosome. Y chromosome–positive cells were abundant within the plaque and were also observed in the adventitia but were not observed in the media (Figure 2A and 2B). Correlative immunofluorescence staining with antibodies against Mac-2, smooth muscle α-actin, or vascular cell adhesion molecule-1 confirmed that the majority of the lesional Y chromosome–positive cells were macrophages (Online Figure II and data not shown). Quantification of the number of Y chromosome–positive cells in the plaques revealed a significant 47% decrease in bone marrow–derived macrophage accumulation in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice (Figure 2C, gray bars). Furthermore, the decrease in bone marrow–derived macrophages was responsible for the decrease in total cell number that was observed in the aortic sinus lesions of Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice (467±53 versus 324±24; Figure 2C, sum of black and gray bars). In contrast, bone marrow–specific deletion of DDR1 had no effect on the number of host-derived resident vessel wall cells (Figure 2C, black bars). Taken together with the reduction in lesion size, these data suggest that deficiency of DDR1 in bone marrow–derived cells specifically limits the accumulation of macrophages in the developing plaques, which results in the attenuation of lesion growth.
The net accumulation of macrophages in the atherosclerotic plaque is influenced by macrophage infiltration into the lesion and cell death within the lesion. To determine whether the reduction in macrophage number in Ddr1−/−→+/+ plaques was caused by an increase in cell death, we measured the percentage of apoptotic cells in sections of the aortic sinus using TUNEL. The percentage of TUNEL-labeled cells was significantly decreased in Ddr1−/−→+/+ mice compared to Ddr1+/+→+/+ mice (Figure 2D). Therefore, these data are consistent with the hypothesis that the reduction in plaque macrophages observed in Ddr1−/−→+/+ mice was the result of impaired infiltration into the lesion and not attributable to increased cell death.
Matrix Composition Was Similar in Ddr1−/−→+/+ and Ddr1+/+→+/+ Plaques
To evaluate changes in matrix composition of the atherosclerotic plaques from Ddr1+/+→+/+ and Ddr1−/−→+/+ mice, we stained serial sections of aortic sinus with Verhoeff’s van Gieson and picrosirius red to identify elastin and collagen respectively (Figure 3). Despite the significant reduction in plaque size, measurement of the percentage of lesion area occupied by collagen or elastin demonstrated no difference between groups. Similarly, using the LC-PolScope, which detects the retardance of polarized light by fibrillar collagens, we observed comparable fibrillar collagen content of plaques between transplant groups (Figure 3).
Deficiency of DDR1 Limits the Infiltration of BrdUrd-Labeled Monocytes/Macrophages in the Developing Fatty Streak
Because our findings on well-developed atherosclerotic plaques indicated reduced macrophage accumulation in the Ddr1−/−→+/+ lesions that could be attributed to decreased macrophage infiltration, we wished to determine whether this deficit was also present at the earliest stages of lesion development: the formation of the fatty streak. The fatty streak provides a simplified in vivo model to study the role of DDR1 in macrophage infiltration because the majority of lesional cells are macrophage foam cells and there is little SMC involvement or lesion fibrosis. We performed BrdUrd pulse labeling in Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice fed an atherogenic diet for 2 weeks. Mice were given a single pulse injection of BrdUrd, which labeled a cohort of proliferating cells in the bone marrow and allowed us to track their subsequent infiltration and accumulation in the atherosclerotic intima using en face confocal microscopy.
Fasting plasma lipids assayed after 2 weeks on the atherogenic diet were comparable between the two genotypes (Online Table II), and the proportion of BrdUrd-positive monocytes in the circulation and the bone marrow were also similar between groups (Figure 4A and 4B). Measurement of the percentage of ascending aortic surface area occupied by fatty streak was carried out by en face confocal imaging, which revealed a significant decrease in lesion size in Ddr1−/−; Ldlr−/− mice compared to Ddr1+/+;Ldlr−/− mice (Figure 4C). These data provide the first evidence of a role for DDR1 as a regulator of fatty streak formation. Independent of the change in lesion size, the deficiency of DDR1 also limited the infiltration of BrdUrd labeled monocytes/macrophages into the fatty streak. Figure 5A and 5B show the extent of oil red O–stained fatty streak, and Figure 5C and 5D show the BrdUrd-labeled cells in the lesions. Ddr1+/+;Ldlr−/− mice exhibited a significant increase in the number of BrdUrd+ cells per unit of lesion area between 2 and 24 hours (Figure 5E), indicating that an accumulation of labeled macrophages occurred during this time period. By contrast, the number of BrdUrd+ cells per unit of lesion area in the Ddr1−/−;Ldlr−/− mice was unchanged between 2 and 24 hours (Figure 5E). Taken together, these data suggest that macrophage infiltration in the fatty streak was impaired in Ddr1−/−;Ldlr−/− mice, which resulted in reduced fatty streak lesion size.
To determine whether the attenuation of macrophage infiltration in DDR1-deficient animals also occurred in response to a general inflammatory stimulus, we measured macrophage accumulation in the peritoneum after an injection of thioglycollate. The percentage of macrophages in the peritoneal lavage was 12.3±2.3% in Ddr1−/−;Ldlr−/− mice compared to 15.5±1% in Ddr1+/+;Ldlr−/− mice; however, the difference was not statistically significant.
DDR1 Regulates Macrophage Infiltration by Promoting the Interaction With and Invasion of Type IV Collagen Matrices
Emerging evidence supports a role for DDR1 as a mediator of matrix invasion in multiple cell types.2 Therefore, to further investigate the mechanisms behind the reduction in macrophage accumulation, we assayed the adhesion and invasion of peritoneal macrophages from Ddr1+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− mice through type IV collagen matrices in vitro. Type IV collagen is a principal component of the endothelial basement membrane and likely the first collagenous barrier to be encountered by a macrophage invading the plaque. Adhesion of Ddr1−/− macrophages to type IV collagen was significantly reduced by 51% compared to Ddr1+/+ cells (Figure 6A). Next, we assayed the monocyte chemoattractant protein-1–dependant chemotaxis of Ddr1+/+ or Ddr1−/− peritoneal macrophages through type IV collagen and found that Ddr1−/− macrophages exhibited a 50% reduction in chemotactic invasion though type IV collagen compared to Ddr1+/+ macrophages (Figure 6B). These data suggest that deficiency of DDR1 limits the ability of macrophages to adhere to and invade through collagen matrix barriers, key steps in their infiltration and accumulation in the arterial intima.
Using a bone marrow transplantation approach, we have identified an independent role for DDR1 expressed on macrophages in contributing to atherosclerotic plaque development by regulating macrophage infiltration and accumulation in the plaque. The reduction in lesion size and cellularity in Ddr1−/−→+/+ mice was attributable to decreased bone marrow–derived macrophage accumulation and was associated with impaired adhesion and invasion through type IV collagen matrices by Ddr1−/− macrophages. Furthermore, we have determined that DDR1-dependant macrophage accumulation is also critical for the formation of the fatty streak, one of the earliest stages of lesion development. Taken together, our data suggest that DDR1 expression on macrophages, by facilitating the interaction with and invasion through type IV collagen–rich matrices such as the endothelial basement membrane, mediates macrophage accumulation and contributes to lesion growth at multiple stages.
We show that deletion of DDR1 on bone marrow–derived macrophages is sufficient to result in decreased plaque growth at both early (2 weeks) and late (12 weeks) stages of plaque development. That the reduction in plaque size is a consequence of reduced macrophage accumulation is evidenced by comparing the specific decrease in donor-derived macrophage cell number (47% decrease), and the parallel and equivalent reduction in plaque size (44% decrease). Furthermore, even at the earliest stages of plaque development, deficiency of DDR1 limits the infiltration of BrdUrd-labeled monocytes/macrophages into the lesion. By allowing us to dissect out the relative role of DDR1 on macrophages versus SMCs in plaque growth, these novel findings expand on our previous work examining the effects of systemic deletion of DDR1 during atherogenesis10 and point to an important and independent role for DDR1 on macrophage infiltration to the atherosclerotic plaque.
Another very important finding in our present study is the discovery that there is a reduction in fatty streak lesion size in Ddr1−/−;Ldlr−/− mice, which provides the first evidence of a role for DDR1 in one of the earliest stages of atherogenesis. Moreover, these data are consistent with our previous work showing attenuation of much more advanced lesions in both the Ddr1−/−;Ldlr−/− mice at 12 and 24 weeks10 and in the Ddr1−/−→+/+ chimeras at 12 weeks (present study). Taken together, our data support the notion that deficiency of DDR1 in macrophages early in atherogenesis may have long-term consequences for disease progression at later time points and underscores the importance of DDR1 as a potential target in the long-term management of atherosclerotic disease.
These results prompted us to investigate the mechanisms whereby DDR1 promotes macrophage infiltration and accumulation within the plaque. Because DDR1 is a collagen receptor, it was logical to hypothesize that newly recruited monocytes would use DDR1 to adhere to and traverse the collagen-rich plaque matrix. Indeed, we found that DDR1-deficient macrophages exhibited a reduced capacity to interact with and traverse a type IV collagen matrix. It is likely that the failure of macrophages to traverse the matrix is related to the attenuation of MMP production, because we have shown that MMP expression is reduced in Ddr1−/− macrophages,10 and others have reported that DDR1 overexpression in a macrophage cell line enhanced type I collagen matrix invasion.13 Furthermore, collagen signaling has been shown to regulate the production of MMP-1 and MMP-9 in human macrophages.14–16 These data strongly suggest that the regulation of MMP expression is the mechanism for DDR1-mediated matrix invasion. Taken together, these findings suggest that DDR1 deficiency in macrophages limits macrophage invasion of the endothelial basement membrane and plaque extracellular matrix.
To assess whether DDR1 deficiency also limited macrophage infiltration to a more general inflammatory stimulus, we assayed peritoneal macrophage accumulation after an injection of thioglycollate. We observed a trend toward a reduction in peritoneal macrophage accumulation in DDR1-deficient mice; however, the reduction was not statistically significant and was not as great as we observed in the atherosclerotic plaque. Recently, Voisin et al demonstrated that monocytes preferentially traverse the basement membrane of postcapillary venules at sites of low matrix protein expression, termed low expression regions (LERs). Moreover, their data suggest that monocyte transmigration at LERs does not require proteases.17 Because LERs have not been demonstrated in the matrix rich subendothelial basement membrane of the aortic intima, it is possible that protease dependant transmigration at this arterial location could account for the differences observed between macrophage infiltration in atherosclerotic plaques versus peritoneal inflammation.
Despite the dramatic change in plaque size and cell accumulation, the atherosclerotic plaques from Ddr1−/−→+/+ chimeric mice exhibited no difference in the proportion of lesion area occupied by matrix molecules compared to Ddr1+/+→+/+ controls. By contrast, in previous work examining the advanced plaques of Ddr1−/−;Ldlr−/− mice (with systemic deletion of DDR1), we observed an acceleration of collagen and elastin deposition and increased fibrosis compared to Ddr1+/+;Ldlr−/− mice, and we also documented increased collagen and elastin expression by DDR1-deficient SMCs in vitro.10 This suggests that enhanced matrix accumulation is the result of DDR1 deletion in SMCs alone, and that macrophage DDR1 deficiency does not contribute to the enhanced matrix accumulation observed in the lesions of Ddr1−/−;Ldlr−/− mice.
Emerging evidence supports a role for DDR1 in leukocyte accumulation in chronic inflammatory diseases such as renal18 and pulmonary fibrosis.19 Using bone marrow transplantation, we demonstrate, for the first time, an independent role for macrophage DDR1 in atherosclerotic plaque development. Our data support a model whereby DDR1 facilitates the invasion and accumulation of monocytes/macrophages in the arterial intima by mediating the interaction with and invasion through the type IV collagen–rich endothelial basement membrane, resulting in reduced macrophage accumulation, decreased intimal inflammation, and the attenuation of atherogenesis.
We thank Dr Phillip Connelly and Graham Maguire for assistance with measurements of plasma lipoproteins and triglycerides; Dr Jeremy Squire for advice on the fluorescence in situ hybridization; and Dr Philip Marsden and Brent Steer for assistance with the real-time PCR.
Sources of Funding
This study was funded by Canadian Institutes of Health Research grant MOP37847 and Heart and Stroke Foundation of Ontario grants NA6069 and T6734 (to M.P.B.). M.P.B. and M.C. are Career Investigators 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 Malcolm Silver Scholarship in Cardiovascular Studies. K.B. was supported by a Masters Award from the Heart and Stroke Foundation of Ontario. E.W. was supported by a John D. Schultz Scholarship from the Heart and Stroke Foundation of Ontario.
Original received August 27, 2008; resubmission received August 14, 2009; revised resubmission received September 28, 2009; accepted October 2, 2009.
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