Collagens constitute a major portion of the extracellular matrix in the atherosclerotic plaque, where they contribute to the strength and integrity of the fibrous cap, and also modulate cellular responses via specific receptors and signaling pathways. This review focuses on the diverse roles that collagens play in atherosclerosis; regulating the infiltration and differentiation of smooth muscle cells and macrophages; controlling matrix remodeling through feedback signaling to proteinases; and influencing the development of atherosclerotic complications such as plaque rupture, aneurysm formation and calcification. Expanding our understanding of the pathways involved in cell-matrix interactions will provide new therapeutic targets and strategies for the diagnosis and treatment of atherosclerosis.

Cardiovascular disease (CVD) and cancer are leading causes of mortality and morbidity worldwide. Strategies to improve their treatment and prevention are global priorities and major focus of World Health Organization’s joint prevention programs. Emerging evidence suggests that modifiable risk factors including diet, sedentary lifestyle, obesity and tobacco use are central to the pathogenesis of both diseases and are reflected in common genetic, cellular, and signaling mechanisms. Understanding this important biological overlap is critical and may help identify novel therapeutic and preventative strategies for both disorders. In this review, we will discuss the shared genetic and molecular factors central to CVD and cancer and how the strategies commonly used for the prevention of atherosclerotic vascular disease can be applied to cancer prevention.

Abstract-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 ϩ/ϩ ;Ldlr Ϫ/Ϫ and Ddr1 Ϫ/Ϫ ;Ldlr Ϫ/Ϫ 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 Ϫ/Ϫ ;Ldlr Ϫ/Ϫ plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1 ϩ/ϩ ;Ldlr Ϫ/Ϫ plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1 Ϫ/Ϫ 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 Ϫ/Ϫ ;Ldlr Ϫ/Ϫ 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 Ϫ/Ϫ ;Ldlr Ϫ/Ϫ 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 Ⅲ discoidin domain receptor 1 Ⅲ collagen Ⅲ inflammation Ⅲ macrophage A therosclerosis 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 ...

Remodeling of injured blood vessels is dependent on smooth muscle cells and matrix metalloproteinase activity. Doxycycline is a broad spectrum matrix metalloproteinase inhibitor that is under investigation for the treatment of acute coronary syndromes and aneurysms. In the present study, we examine the mechanisms by which doxycycline inhibits smooth muscle cell responses using a series of in vitro assays that mimic critical steps in pathological vascular remodeling. Doxycycline treatment dramatically increased smooth muscle cell adhesion to the substrate, as evidenced by interference reflection microscopy and immunostaining for paxillin and phosphotyrosine. Cell aggregation was also potentiated after treatment with doxycycline. Treatment with 104 mumol/L doxycycline reduced thymidine uptake by 58% compared with untreated cells (P < 0.05) and inhibited closure of a scrape wound made in a smooth muscle cell monolayer by 20% (P < 0.05). Contraction of a three-dimensional collagen gel was used as an in vitro model for constrictive vessel remodeling, demonstrating that treatment with 416 mumol/L doxycycline for 12 hours inhibited collagen gel remodeling by 37% relative to control (P < 0.05). In conclusion, we have shown that doxycycline treatment leads to dramatically increased smooth muscle cell adhesion, which in turn might limit responses in pathological vascular remodeling.

Key Words: atherosclerosis Ⅲ discoidin domain receptor Ⅲ extracellular matrix Ⅲ collagen Ⅲ smooth muscle cell T he 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 macrophages 3-7 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 Ϫ/Ϫ mouse model. Atherosclerotic plaques that formed in mice doubly deficient in DDR1 and LDLR (Ddr1 Ϫ/Ϫ ;Ldlr Ϫ/Ϫ ) 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 ϩ/ϩ ;Ldlr Ϫ/Ϫ ). 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 Ϫ/Ϫ mice. 12 This re-

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 Key Words: atherosclerosis Ⅲ discoidin domain receptor Ⅲ collagen Ⅲ macrophage Ⅲ inflammation T he accumulation of macrophages in the arterial intima is a critical event in atherosclerotic plaque development. Macrophage accumulation depends on a series of welldefined 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 regula-

Intimal calcification is a feature of advanced atherosclerotic disease that predicts a two-to eightfold increase in the risk of coronary events. Type I collagen promotes vascular smooth muscle cell-mediated calcification, although the mechanism by which this occurs is unknown. The discoidin domain receptor 1 (DDR1) is a collagen receptor that is emerging as a critical mediator of atherosclerosis. To determine whether DDR1 is involved in intimal calcification, we fed male Ddr1 ؊/؊ ;Ldlr ؊/؊ and Ddr1 ؉/؉ ;Ldlr ؊/؊ mice an atherogenic diet for 6, 12, or 24 weeks. DDR1 deficiency significantly reduced the calcium content of the aortic arch, and microcomputed tomography demonstrated a significant decrease in hydroxyapatite deposition after 24 weeks of atherogenic diet. Reduced calcification was correlated with decreases in macrophage accumulation and tumor necrosis factor ␣ staining, suggesting that the reduction in calcification was in part due to decreased inflammation. The chondrogenic markers type II collagen, type X collagen , and Sox-9 were expressed within the mineralized foci. An in vitro assay performed with vascular smooth muscle cells revealed that DDR1 was required for cell-mediated calcification of the matrix , and Ddr1 ؉/؉ smooth muscle cells expressed more alkaline phosphatase activity, whereas Ddr1 ؊/؊ smooth muscle cells expressed elevated levels of mRNA for nucleotide pyrophosphatase phosphodiesterase 1, an inhibitor of tissue mineralization. Taken together, our results demonstrate that DDR1 mediates an important mechanism for atherosclerotic calcification. (Am J Pathol

Recent research has shown that the tetracycline antibiotics are pluripotent drugs that inhibit the activity of matrix metalloproteinases (MMPs) and affect many cellular functions including proliferation, migration, and matrix remodeling. We have shown that doxycycline inhibits MMP activity and intimal thickening after injury of the rat carotid artery, however we do not know whether these effects are because of the antibiotic, anti-MMP, or other actions of doxycycline. Recently, chemically modified tetracyclines have been synthesized that lack antibiotic activity but retain anti-MMP activity (CMT-3), or lack both antibiotic and anti-MMP activity (CMT-5). In the current study we have assessed the effects of treatment with CMT-3 or CMT-5 on intimal thickening after balloon catheter injury of the rat carotid artery. Rats were treated by oral gavage with 15 mg/kg/day CMT-3 or CMT-5. CMT-3 significantly reduced smooth muscle cell (SMC) proliferation in both the medial and intimal layers of the injured rat carotid artery compared to CMT-5. Furthermore, CMT-3 inhibited SMC migration from the media to the intima by 86% at 4 days after injury. CMT-3 also decreased MMP-2 activity. Finally, we found that CMT-3 treatment resulted in a significant reduction in intimal cross-sectional area from 0.23 +/- 0.01 mm(2) in the CMT-5 control group to 0.19 +/- 0.01 mm(2). There was also a reduction in elastin and collagen accumulation within the intima. We conclude that CMT-3 attenuated intimal thickening after arterial injury by inhibiting SMC proliferation, migration and MMP activity, and accumulation of extracellular matrix. The inhibitory effects of CMT-3 were independent of the antibiotic properties, but were dependent on the anti-MMP activity of the tetracycline family.