IntroductionAtherosclerosis and restenosis are characterized by the development of a thickened neointimal layer in the blood vessel wall. Smooth muscle cells (SMCs) are activated after arterial injury and contribute to neointimal lesion development through proliferation, migration, and ECM synthesis. Recent research suggests that the ECM is not simply an inert scaffold, but instead there are dynamic interactions between cells and matrix that contribute to SMC responses. After arterial injury, SMCs synthesize the fibrillar type I and III collagens (1, 2) and the short-chain type VIII collagen (3-6). Recently, the discoidin domain receptor tyrosine kinases (DDRs) were shown to function as collagen receptors and to increase matrix metalloproteinase (MMP) production in a fibrosarcoma cell line (7,8). The MMPs (including MMP-1, MMP-2, MMP-3, MMP-9, and MT1-MMP) are upregulated after injury and facilitate SMC migration in the vessel wall (9-12). Given the role of the DDRs in mediating interactions with collagen and stimulating MMP synthesis, we posit here that the DDRs are important mediators of the SMC response to injury.The DDRs are distinguished by an extracellular domain of 160 amino acids that is homologous to the Dictyostelium discoideum protein discoidin-I (13). There are two distinct gene products, DDR1 and DDR2, and DDR1 appears in three alternative splice variants, 1a, 1b, and 1c (13). DDR1 is widely expressed during embryonic development and in adult tissues, particularly in the epithelium of skin, kidney, gut, and brain, and the splice variant DDR1b increases considerably during postnatal development. DDR2 is restricted to skeletal muscle, heart, and connective tissues (13). Strikingly high levels of DDR1 and 2 are seen in fastgrowing invasive mammary, ovarian, and lung tumors (14), in keeping with the increased proliferative rates and MMP production in these tumors. To date, one abstract has reported DDR1 and DDR2 expression in atherosclerotic lesions of nonhuman primates fed a high-cholesterol diet (15); however, nothing is known about the function of DDRs in the vascular system. In the current study we have examined DDR1, the most widely expressed DDR in adult mammals.The arterial collagens that are upregulated after injury can be classified broadly into two categories based on their structure; fibril-forming (type I and III) and short-chain (type VIII) collagens. Tissue-culture studies show that type I collagen affects SMC growth and migration (16)(17)(18). Type VIII collagen is a shortchain collagen expressed during active remodeling in angiogenesis (19), embryonic development of the heart (20), and glomerulonephritis (21). We and others have shown that it is dramatically upregulated following experimental arterial injury (3-5) and in human atherosclerotic plaques (6), and we have shown The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain...

Smooth muscle cell (SMC) interactions with collagen mediate cell migration during the pathogenesis of atherosclerosis and restenosis. Discoidin domain receptors (DDRs) have been identified as novel collagen receptors. We used aortic SMCs from wild-type and DDR1؊/؊ mice to evaluate the function of the DDR1 in regulating migration. DDR1؊/؊ SMCs exhibited impaired attachment to and migration toward a type I collagen substrate. Matrix metalloproteinase-2 (MMP-2) and MMP-9 activities were concomitantly reduced in these cells. Transfection of a full-length cDNA for DDR1b rescued these deficits, whereas kinase-dead mutants of DDR1 restored attachment but not migration and MMP production. These results suggest that active DDR1 kinase is a central mediator of SMC migration. Interactions between smooth muscle cells (SMCs) and collagen are important in cell migration during the pathogenesis of atherosclerosis and restenosis. The synthesis of several collagens, including fibrillar type I and III and short-chain type VIII, is upregulated after injury. 1,2 These collagens act as chemotactic factors for SMCs, and migration through the extracellular matrix is facilitated when type VIII collagen triggers matrix metalloproteinase (MMP) synthesis. 3,4 The discoidin domain receptors (DDRs) are two novel collagen receptors distinguished by an extracellular domain homologous to discoidin-I in Dictyostelium discoideum and by a catalytic tyrosine kinase domain in the cytoplasmic tail. The activation of DDR2 by collagen results in increased production of MMP-1 5 and MMP-2, 6 suggesting an important role for this receptor in matrix degradation. Targeted deletion of the DDR1 gene in mice resulted in severe defects in placental implantation and mammary gland development, 7 whereas DDR2 Ϫ/Ϫ mice exhibit skeletal malformation and delayed wound healing, 8 suggesting potential roles in migration and matrix degradation for both receptors. DDR1 was expressed on migrating SMCs after balloon catheter injury of the rat carotid artery, and intimal thickening after arterial injury was reduced in the DDR1 Ϫ/Ϫ mouse. 9 In the present study, we have investigated the role of DDR1 in SMC migration by transfecting DDR1 Ϫ/Ϫ SMCs with full-length or kinase-mutated DDR1 constructs to determine whether kinase activity is required for migration and MMP production. Materials and MethodsSMCs were isolated from the aortas of DDR1 Ϫ/Ϫ mice and wild-type littermates (mixture of 129/Sv and ICR strains). Knockout cells were transfected with the full-length cDNA of DDR1b, with a truncated form of DDR1b that lacks the catalytic domain (K529*) or with a kinase-dead form of DDR1a containing a lysine to alanine mutation at position 618 in the catalytic domain (K618A). 10 SMC attachment was measured by toluidine blue staining; chemotaxis chamber migration assays and gelatin zymograms were performed using methods we have described. 3 To localize the gelatinolytic activity in SMC cultures, the cells were overlaid with FITC-labeled DQ gelatin that is intermolecularly quenched ...

Type VIII collagen is a matrix protein expressed in a number of tissues undergoing active remodeling, including injured arteries during neointimal formation and in human atherosclerotic plaques; however, very little is known about its function. We have investigated whether the type VIII collagen stimulates smooth muscle cell (SMC) migration and invasion by binding to integrin receptors and up-regulating matrix metalloproteinase (MMP) production. SMCs attached to plates coated with type VIII collagen in a dose-dependent manner, with maximal attachment occurring with coating solutions containing 25 microgram/ml collagen. Type VIII collagen at 100 microgram/ml stimulated an 83-fold increase in the migration of SMCs in a chemotaxis chamber. Antibodies against beta1 integrin receptors prevented attachment and migration of SMCs. Antibodies against alpha1 or alpha2 integrins reduced attachment of SMCs to type VIII collagen by 29% and 77%, respectively. We found that SMCs grown from the rat neointima, but not medial SMCs, increased their production of MMP-2 and -9 on adherence to type VIII collagen. This suggests that there is an important difference in phenotype between intimal and medial SMCs and that intimal SMCs have distinct matrix-dependent signaling mechanisms. Our findings suggest that type VIII collagen deposited in vascular lesions functions to promote SMC attachment and chemotaxis, and signals through integrin receptors to stimulate MMP synthesis, all of which are important mechanisms used in cell migration and invasion.

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 ...

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.

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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.

Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.