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Collagens, Integrins, and the Discoidin Domain Receptors in Arterial Occlusive Disease
BRIEF REVIEWS Collagens, Integrins, and the Discoidin Domain Receptors in Arterial Occlusive Disease Christopher D. Franco, Guangpei Hou, and Michelle P. Bendeck*
The collagen matrix constitutes a major portion of the vascular extracellular matrix and imparts blood vessels with tensile strength and, even more important, modulates smooth muscle cell (SMC) responses via specific receptors and signaling pathways. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the receptors that mediate these processes. Better understanding of the pathways involved in cell matrix interactions promises to provide novel therapeutic targets and treatment strategies for the prevention of arterial occlusive diseases such as atherosclerosis and restenosis. (Trends Cardiovasc Med 2002;12:143–148). © 2002, Elsevier Science Inc.
The extracellular matrix (ECM) is a complex mixture of proteins and proteoglycans that provides a scaffold for cells and contributes to the mechanical strength of tissues. The fibrillar collagens are a major component of the vascular matrix, and the most abundant are types I and III collagen, which form an interconnected network of cross-banded fibers
Christopher D. Franco, Guangpei Hou, and Michelle P. Bendeck are at the Departments of Laboratory Medicine and Pathobiology and Medicine, University of Toronto, Ontario, Canada. * Address correspondence to: Michelle P. Bendeck, Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, Room 6217, Toronto, ON M5S 1A8 Canada. Tel.: (11) 416-946-7133; fax: (11) 416-9785959; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
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along with smaller amounts of type VI and XVIII collagen associated with the fibers and the elastic lamellae in the blood vessel wall. Network-forming type IV collagen is a major component of the basement membrane that lies beneath the endothelium and surrounds medial smooth muscle cells (SMCs). The adventitia contains abundant type I and III collagen surrounding the resident fibroblasts (Raines 2000). Once seen as merely the building blocks of tissue architecture, recent research has suggested that the dynamic interactions between cells and matrix control many processes including proliferation, migration, and survival (Schwartz 2001). Furthermore, recent findings have revealed that matrix composition is altered during the progression of diseases such as atherosclerosis and restenosis, and these changes can have a profound effect on vessel structure and
cellular responses. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the relationship to the pathogenesis of arterial occlusive diseases. • Integrin-Mediated Cell–Collagen Interactions The influence of the ECM is exerted on cells via cell-surface receptors. Integrins are a family of heterodimeric transmembrane glycoproteins composed of a and b subunits that link cells to the ECM. Integrins activate intracellular signaling pathways by recruiting cytoplasmic tyrosine kinases and adapter proteins to focal adhesion sites at the cell membrane (Schwartz 2001). Several members of the integrin family that are expressed in blood vessels bind to collagen, namely a1b1, a2b1, and avb3. a1b1 and a2b1 are the main fibrillar collagen receptors (Heino 2000). The avb3 integrin binds gelatin, the denatured form of type I collagen (Davis 1992). Although the a2b1 and a1b1 integrin receptors bind the same collagen ligands, they stimulate distinct signaling pathways and regulate different functions. This review will concentrate on studies of SMCs and fibroblasts, because these cells are present in the vessel wall and may contribute to constrictive vessel remodeling in atherosclerosis and restenosis (see below). The a2b1 integrin mediates collagen gel contraction by skin fibroblasts and SMCs (Langholz et al. 1995, Lee et al. 1995). This receptor also increases type I collagen and matrix metalloproteinase (MMP) production, via p38 kinase signaling, in skin fibroblasts cultured in three-dimensional collagen gels (Ravanti et al. 1999). In contrast, ligation of the a1b1 integrin in fibroblasts leads to a marked downregulation of type I collagen synthesis (Langholz et al. 1995), and a1-null fibroblasts from knockout mice lose the ability to downregulate
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collagen synthesis (Gardner et al. 1999). In osteosarcoma cells transfected with integrin receptors, a2b1 integrins compete with a1b1 for collagen ligand, thereby abrogating the negative effects of a1b1 on collagen synthesis; however, the physiologic significance of this response in other cell types is not known (Riikonen et al. 1995). Activation of the a1b1 receptor in fibroblasts also stimulates cell proliferation, signaling via the adapter protein Shc and activation of Ras and Erk (Pozzi et al. 1998). Studying the importance of these two collagen receptors in the vasculature is complicated by the fact that vascular SMCs in vivo express mostly a1b1 receptor with only negligible levels of a2b1, whereas cultured SMCs express only the a2b1 integrin and no a1b1 (Gotwals et al. 1996, Skinner et al. 1994). Given that the rate of matrix turnover in normal blood vessels is incredibly slow, it is most likely that expression of the a2b1 collagen receptor, which can mediate migration, collagen turnover, and gel contraction, occurs only during periods of active remodeling, such as in development and wound repair. The reciprocal effects of a1b1 and a2b1 integrin receptors suggest that there is a coordinated mechanism regulating collagen synthesis and degradation that can sense and effect changes in the composition of the matrix. • Novel Collagen Receptors: The Discoidin Domain Receptor Tyrosine Kinases A new class of collagen receptors was discovered recently—the discoidin domain receptor (DDR) tyrosine kinases. They are the first receptor tyrosine kinases identified that bind directly to the ECM (Shrivastava et al. 1997, Vogel et al. 1997). DDRs are characterized by an extracellular domain homologous to discoidin-1, a lectin in Dictyostelium discoideum that mediates intercellular adhesion. Discoidin domains are found in a variety of proteins that are secreted or associated with the cell surface, for example, blood coagulation factors V and VIII, the matrix-binding proteins Del-1 and aortic carboxypeptidase-like protein (ACLP), the neuropilin receptor (a receptor for VEGF165), and the trkA receptor (Vogel 1999). There are two distinct genes for DDR1 and DDR2. DDR1 binds collagens I
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through V and VIII, whereas DDR2 only responds to the fibrillar collagens types I and III (Hou et al. 2001, Shrivastava et al. 1997, Vogel 1999). DDR1 appears in several distinct splice variants (Alves et al. 2001) and is widely expressed during development and in adult tissues such as kidney, gut, brain, cornea and skin epithelium; and in corneal and dermal fibroblasts. The DDR1a splice variant is expressed by macrophages infiltrating tumors (Kamohara et al. 2001). DDR1 mRNA expression in monocytes is upregulated by tumor necrosis factor a, interleukin-1b, granulocyte macrophagecolony stimulating factor (GM-CSF), and lipopolysaccharide (LPS) (Kamohara et al. 2001), but very little is known about regulation in other cell types or tissues. The expression pattern of DDR2 is more restricted, but it is expressed in skeletal muscle, heart, blood vessels, and connective tissues (Vogel 1999). In contrast to the integrin receptors, most DDRs contain an active tyrosine kinase in their cytoplasmic domain; the receptor dimerizes after collagen binding, and induces transphosphorylation of the cytoplasmic domains (Curat et al. 2001). The extracellular discoidin domain is necessary and sufficient for collagen binding, whereas the entire extracellular domain is necessary for transmembrane signaling (Curat et al. 2001). Initial studies indicated that the kinetics of DDR phosphorylation were very slow, with maximal activation after 18 h of continuous exposure to collagen. Furthermore, because the receptors were not downregulated, the signal could persist for very long periods of time (Vogel et al. 1997). However, more recent studies have demonstrated that in a monocytic cell line transfected with either DDR1a or DDR1b, or in hepatic stellate cells expressing DDR2, the receptors were phosphorylated as early as 60 min after collagen stimulation, so in some cells activation may be much faster (Kamohara et al. 2001, Olaso et al. 2001a). Little is known about postreceptor signal transduction by the DDRs. It has been shown that the adapter proteins Shc (Vogel et al. 1997) and fibroblast growth factor (FGF) receptor substrate-2 (Foehr et al. 2000) bind to the juxtamembrane region of DDR1; however, the rasmitrogen-activated protein kinase pathway is not activated. DDR1 is activated in the presence of blocking antibodies to
the b1 integrin receptor, indicating that the DDRs can participate in integrinindependent signaling responses (Vogel et al. 2000). Repression of Wnt signaling impairs DDR1 phosphorylation and potentiates migration of mammary carcinoma cells on collagen, suggesting important but poorly defined links between these two signaling pathways (Jonsson and Andersson 2001). Prolonged stimulation of DDR2 has been associated with the upregulation of MMP-1 expression, suggesting an important role for this receptor in regulating collagen matrix degradation and reorganization (Vogel et al. 1997). The DDR1a isoform also plays a critical role in mediating monocyte migration through collagen gels, a process dependent on proteolytic activity (Kamohara et al. 2001). Two recent publications reported the effects of deletion of the DDR1 or DDR2 genes in mice. Targeted deletion of the DDR1 gene resulted in dwarfism and severe defects in placental implantation and mammary gland development (Vogel et al. 2001), whereas DDR2null mice also exhibited dwarfism, skeletal malformation, and delayed wound healing (Labrador et al. 2001). Bone and mammary morphogenesis and skin wound healing are critically dependent on the coordinated activity of MMPs, supporting the hypothesis that the control of MMP expression by the DDRs is of profound physiological significance. Other studies using soluble receptor domains as binding antagonists, or expressing kinase-dead dominant negative DDR1 receptors, suggest that the DDRs play important roles in the differentiation of neurons (Bhatt et al. 2000), myoblasts (Vogel et al. 2000), and mammary cells in vitro (Jonsson and Andersson 2001). • The Role of Collagens in the Pathogenesis of Arterial Occlusive Diseases Atherosclerosis and restenosis are characterized by a marked reduction in vessel lumen area due to encroachment by the atherosclerotic plaque or restenotic neointima, and by arterial shrinkage due to constrictive remodeling. The neointima is composed of SMCs that synthesize an abundant ECM, and of macrophages that accumulate lipids. Arterial lesions are rich in matrix, with collagen constituting up to 60% of the total TCM Vol. 12, No. 4, 2002
plaque protein (Rekhter 1999). Recent research suggests that the collagen matrix is not simply an inert scaffold, but that instead after injury there is a dynamic interplay involving matrix degradation and synthesis, with collagens regulating many cellular responses that contribute to arterial occlusion. Matrix-degrading proteinases are upregulated following vascular injury and modify the ECM, with dramatic consequences for SMCs. Degradation of collagen and gelatin by MMP-1, MMP-2, MMP-3, MMP-9, and MT1-MMP facilitates SMC migration after arterial injury by allowing the SMC to clear a path through the surrounding matrix (Bendeck et al. 1996a, Lijnen et al. 1999, Zempo et al. 1996). Collagenases cleave collagen at a single site within the triple helix, between Gly775 and Leu/Ile776, producing two distinct collagen fragments that are three fourths and one fourth the length of the collagen monomer, respectively (Welgus et al. 1982). Polyclonal antibodies raised against the neoepitopes derived after cleavage have allowed the immunohistochemical detection of collagen degradation in situ in the atherosclerotic plaque (Sukhova et al. 1999). As atherosclerosis progresses, local collagen degradation can also cause mechanical weakening of the atherosclerotic plaque and lead to plaque rupture (Rekhter et al. 2000). Therefore, the regulation of MMP expression and activity represents an important therapeutic target for atherosclerosis and restenosis. Proteolytic digestion of matrix within the vessel wall leads to the generation of degradation products that have quite distinct effects compared with the normal intact matrix molecules. Responses to denatured or degraded collagen are mediated at least in part by cryptic integrin binding sites within collagen, resulting in a switch of binding to the avb3 integrin (Davis 1992). When grown on polymerized type I collagen (mimicking intact type I collagen), SMCs are arrested in the G1 phase of the cell cycle owing to upregulation of p21, which suppresses cyclin-dependent kinase 2 and cyclin E kinase activities (Koyama et al. 1996). In contrast, cells plated on monomeric type I collagen (mimicking degraded collagen) proliferate rapidly (Koyama et al. 1996). Type I collagen degradation products stimulate the expression of tenascin C, a matrix glycoprotein itself important in TCM Vol. 12, No. 4, 2002
controlling SMC proliferation in remodeling arteries (Jones et al. 1999). Collagenase-degraded collagen fragments induce the rapid disassembly of focal adhesion complexes in SMCs, facilitating release from the matrix and cell migration (Carragher et al. 1999). Current work using DNA array screening suggests that many other genes are overexpressed in SMCs plated on monomeric collagen, including actin-binding proteins, signaling molecules, other matrix components, and genes of unknown function (Ichii et al. 2001), and this will provide the basis for much future study. New matrix molecules are synthesized after arterial injury, resulting in dramatic changes in the composition of the matrix in contact with SMCs. Type VIII collagen is upregulated in several animal models of arterial injury and atherosclerosis, coincident with SMC migration and co-localized with areas of lipid deposition and macrophage accumulation (Bendeck et al. 1996b, Plenz et al. 1999, Sibinga et al. 1997). Type VIII collagen serves as a chemotactic factor for SMCs and provides a matrix favorable for migration (cells adhere less strongly than to other matrix molecules). In addition, it stimulates SMC production of MMP-2 and MMP-9, which facilitates migration (Hou et al. 2000). In vitro, blocking the elaboration of collagen prevents SMC migration (Rocnik et al. 1998). It is tempting to speculate that the synthesis of new type VIII collagen is important in this regard; however, in the study cited, total collagen synthesis was blocked, precluding the identification of the particular collagen type(s) affected. Type I collagen also contributes to the regulation of SMC migration in vitro (Gotwals et al. 1996, Skinner et al. 1994). Furthermore, SMCs must be able to degrade collagen in order to move across or invade through collagen gels (Kandu et al. 2000, Li et al. 1999). Taken together, this suggests that continual modulation of collagen elaboration and degradation is necessary to permit SMC migration, implicating complex feedback mechanisms in the control of this process. This precise modulation and feedback is effected through the cellular collagen receptors, and the expression of b1 and b3 integrin receptors on SMCs is upregulated after vascular injury (Bendeck et al. 2000, Gotwals et al. 1996). Blocking the avb3 integrin receptor following arterial in-
jury dramatically attenuates SMC migration, MMP synthesis, and intimal thickening (Bendeck et al. 2000, Bendeck and Nakada 2001, Byzova et al. 1998). However, at present it is not possible to distinguish between the effects of denatured collagen versus other ligands such as osteopontin, vitronectin, and tenascin, which also bind the avb3 receptor. Finally, collagen deposition has been linked to constrictive remodeling in atherosclerosis and after balloon angioplasty (Pasterkamp et al. 2000). The underlying mechanisms are not completely understood, but probably involve the integrin-mediated contraction of collagen networks by intimal and/or medial SMCs and/or adventitial fibroblasts (Travis et al. 2001). Also, MMP activity may be important for breaking cell-matrix contacts, permitting reorganization of the ECM and adaptation to a decreased vessel diameter (Sierevogel et al. 2001). It is logical to postulate that de novo collagen synthesis and the maturation and crosslinking of collagen fibrils into a condensed network may also be involved, but studies on collagen synthesis and cross-linking in vascular remodeling have not yet been done. There is very little known about the roles of integrins or DDRs in mediating these remodeling processes in vivo. • The Role of DDR1 in Arterial Wound Repair In contrast to the integrins, very little is known about the expression or function of DDRs in the cardiovascular system. There are no gross abnormalities in cardiac or vascular structure in DDR1- or DDR2-null mice; however, many genes are upregulated and contribute to SMC phenotypic modulation only after arterial injury. One abstract has reported DDR1 and DDR2 expression in atherosclerotic lesions from nonhuman primates fed a highcholesterol diet (Carragher et al. 2000) Because DDR1 is a novel collagen receptor and is known to regulate MMP production, we investigated the expression and function of DDR1 in the vasculature before and after arterial injury (Hou et al. 2001). Using the well-characterized rat carotid balloon catheter injury model, we showed that there was minimal expression of DDR1 in the uninjured carotid, but DDR1 mRNA and protein expression were increased as early as 2 days after injury in the medial SMCs
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immediately adjacent to the lumen, with continued receptor expression in the neointima at 14 days and later (Figure 1). We hypothesized that this pattern of expression was linked to the process of neointimal formation that involves SMC proliferation, expression of MMPs to degrade the ECM, and migration from the media to the intima. Therefore, in vitro assays were used to mimic critical steps in migration and intimal formation, and aortic SMCs harvested from DDR1-null and wild-type mice were studied. DDR1null SMCs showed reduced attachment to, proliferation on, and chemotaxis towards collagen types I and VIII compared with wild-type SMCs (Hou et al. 2001). These experiments were the first to demonstrate a functional role for the DDR1 in mediating cell proliferation and migration. We also found that MMP-2 and MMP-9 activity were dramatically downregulated in DDR1-null SMCs, suggesting that the DDR1 plays an important role regulating baseline levels of cellular MMP activity; thus, it may impact upon cell migration through the
Figure 1. Discoidin domain receptor 1 (DDR1) immunostaining was increased after injury of the rat carotid artery. Control uninjured carotid artery (A). DDR1 was expressed by smooth muscle cells (SMCs) immediately adjacent to the lumen at 2 days after injury (B); by SMCs newly migrated onto the vessel surface at 4 days (C); and in SMCs of the thickened neointima at 14 days (D). Original magnification 3600. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 2.
Figure 2. The neointimal thickening response after vascular injury was decreased in the discoidin domain receptor 1 (DDR1)-null mouse. Area of the intima (A), and media (B) were measured on carotid cross-sections by morphometric digital image analysis. Values are mean 6 SEM. *Intimal area is significantly decreased in DDR1-null compared with control mice, P , .05. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 7.
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Figure 3. Intimal collagen deposition was reduced in the discoidin domain receptor 1 (DDR1)-null mouse. Picrosirius red staining and polarized light microscopy were used to assess the deposition of refringent collagen in the vessel wall. Original magnification 3400. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 7.
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ECM (Hou et al. 2001). Subsequently, two studies were published showing that the DDR2 also plays an important role in mediating hepatic stellate cell (Olaso et al. 2001a), and fibroblast (Olaso et al. 2001b) migration and proliferation by MMP-2 dependant mechanisms. The in vitro results with SMCs led us to predict a critical impairment in neointimal formation after vascular injury, so we injured the carotid arteries of DDR1-null and wild-type mice, using a copper wire to remove the endothelium. We found that the neointimal area after vascular injury was reduced by 70% in the DDR1-null mice compared with the wild-type mice (Figure 2). Furthermore, accumulation of collagen in the intima was significantly inhibited in the DDR1null mice (Figure 3). The latter observation suggests that the DDR1 functions as a collagen sensor that is activated by various types of collagen and is an essential part of the cellular machinery that triggers ECM degradation and renewal. Thus, our results support the hypothesis that DDR1 plays an important role as a collagen receptor in arterial wound repair. In the absence of DDR1, decreased SMC proliferation, migration, MMP, and collagen synthesis result in the attenuation of intimal thickening.
lagen scaffold of the abdominal aorta, and the roles of the DDRs in this process have not yet been addressed. Finally, we are far from having a clear picture of DDR signaling pathways. The integration of these pathways with other receptors controlling cell adhesion, differentiation, and growth—particularly integrins, cadherins, and the Wnt pathway—is an important area for future study. • Conclusions Neointimal thickening and constrictive remodeling are major determinants of arterial occlusive disease. Research over the past decade has shown inarguably that changes in the structure and composition of the extracellular matrix play a very important role in activating SMC responses in injured vessels. The collagens and their cellular receptors, the DDRs, and the integrins are key mediators central to these processes of arterial repair. With rapidly accumulating knowledge of these mechanisms, targets for intervention and therapeutic strategies can be identified for the prevention of arterial occlusive diseases, and later complications such as plaque rupture and aneurysm formation. • Acknowledgments
• Future Studies Mechanical injury of the mouse carotid is a simple model that provides valuable information on the SMC responses after arterial injury; however, there is little inflammation and no lipid infiltration in this model. Future research will involve crossing the DDR1-null mice with atherosclerosis-prone strains such as the apolipoprotein e-null or low-density lipoprotein receptor-null mice. The atherosclerotic mice exhibit progressive accumulation of macrophages, SMCs, and lipid-laden foam cells in the subendothelial intimal space, with development of a necrotic core and fibrous cap closely resembling the human atherosclerotic lesion. Using the atherosclerotic mouse models will also allow for analysis of later plaque complications such as hemorrhage and rupture, which are important pathologic processes involving destabilization of the collagen matrix by MMP activity. Aneurysm formation is another long-term complication of atherosclerosis that involves degradation of the elastin and colTCM Vol. 12, No. 4, 2002
This research was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Ontario, and a Premier’s Research Excellence Award to M. B. C. F. was supported by a studentship from the Heart and Stroke Foundation of Ontario.
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The collagen matrix constitutes a major portion of the vascular extracellular matrix and imparts blood vessels with tensile strength and, even more important, modulates smooth muscle cell (SMC) responses via specific receptors and signaling pathways. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the receptors that mediate these processes. Better understanding of the pathways involved in cell matrix interactions promises to provide novel therapeutic targets and treatment strategies for the prevention of arterial occlusive diseases such as atherosclerosis and restenosis. (Trends Cardiovasc Med 2002;12:143–148). © 2002, Elsevier Science Inc.
The extracellular matrix (ECM) is a complex mixture of proteins and proteoglycans that provides a scaffold for cells and contributes to the mechanical strength of tissues. The fibrillar collagens are a major component of the vascular matrix, and the most abundant are types I and III collagen, which form an interconnected network of cross-banded fibers
Christopher D. Franco, Guangpei Hou, and Michelle P. Bendeck are at the Departments of Laboratory Medicine and Pathobiology and Medicine, University of Toronto, Ontario, Canada. * Address correspondence to: Michelle P. Bendeck, Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, Room 6217, Toronto, ON M5S 1A8 Canada. Tel.: (11) 416-946-7133; fax: (11) 416-9785959; e-mail: [email protected]. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
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along with smaller amounts of type VI and XVIII collagen associated with the fibers and the elastic lamellae in the blood vessel wall. Network-forming type IV collagen is a major component of the basement membrane that lies beneath the endothelium and surrounds medial smooth muscle cells (SMCs). The adventitia contains abundant type I and III collagen surrounding the resident fibroblasts (Raines 2000). Once seen as merely the building blocks of tissue architecture, recent research has suggested that the dynamic interactions between cells and matrix control many processes including proliferation, migration, and survival (Schwartz 2001). Furthermore, recent findings have revealed that matrix composition is altered during the progression of diseases such as atherosclerosis and restenosis, and these changes can have a profound effect on vessel structure and
cellular responses. This review is focused on the interactions of SMCs with the collagen matrix, how these interactions are involved in sensing the local environment, and the relationship to the pathogenesis of arterial occlusive diseases. • Integrin-Mediated Cell–Collagen Interactions The influence of the ECM is exerted on cells via cell-surface receptors. Integrins are a family of heterodimeric transmembrane glycoproteins composed of a and b subunits that link cells to the ECM. Integrins activate intracellular signaling pathways by recruiting cytoplasmic tyrosine kinases and adapter proteins to focal adhesion sites at the cell membrane (Schwartz 2001). Several members of the integrin family that are expressed in blood vessels bind to collagen, namely a1b1, a2b1, and avb3. a1b1 and a2b1 are the main fibrillar collagen receptors (Heino 2000). The avb3 integrin binds gelatin, the denatured form of type I collagen (Davis 1992). Although the a2b1 and a1b1 integrin receptors bind the same collagen ligands, they stimulate distinct signaling pathways and regulate different functions. This review will concentrate on studies of SMCs and fibroblasts, because these cells are present in the vessel wall and may contribute to constrictive vessel remodeling in atherosclerosis and restenosis (see below). The a2b1 integrin mediates collagen gel contraction by skin fibroblasts and SMCs (Langholz et al. 1995, Lee et al. 1995). This receptor also increases type I collagen and matrix metalloproteinase (MMP) production, via p38 kinase signaling, in skin fibroblasts cultured in three-dimensional collagen gels (Ravanti et al. 1999). In contrast, ligation of the a1b1 integrin in fibroblasts leads to a marked downregulation of type I collagen synthesis (Langholz et al. 1995), and a1-null fibroblasts from knockout mice lose the ability to downregulate
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collagen synthesis (Gardner et al. 1999). In osteosarcoma cells transfected with integrin receptors, a2b1 integrins compete with a1b1 for collagen ligand, thereby abrogating the negative effects of a1b1 on collagen synthesis; however, the physiologic significance of this response in other cell types is not known (Riikonen et al. 1995). Activation of the a1b1 receptor in fibroblasts also stimulates cell proliferation, signaling via the adapter protein Shc and activation of Ras and Erk (Pozzi et al. 1998). Studying the importance of these two collagen receptors in the vasculature is complicated by the fact that vascular SMCs in vivo express mostly a1b1 receptor with only negligible levels of a2b1, whereas cultured SMCs express only the a2b1 integrin and no a1b1 (Gotwals et al. 1996, Skinner et al. 1994). Given that the rate of matrix turnover in normal blood vessels is incredibly slow, it is most likely that expression of the a2b1 collagen receptor, which can mediate migration, collagen turnover, and gel contraction, occurs only during periods of active remodeling, such as in development and wound repair. The reciprocal effects of a1b1 and a2b1 integrin receptors suggest that there is a coordinated mechanism regulating collagen synthesis and degradation that can sense and effect changes in the composition of the matrix. • Novel Collagen Receptors: The Discoidin Domain Receptor Tyrosine Kinases A new class of collagen receptors was discovered recently—the discoidin domain receptor (DDR) tyrosine kinases. They are the first receptor tyrosine kinases identified that bind directly to the ECM (Shrivastava et al. 1997, Vogel et al. 1997). DDRs are characterized by an extracellular domain homologous to discoidin-1, a lectin in Dictyostelium discoideum that mediates intercellular adhesion. Discoidin domains are found in a variety of proteins that are secreted or associated with the cell surface, for example, blood coagulation factors V and VIII, the matrix-binding proteins Del-1 and aortic carboxypeptidase-like protein (ACLP), the neuropilin receptor (a receptor for VEGF165), and the trkA receptor (Vogel 1999). There are two distinct genes for DDR1 and DDR2. DDR1 binds collagens I
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through V and VIII, whereas DDR2 only responds to the fibrillar collagens types I and III (Hou et al. 2001, Shrivastava et al. 1997, Vogel 1999). DDR1 appears in several distinct splice variants (Alves et al. 2001) and is widely expressed during development and in adult tissues such as kidney, gut, brain, cornea and skin epithelium; and in corneal and dermal fibroblasts. The DDR1a splice variant is expressed by macrophages infiltrating tumors (Kamohara et al. 2001). DDR1 mRNA expression in monocytes is upregulated by tumor necrosis factor a, interleukin-1b, granulocyte macrophagecolony stimulating factor (GM-CSF), and lipopolysaccharide (LPS) (Kamohara et al. 2001), but very little is known about regulation in other cell types or tissues. The expression pattern of DDR2 is more restricted, but it is expressed in skeletal muscle, heart, blood vessels, and connective tissues (Vogel 1999). In contrast to the integrin receptors, most DDRs contain an active tyrosine kinase in their cytoplasmic domain; the receptor dimerizes after collagen binding, and induces transphosphorylation of the cytoplasmic domains (Curat et al. 2001). The extracellular discoidin domain is necessary and sufficient for collagen binding, whereas the entire extracellular domain is necessary for transmembrane signaling (Curat et al. 2001). Initial studies indicated that the kinetics of DDR phosphorylation were very slow, with maximal activation after 18 h of continuous exposure to collagen. Furthermore, because the receptors were not downregulated, the signal could persist for very long periods of time (Vogel et al. 1997). However, more recent studies have demonstrated that in a monocytic cell line transfected with either DDR1a or DDR1b, or in hepatic stellate cells expressing DDR2, the receptors were phosphorylated as early as 60 min after collagen stimulation, so in some cells activation may be much faster (Kamohara et al. 2001, Olaso et al. 2001a). Little is known about postreceptor signal transduction by the DDRs. It has been shown that the adapter proteins Shc (Vogel et al. 1997) and fibroblast growth factor (FGF) receptor substrate-2 (Foehr et al. 2000) bind to the juxtamembrane region of DDR1; however, the rasmitrogen-activated protein kinase pathway is not activated. DDR1 is activated in the presence of blocking antibodies to
the b1 integrin receptor, indicating that the DDRs can participate in integrinindependent signaling responses (Vogel et al. 2000). Repression of Wnt signaling impairs DDR1 phosphorylation and potentiates migration of mammary carcinoma cells on collagen, suggesting important but poorly defined links between these two signaling pathways (Jonsson and Andersson 2001). Prolonged stimulation of DDR2 has been associated with the upregulation of MMP-1 expression, suggesting an important role for this receptor in regulating collagen matrix degradation and reorganization (Vogel et al. 1997). The DDR1a isoform also plays a critical role in mediating monocyte migration through collagen gels, a process dependent on proteolytic activity (Kamohara et al. 2001). Two recent publications reported the effects of deletion of the DDR1 or DDR2 genes in mice. Targeted deletion of the DDR1 gene resulted in dwarfism and severe defects in placental implantation and mammary gland development (Vogel et al. 2001), whereas DDR2null mice also exhibited dwarfism, skeletal malformation, and delayed wound healing (Labrador et al. 2001). Bone and mammary morphogenesis and skin wound healing are critically dependent on the coordinated activity of MMPs, supporting the hypothesis that the control of MMP expression by the DDRs is of profound physiological significance. Other studies using soluble receptor domains as binding antagonists, or expressing kinase-dead dominant negative DDR1 receptors, suggest that the DDRs play important roles in the differentiation of neurons (Bhatt et al. 2000), myoblasts (Vogel et al. 2000), and mammary cells in vitro (Jonsson and Andersson 2001). • The Role of Collagens in the Pathogenesis of Arterial Occlusive Diseases Atherosclerosis and restenosis are characterized by a marked reduction in vessel lumen area due to encroachment by the atherosclerotic plaque or restenotic neointima, and by arterial shrinkage due to constrictive remodeling. The neointima is composed of SMCs that synthesize an abundant ECM, and of macrophages that accumulate lipids. Arterial lesions are rich in matrix, with collagen constituting up to 60% of the total TCM Vol. 12, No. 4, 2002
plaque protein (Rekhter 1999). Recent research suggests that the collagen matrix is not simply an inert scaffold, but that instead after injury there is a dynamic interplay involving matrix degradation and synthesis, with collagens regulating many cellular responses that contribute to arterial occlusion. Matrix-degrading proteinases are upregulated following vascular injury and modify the ECM, with dramatic consequences for SMCs. Degradation of collagen and gelatin by MMP-1, MMP-2, MMP-3, MMP-9, and MT1-MMP facilitates SMC migration after arterial injury by allowing the SMC to clear a path through the surrounding matrix (Bendeck et al. 1996a, Lijnen et al. 1999, Zempo et al. 1996). Collagenases cleave collagen at a single site within the triple helix, between Gly775 and Leu/Ile776, producing two distinct collagen fragments that are three fourths and one fourth the length of the collagen monomer, respectively (Welgus et al. 1982). Polyclonal antibodies raised against the neoepitopes derived after cleavage have allowed the immunohistochemical detection of collagen degradation in situ in the atherosclerotic plaque (Sukhova et al. 1999). As atherosclerosis progresses, local collagen degradation can also cause mechanical weakening of the atherosclerotic plaque and lead to plaque rupture (Rekhter et al. 2000). Therefore, the regulation of MMP expression and activity represents an important therapeutic target for atherosclerosis and restenosis. Proteolytic digestion of matrix within the vessel wall leads to the generation of degradation products that have quite distinct effects compared with the normal intact matrix molecules. Responses to denatured or degraded collagen are mediated at least in part by cryptic integrin binding sites within collagen, resulting in a switch of binding to the avb3 integrin (Davis 1992). When grown on polymerized type I collagen (mimicking intact type I collagen), SMCs are arrested in the G1 phase of the cell cycle owing to upregulation of p21, which suppresses cyclin-dependent kinase 2 and cyclin E kinase activities (Koyama et al. 1996). In contrast, cells plated on monomeric type I collagen (mimicking degraded collagen) proliferate rapidly (Koyama et al. 1996). Type I collagen degradation products stimulate the expression of tenascin C, a matrix glycoprotein itself important in TCM Vol. 12, No. 4, 2002
controlling SMC proliferation in remodeling arteries (Jones et al. 1999). Collagenase-degraded collagen fragments induce the rapid disassembly of focal adhesion complexes in SMCs, facilitating release from the matrix and cell migration (Carragher et al. 1999). Current work using DNA array screening suggests that many other genes are overexpressed in SMCs plated on monomeric collagen, including actin-binding proteins, signaling molecules, other matrix components, and genes of unknown function (Ichii et al. 2001), and this will provide the basis for much future study. New matrix molecules are synthesized after arterial injury, resulting in dramatic changes in the composition of the matrix in contact with SMCs. Type VIII collagen is upregulated in several animal models of arterial injury and atherosclerosis, coincident with SMC migration and co-localized with areas of lipid deposition and macrophage accumulation (Bendeck et al. 1996b, Plenz et al. 1999, Sibinga et al. 1997). Type VIII collagen serves as a chemotactic factor for SMCs and provides a matrix favorable for migration (cells adhere less strongly than to other matrix molecules). In addition, it stimulates SMC production of MMP-2 and MMP-9, which facilitates migration (Hou et al. 2000). In vitro, blocking the elaboration of collagen prevents SMC migration (Rocnik et al. 1998). It is tempting to speculate that the synthesis of new type VIII collagen is important in this regard; however, in the study cited, total collagen synthesis was blocked, precluding the identification of the particular collagen type(s) affected. Type I collagen also contributes to the regulation of SMC migration in vitro (Gotwals et al. 1996, Skinner et al. 1994). Furthermore, SMCs must be able to degrade collagen in order to move across or invade through collagen gels (Kandu et al. 2000, Li et al. 1999). Taken together, this suggests that continual modulation of collagen elaboration and degradation is necessary to permit SMC migration, implicating complex feedback mechanisms in the control of this process. This precise modulation and feedback is effected through the cellular collagen receptors, and the expression of b1 and b3 integrin receptors on SMCs is upregulated after vascular injury (Bendeck et al. 2000, Gotwals et al. 1996). Blocking the avb3 integrin receptor following arterial in-
jury dramatically attenuates SMC migration, MMP synthesis, and intimal thickening (Bendeck et al. 2000, Bendeck and Nakada 2001, Byzova et al. 1998). However, at present it is not possible to distinguish between the effects of denatured collagen versus other ligands such as osteopontin, vitronectin, and tenascin, which also bind the avb3 receptor. Finally, collagen deposition has been linked to constrictive remodeling in atherosclerosis and after balloon angioplasty (Pasterkamp et al. 2000). The underlying mechanisms are not completely understood, but probably involve the integrin-mediated contraction of collagen networks by intimal and/or medial SMCs and/or adventitial fibroblasts (Travis et al. 2001). Also, MMP activity may be important for breaking cell-matrix contacts, permitting reorganization of the ECM and adaptation to a decreased vessel diameter (Sierevogel et al. 2001). It is logical to postulate that de novo collagen synthesis and the maturation and crosslinking of collagen fibrils into a condensed network may also be involved, but studies on collagen synthesis and cross-linking in vascular remodeling have not yet been done. There is very little known about the roles of integrins or DDRs in mediating these remodeling processes in vivo. • The Role of DDR1 in Arterial Wound Repair In contrast to the integrins, very little is known about the expression or function of DDRs in the cardiovascular system. There are no gross abnormalities in cardiac or vascular structure in DDR1- or DDR2-null mice; however, many genes are upregulated and contribute to SMC phenotypic modulation only after arterial injury. One abstract has reported DDR1 and DDR2 expression in atherosclerotic lesions from nonhuman primates fed a highcholesterol diet (Carragher et al. 2000) Because DDR1 is a novel collagen receptor and is known to regulate MMP production, we investigated the expression and function of DDR1 in the vasculature before and after arterial injury (Hou et al. 2001). Using the well-characterized rat carotid balloon catheter injury model, we showed that there was minimal expression of DDR1 in the uninjured carotid, but DDR1 mRNA and protein expression were increased as early as 2 days after injury in the medial SMCs
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immediately adjacent to the lumen, with continued receptor expression in the neointima at 14 days and later (Figure 1). We hypothesized that this pattern of expression was linked to the process of neointimal formation that involves SMC proliferation, expression of MMPs to degrade the ECM, and migration from the media to the intima. Therefore, in vitro assays were used to mimic critical steps in migration and intimal formation, and aortic SMCs harvested from DDR1-null and wild-type mice were studied. DDR1null SMCs showed reduced attachment to, proliferation on, and chemotaxis towards collagen types I and VIII compared with wild-type SMCs (Hou et al. 2001). These experiments were the first to demonstrate a functional role for the DDR1 in mediating cell proliferation and migration. We also found that MMP-2 and MMP-9 activity were dramatically downregulated in DDR1-null SMCs, suggesting that the DDR1 plays an important role regulating baseline levels of cellular MMP activity; thus, it may impact upon cell migration through the
Figure 1. Discoidin domain receptor 1 (DDR1) immunostaining was increased after injury of the rat carotid artery. Control uninjured carotid artery (A). DDR1 was expressed by smooth muscle cells (SMCs) immediately adjacent to the lumen at 2 days after injury (B); by SMCs newly migrated onto the vessel surface at 4 days (C); and in SMCs of the thickened neointima at 14 days (D). Original magnification 3600. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 2.
Figure 2. The neointimal thickening response after vascular injury was decreased in the discoidin domain receptor 1 (DDR1)-null mouse. Area of the intima (A), and media (B) were measured on carotid cross-sections by morphometric digital image analysis. Values are mean 6 SEM. *Intimal area is significantly decreased in DDR1-null compared with control mice, P , .05. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 7.
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Figure 3. Intimal collagen deposition was reduced in the discoidin domain receptor 1 (DDR1)-null mouse. Picrosirius red staining and polarized light microscopy were used to assess the deposition of refringent collagen in the vessel wall. Original magnification 3400. Journal of Clinical Investigation by Michelle Bendeck. Copyright 2001 by Journal of Clinical Investigation. Reproduced with permission of Journal of Clinical Investigation in the format Journal via Copyright Clearance Center. See Figure 7.
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ECM (Hou et al. 2001). Subsequently, two studies were published showing that the DDR2 also plays an important role in mediating hepatic stellate cell (Olaso et al. 2001a), and fibroblast (Olaso et al. 2001b) migration and proliferation by MMP-2 dependant mechanisms. The in vitro results with SMCs led us to predict a critical impairment in neointimal formation after vascular injury, so we injured the carotid arteries of DDR1-null and wild-type mice, using a copper wire to remove the endothelium. We found that the neointimal area after vascular injury was reduced by 70% in the DDR1-null mice compared with the wild-type mice (Figure 2). Furthermore, accumulation of collagen in the intima was significantly inhibited in the DDR1null mice (Figure 3). The latter observation suggests that the DDR1 functions as a collagen sensor that is activated by various types of collagen and is an essential part of the cellular machinery that triggers ECM degradation and renewal. Thus, our results support the hypothesis that DDR1 plays an important role as a collagen receptor in arterial wound repair. In the absence of DDR1, decreased SMC proliferation, migration, MMP, and collagen synthesis result in the attenuation of intimal thickening.
lagen scaffold of the abdominal aorta, and the roles of the DDRs in this process have not yet been addressed. Finally, we are far from having a clear picture of DDR signaling pathways. The integration of these pathways with other receptors controlling cell adhesion, differentiation, and growth—particularly integrins, cadherins, and the Wnt pathway—is an important area for future study. • Conclusions Neointimal thickening and constrictive remodeling are major determinants of arterial occlusive disease. Research over the past decade has shown inarguably that changes in the structure and composition of the extracellular matrix play a very important role in activating SMC responses in injured vessels. The collagens and their cellular receptors, the DDRs, and the integrins are key mediators central to these processes of arterial repair. With rapidly accumulating knowledge of these mechanisms, targets for intervention and therapeutic strategies can be identified for the prevention of arterial occlusive diseases, and later complications such as plaque rupture and aneurysm formation. • Acknowledgments
• Future Studies Mechanical injury of the mouse carotid is a simple model that provides valuable information on the SMC responses after arterial injury; however, there is little inflammation and no lipid infiltration in this model. Future research will involve crossing the DDR1-null mice with atherosclerosis-prone strains such as the apolipoprotein e-null or low-density lipoprotein receptor-null mice. The atherosclerotic mice exhibit progressive accumulation of macrophages, SMCs, and lipid-laden foam cells in the subendothelial intimal space, with development of a necrotic core and fibrous cap closely resembling the human atherosclerotic lesion. Using the atherosclerotic mouse models will also allow for analysis of later plaque complications such as hemorrhage and rupture, which are important pathologic processes involving destabilization of the collagen matrix by MMP activity. Aneurysm formation is another long-term complication of atherosclerosis that involves degradation of the elastin and colTCM Vol. 12, No. 4, 2002
This research was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Ontario, and a Premier’s Research Excellence Award to M. B. C. F. was supported by a studentship from the Heart and Stroke Foundation of Ontario.
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