Guangpei Hou

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 receptors (DDRs) are a novel class of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinase (MMP) production, but little is known about their expression and function in the vasculature. We posited a critical role for the DDRs controlling smooth muscle cell migration and proliferation and thus repair following arterial injury. Smooth muscle cells were isolated from the aortas of mice with a targeted deletion of the DDR1 gene (DDR1-null) and studied in culture using models that mimic critical steps in neointimal thickening. Our studies suggest that DDR1 plays an important role in regulating attachment to collagen, chemotaxis, proliferation, and MMP production in smooth muscle cells. Following mechanical injury to the carotid arteries, cross-sectional area of the neointima was significantly lower in DDR1-null mice than in wild-type mice. There was also a significant decrease in collagen deposition in the injured arteries of the DDR1-null mice. Our results support the hypothesis that DDR1 plays an important role as a collagen receptor, mediating intimal thickening after vascular injury.

Tyrosine Kinase Activity of Discoidin Domain Receptor 1 Is Necessary for Smooth Muscle Cell Migration and Matrix Metalloproteinase Expression

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.

Discoidin Domain Receptor 1 (Ddr1) Deletion Decreases Atherosclerosis by Accelerating Matrix Accumulation and Reducing Inflammation in Low-Density Lipoprotein Receptor–Deficient Mice

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+/+;Ldlr−/− and Ddr1−/−;Ldlr−/− 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−/−;Ldlr−/− plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1+/+;Ldlr−/− plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1−/− smooth muscle cells in vitro, revealed increased mRNA levels for procollagen &agr;1(I) and &agr;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−/−;Ldlr−/− 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−/−;Ldlr−/− 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.

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.

Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) maintain the ability to modulate their phenotype in response to changing environmental stimuli. This phenotype modulation plays a critical role in the development of most vascular disease states. In these studies, stimulation of cultured vascular smooth muscle cells with platelet-derived growth factor resulted in marked induction of c-jun expression, which was attenuated by protein kinase Cδ and calcium/calmodulin-dependent protein kinase inhibition. Given that these signaling pathways have been shown to relieve the repressive effects of class II histone deacetylases (HDACs) on myocyte enhancer factor (MEF) 2 proteins, we ectopically expressed HDAC4 and observed repression of c-jun expression. Congruently, suppression of HDAC4 by RNA interference resulted in enhanced c-jun expression. Consistent with these findings, mutation of the MEF2 cis-element in the c-jun promoter resulted in promoter activation during quiescent conditions, suggesting that the MEF2 cis-element functions as a repressor in this context. Furthermore, we demonstrate that protein kinase A attenuates c-Jun expression by promoting the formation of a MEF2·HDAC4 repressor complex by inhibiting salt-inducible kinase 1. Finally, we document a physical interaction between c-Jun and myocardin, and we document that forced expression of c-Jun represses the ability of myocardin to activate smooth muscle gene expression. Thus, MEF2 and HDAC4 act to repress c-Jun expression in quiescent VSMCs, protein kinase A enhances this repression, and platelet-derived growth factor derepresses c-Jun expression through calcium/calmodulin-dependent protein kinases and novel protein kinase Cs. Regulation of this molecular “switch” on the c-jun promoter may thus prove critical for toggling between the activated and quiescent VSMC phenotypes.

Migration and Growth Are Attenuated in Vascular Smooth Muscle Cells With Type VIII Collagen-Null Alleles

Objective—Type VIII collagen is upregulated after vascular injury and in atherosclerosis. However, the role of type VIII collagen endogenously expressed by smooth muscle cells (SMCs) and in the context of the vascular matrix microenvironment, which is rich in type I collagen, is not known. To address this, we have compared aortic SMCs from wild-type (WT) mice to SMCs from type VIII collagen-deficient (KO) mice when plated on type I collagen. Methods and Results—Type VIII collagen was upregulated after wounding of WT SMCs. KO SMCs exhibited greater adhesion to type I collagen than WT SMCs (optical density [OD595]=0.458±0.044 versus 0.193±0.071). By contrast, the WT SMCs spread more (389±75% versus 108±14% increase in cell area), migrated further (total distance 80.6±6.2 &mgr;m versus 64.2±4.4 &mgr;m), and exhibited increased [3H]-thymidine uptake (160 000±22 300 versus 63 100±12 100 counts per minute) when compared with KO SMCs. Gelatin zymograms showed that WT SMCs expressed latent matrix metalloproteinase 2, whereas KO SMCs did not. Addition of exogenous type VIII collagen returned levels of KO SMC adhesion (OD595=0.316±0.038), migration (79.5±5.8 &mgr;m), and latent matrix metalloproteinase 2 expression to levels comparable to WT SMCs. Conclusions—This study suggests that SMCs can modify the matrix microenvironment by producing type VIII collagen, using it to overlay type I collagen, and generating a substrate favorable for migration.

Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

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 inxa0vivo. 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.

Increased Cell and Matrix Accumulation During Atherogenesis in Mice With Vessel Wall–Specific Deletion of Discoidin Domain Receptor 1

Rationale: Discoidin domain receptor (DDR)1 is a collagen receptor expressed on both smooth muscle cells (SMCs) and macrophages, where it plays important roles regulating cell and matrix accumulation during atherogenesis. Systemic deletion of DDR1 resulted in attenuated plaque growth but accelerated matrix accumulation in LDLR-deficient mice. Deletion of DDR1 solely on bone marrow–derived cells resulted in decreased macrophage accumulation and plaque growth but no change in matrix accumulation. Objective: These findings led us to hypothesize that accelerated matrix accumulation was attributable to the increased synthetic ability of Ddr1−/− resident vascular wall SMCs. Methods and Results: We used bone marrow transplantation to generate chimeric mice and investigate the role of SMC DDR1 during atherogenesis. Mice with deficiency of DDR1 in vessel wall–derived cells (Ddr1+/+→−/−) or control mice (Ddr1+/+→+/+) were fed an atherogenic diet for 12 weeks. We observed a 3.8-fold increase in the size of aortic sinus plaques in Ddr1+/+→−/− compared to Ddr1+/+→+/+ mice. This was attributed to pronounced accumulation of collagen, elastin, proteoglycans, and fibronectin and resulted in a thickened fibrous cap. The enhanced matrix accumulation decreased the proportion of plaque area occupied by cells but was associated with a shift in the cellular composition of the lesions toward increased numbers of vessel wall–derived SMCs compared to bone marrow–derived macrophages. In vitro studies confirmed that Ddr1−/− SMCs expressed more matrix, proliferated more, and migrated farther than Ddr1+/+ SMCs. Conclusions: DDR1 expression on resident vessel wall SMCs limits proliferation, migration and matrix accumulation during atherogenesis.

Collagens, Integrins, and the Discoidin Domain Receptors in Arterial Occlusive Disease

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.

PKA regulated assembly of a MEF2/HDAC4 repressor complex controls C-jun expression in vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) maintain the ability to modulate their phenotype in response to changing environmental stimuli. This phenotype modulation plays a critical role in the development of most vascular disease states. In these studies, stimulation of cultured vascular smooth muscle cells with platelet-derived growth factor resulted in marked induction of c-jun expression, which was attenuated by protein kinase Cδ and calcium/calmodulin-dependent protein kinase inhibition. Given that these signaling pathways have been shown to relieve the repressive effects of class II histone deacetylases (HDACs) on myocyte enhancer factor (MEF) 2 proteins, we ectopically expressed HDAC4 and observed repression of c-jun expression. Congruently, suppression of HDAC4 by RNA interference resulted in enhanced c-jun expression. Consistent with these findings, mutation of the MEF2 cis-element in the c-jun promoter resulted in promoter activation during quiescent conditions, suggesting that the MEF2 cis-element functions as a repressor in this context. Furthermore, we demonstrate that protein kinase A attenuates c-Jun expression by promoting the formation of a MEF2·HDAC4 repressor complex by inhibiting salt-inducible kinase 1. Finally, we document a physical interaction between c-Jun and myocardin, and we document that forced expression of c-Jun represses the ability of myocardin to activate smooth muscle gene expression. Thus, MEF2 and HDAC4 act to repress c-Jun expression in quiescent VSMCs, protein kinase A enhances this repression, and platelet-derived growth factor derepresses c-Jun expression through calcium/calmodulin-dependent protein kinases and novel protein kinase Cs. Regulation of this molecular “switch” on the c-jun promoter may thus prove critical for toggling between the activated and quiescent VSMC phenotypes.