Christopher Franco

Collagens in the progression and complications of atherosclerosis

Abstract 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.

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.

A nonantibiotic chemically modified tetracycline (CMT-3) inhibits intimal thickening.

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.

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.

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.

Discoidin Domain Receptor-1 Deficiency Attenuates Atherosclerotic Calcification and Smooth Muscle Cell-Mediated Mineralization

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 alpha 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.

Inward Remodeling of the Rabbit Aorta Is Blocked by the Matrix Metalloproteinase Inhibitor Doxycycline

Constrictive arterial remodeling accounts for a significant proportion of lumen loss in atherosclerotic progression and postangioplasty stenosis. Recent research suggests that constrictive remodeling is mediated by turnover of the extracellular matrix. We hypothesized that remodeling could be attenuated by treatment with the safe, effective matrix metalloproteinase (MMP) inhibitor doxycycline. Female rabbit abdominal aortas were denuded using a 4-Fr balloon embolectomy catheter, and reinjured 3 weeks later. Treatment with 30 mg/kg/day doxycycline was begun the day before the second injury. At 6 weeks after injury, lumen area measured 13.1 ± 1.2 mm2 in controls compared to 17.5 ± 1.6 mm2 in doxycycline-treated rabbits (p = 0.05). At 4 days after injury, MMP-2 activity was increased compared to uninjured controls, but doxycycline treatment reduced MMP-2 activity. Doxycycline treatment also inhibited fibrillar collagen deposition in the intima by 87% as detected by polarized light microscopy. Doxycycline was an effective inhibitor of constrictive arterial remodeling in the rabbit aorta. Treatment reduced MMP activity and attenuated the deposition of extracellular matrix particularly in the intima.

Targeted deletion of discoidin domain receptor 1 (Ddr1) decreases atherosclerosis, reduces inflammation and accelerates matrix accumulation in LDL receptor deficient mices

Background: Collagens are abundant within the atherosclerotic plaque where they contribute to lesion volume, mechanical stability, and influence cellular behaviour. The discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds multiple collagen subtypes, has been observed in atheromata of non-human primates, but its function during atherogenesis is unclear. Methods: To examine the role of DDR1 during atherosclerotic plaque development, we generated Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice and fed them an atherogenic diet for 12 or 24 weeks. Results: Targeted deletion of Ddr1 resulted in a 50-60% reduction in atherosclerotic lesion area in the descending aorta at both 12 and 24 weeks. Atherosclerotic plaques from Ddr1-/-;Ldlr-/- mice demonstrated a 49% decrease in the area occupied by macrophages at 12 weeks, however, plaque SMC content was unchanged. We also observed accelerated deposition of fibrillar collagen and elastin in Ddr1-/-;Ldlr-/- plaques with a 36% and 45% increase at 12 weeks, respectively. Ddr1-/-;Ldlr-/- mice also demonstrated an early reduction in situ gelatinolytic activity in lesions and gelatin zymography revealed reduced MMP-2 activity at 12 weeks. Finally, mRNA expression analysis of laser microdissected plaques demonstrated enhanced expression of type I collagen and elastin, and reduced collagenase expression at 12 weeks. Moreover, mRNA expression of both MCP-1 and VCAM-1 was reduced in Ddr1-/-;Ldlr-/- plaques suggesting a novel role for DDR1 in the regulation of vascular inflammation. Conclusion: Our data support a role for DDR1 as a positive regulator of atherosclerosis; capable of influencing both inflammation and matrix turnover early in plaque development, and when inhibited can result in a persistent reduction in atherosclerosis.