▼ 

In atherosclerosis, the expression of discoidin domain receptor 1 (DDR1) promotes macrophage accumulation in the arterial vessel wall in hyper-cholesterolemic mice; however, the mechanism involved remains elusive. DDR1 has been hypothesized to enhance macrophage accumulation by mediating the phenotypic polarization of these leukocytes. We report here that DDR1-expressing and DDR1-null macrophages displayed incongruent cytokine profiles in the in vivo plaque. In vitro, DDR1 blunted the up-regulation of anti-inflammatory genes in peritoneal macrophages following IL-4 stimulation. Moreover, DDR1 correlated with a relative decrease in the mRNA expression of select MMPs and TIMP-2, suggestive of a reduced matrix remodeling efficiency that may contribute to accelerated plaque expansion. Our findings preliminarily demonstrate that DDR1 effects an overall pro-inflammatory status in macrophages by dampening their adaptability to anti-inflammatory stimuli. Further effort to determine how DDR1 fine-tunes macrophage polarization will be relevant to the engineering of effective treatment for atherosclerosis and other inflammatory diseases.

M.A.S.

Advisors/Committee Members: Rocheleau, Jonathan V., Bendeck, Michelle P., Biomedical Engineering.

▼ 

Introduction: Activation of Runt Related Transcription Factor 2 (RUNX2) is required for transdifferentiation of Vascular Smooth Muscle Cells (VSMCs) into a calcifying osteoblast-like phenotype. Our lab showed that deletion of Discoidin Domain Receptor 1 (Ddr1), decreased atherosclerotic vascular calcification in the Ldlr-/- mouse. Hypothesis: DDR1 regulates RUNX2 activity by affecting microtubule organization during VSMC mediated calcification. Results: Ddr1-/- VSMCs show reduced RUNX2 activity when compared to Ddr1+/+ VSMCs. Addition of the microtubule-destabilizing agent nocodazole inhibited both RUNX2 activity and nuclear localization in Ddr1+/+ VSMCs. Addition of the microtubule-stabilizing agent taxol rescued RUNX2 nuclear localization in Ddr1-/- VSMCs. Despite this, Taxol was unable to rescue RUNX2 activity as it eliminated activity in both genotypes. Conclusion: These findings indicate that under osteogenic conditions, Ddr1 deletion impedes the dynamic instability required for the maintenance of microtubule architecture. This prevents RUNX2 nuclear localization and transcriptional activation in VSMCs.

MAST

Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.

▼ N-cadherin mediates cell-cell contacts in vascular smooth muscle cells (VSMCs) and regulates VSMC behaviours. Discoidin domain receptor 1 (DDR1) is a collagen binding receptor also implicated in these processes. Previous studies showed that both N-cadherin and DDR1 are upregulated after vascular injury, but it is not known whether the two molecules are associated. The ability to establish proper N-cadherin junctions is important in regulating VSMC behaviour. In my studies, I found that N-cadherin could associate with DDR1, and that this association was increased during VSMC migration and wound closure. I found that N-cadherin was reduced in lipid rafts and mislocalized from cell-cell junctions in the absence of DDR1. Disruption of lipid rafts by cholesterol oxidase or methyl-β-cyclodextrin removed N-cadherin from lipid rafts of DDR1+/+ VSMCs and disrupted N-cadherin contacts, and these effects were reversed by cholesterol rescue that restored lipid rafts. Knockdown of DDR1 by siRNA resulted in mislocalized N-cadherin contacts from cell junctions, and transfection of DDR1-/- VSMCs with full-length DDR1 rescued the formation of N-cadherin junctions. The mislocalization of N-cadherin contacts in DDR1-/- VSMCs was also accompanied by disruption of the actin cytoskeleton. N-cadherin contacts in VSMCs undergo constant remodeling, especially during disease processes. Calcium switch to disrupt and restore contacts did not affect N-cadherin triton solubility in DDR1+/+ and DDR1-/- VSMCs or the association between N-cadherin and DDR1. While collagen stimulation of DDR1 increased the association between N-cadherin and DDR1 in cells with mature, well-established cell contacts, it did not affect N-cadherin localization, its triton solubility, or the association between N-cadherin and DDR1 during contact disruption, restoration, and wound closure. Rho GTPases are master regulators of the cytoskeleton and can mediate cytoskeletal processes downstream of cell-cell contacts. Using calcium switch and plating on either IgG or N-cadherin-Fc coated surfaces, active Rho GTPase pull-down assays showed a consistent decrease in Cdc42 and RhoA activity after N-cadherin contact establishment in DDR1+/+ SMCs. Overall, these data reveal that N-cadherin cell-cell contacts in VSMCs can be regulated through interaction with DDR1 and localization in lipid rafts, and that the establishment of N-cadherin contacts can suppress Rho GTPase activity. Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.

▼ 

Atherosclerosis is an inflammatory disease of the cardiovascular system. Discoidin domain receptor 1 is a receptor tyrosine kinase that binds collagens. Previous work in our lab has shown that deleting DDR1 in a mouse model results in attenuation of atherosclerosis, with fewer macrophages in the plaque. The aim of this study was to determine what changes in macrophage behaviour due to the lack of DDR1 was attenuating plaque development. In order to carry out experiments, primary mouse peritoneal macrophages were used. DDR1-deficient macrophages adhered significantly less to type IV collagen and fibronectin compared to DDR1-expressing cells. In addition, when plated on type IV collagen and fibronectin, DDR1-deficient macrophages produced decreased levels of MCP-1 protein, a cytokine known to be important in plaque development, particularly in leukocyte recruitment to plaque. These results suggest that DDR1 is an important mediator in macrophage adhesion to matrix and macrophage cytokine production

MAST

Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.

▼ 

Collagens play an integral role in the progression of atherosclerosis. Collagen synthesis and remodelling influence inflammation, smooth muscle cell (SMC) proliferation and migration, and properties such as plaque tensile strength and mechanical stability. Type VIII collagen is expressed at low levels in the arterial system however, it is dramatically up-regulated in atherosclerosis. The purpose of this thesis is to determine the role of type VIII collagen in the development of atherosclerosis. Our first study uses Col8+/+;Apoe-/- and Col8-/-;Apoe-/- mice on an atherogenic diet to illustrate that type VIII collagen plays a role in formation of the fibrous cap. Deletion of type VIII collagen resulted in reduced SMC and fibrillar type I collagen content and gelatinase activity. Reduced fibrous cap thickness and increased necrotic core size were observed in plaques from these mice. The second study shows that the advanced plaques from mice deficient in type VIII collagen have increased macrophage proliferation and accumulation. Increased features iii associated with plaque instability such as breaks in the elastic lamina and cleaved collagen content were also observed. Plaque stiffness was reduced in Col8-/-;Apoe-/- mice, suggesting that type VIII collagen plays a role in the mechanical integrity of the plaque. In the third study, we use a bone marrow transplantation model to generate chimeric mice with myeloid-specific deletion of type VIII collagen (Col8-/-ď  +/+). We show that plaques from Col8-/-ď  +/+ mice have reduced elastin content compared to control mice. Finally, preliminary work suggests a role for type VIII collagen in the expression of fibrillar collagens. Plaques from Col8-/-;Apoe-/- mice show reduction of type I and III collagen accumulation and a reduction in type III collagen expression is observed in SMCs from Col8-/- mice. In summary, the studies presented here suggest that type VIII collagen confers increased plaque stability by promoting SMC infiltration and fibrosis as well as reducing inflammation. This work underscores the

2017-11-30 00:00:00

Advisors/Committee Members: Bendeck, Michelle P, Laboratory Medicine and Pathobiology.

▼ 

Vascular endothelial cell loss initiates directional migration of medial smooth muscle cells into the arterial intima contributing to in-stent restenosis, atherosclerosis and coronary arterial by-pass graft failure. N-cadherin is a cell-cell adhesion molecule that mediates the interaction between vascular endothelial cells and the innermost smooth muscle cells to stabilize the arterial wall. Upon injury, I reasoned that relocalization of N-cadherin on the inner most smooth muscle cells to the posterior-lateral borders stimulates cell polarization to enable directional migration. Using an in vitro scratch-wound model to stimulate cell polarity and locally remove cell-cell contacts at one pole of smooth muscle cells, I found that N-cadherin localization provides signaling cues via a Cdc42/GSK pathway that promote polarized reorganization of the cytoskeleton and directional cell migration. I also found that N-cadherin was important to functions of lamellipodia at the anterior of migrating cells. In lamellipodia, actin polymerization drives protrusion of the leading edge and coincident, but more posterior, actin depolymerization results in retrograde flow of actin and associated plasma membrane structures. Using live cell imaging, I found that clusters of N-cadherin-GFP repeatedly accumulated at the leading edge specifically at the neck of large pinocytotic vesicles called macropinosomes that were internalized and transported away from the leading edge. This localization is consistent with a role for N-cadherin in closure and scission of vesicles during macropinocytosis. These are the first studies to examine polarity in migrating vascular smooth muscle cells, and advance our understanding concerning cell-cell adhesions in controlling directional cell migration. My results suggest that N-cadherin may serve as a viable target for the treatment of arterial stenosis that would limit smooth muscle cell migration and stabilize the arterial wall. Furthermore, I report on a novel localization and function of N-cadherin in the biogenesis of macropinosomes in the lamellipodia that contribute to cell protrusion.

PhD

Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.

▼ 

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

PhD

Advisors/Committee Members: Bendeck, Michelle, Medical Science.

▼ 

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

PhD

Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.

▼ 

During atherosclerosis and restenosis, there is an extensive amount of collagen synthesis and degradation. Changes in the types of collagen present can have profound effects on vascular smooth muscle cell (SMC) proliferation and migration. Type VIII collagen, which is normally present at low levels within the mature vascular system, is greatly increased during atherogenesis. The central theme of this thesis is to determine the role of type VIII collagen in the pathogenesis of atherosclerosis and restenosis. In the first study, we demonstrated the importance of type VIII collagen in SMC migration and proliferation. SMCs from type VIII collagen-deficient mice display increased adhesion and decreased spreading, migration, and proliferation compared to SMCs from wild-type mice. Treatment of SMCs from type VIII collagen-deficient mice with exogenous type VIII collagen can rescue the defects. In the second study, we determined that type VIII collagen exerts its effects through regulation of MMP-2 expression. Type VIII collagen-deficient SMCs have decreased levels of MMP-2 and are impaired in chemotaxis toward PDGF-BB and in their ability to contract thick collagen gels. We found that decreasing endogenous MMP-2 levels in normal SMCs or adding exogenous collagen to type VIII collagen-deficient SMCs is sufficient to recapitulate the type VIII collagen-deficient or wild-type SMC phenotype, respectively. In the third study, we investigated the contribution of type VIII collagen to intimal hyperplasia after mechanical injury in the mouse. We found that type VIII collagen-deficient mice display a 35% reduction in intimal hyperplasia and attenuated vessel remodeling after femoral artery wire injury, establishing a role for type VIII collagen in restenosis. The results of the work presented in this thesis demonstrate that production of type VIII collagen confers an SMC phenotype with a greater propencity for proliferation and migration. These effects are in part mediated through regulation of MMP-2 expression and activation. We conclude that the increases in type VIII collagen production that occur during atherosclerosis and restenosis contribute to the capacity of SMCs to alter the existing extracellular matrix in a manner which permits enhanced migration.

PhD

Advisors/Committee Members: Bendeck, Michelle, Laboratory Medicine and Pathobiology.