L’anévrysme de l’aorte abdominale (AAA) est une maladie vasculaire consistant en une dilatationde l’aorte abdominale. Son caractère asymptomatique rend importante la recherche debiomarqueurs afin de faciliter la prise en charge des patients.Le but de mes travaux de thèse a été d’étudier spécifiquement les types cellulaires impliqués dansl’AAA.Après obtention d’une banque d’échantillons d’AAA, nous avons localisé ces types cellulaires etrepéré 3 zones d’intérêt : le thrombus intraluminal contenait des neutrophiles, lymphocytes T,mastocytes et macrophages M2. Les macrophages M1 étaient localisés dans l’adventice, avec leslymphocytes B et des mastocytes. La média contenait des cellules musculaires lisses (CML) dont lephénotype variait entre les états sain, anévrysmal et apoptotique.Ces résultats suggérant une implication différente des deux types de macrophages et uneévolution des CML au cours de l’AAA, nous avons isolé ces deux types cellulaires du tissu anévrysmalpar microdissection laser afin d’y étudier l’expression de biomarqueurs potentiels. Nous avons ainsimontré que l’augmentation plasmatique de peroxiredoxine-1 provient des macrophages M1 et quel’augmentation d’ADAMTS5 provient des CML anévrysmales allongées.Nous avons ensuite comparé les protéomes des macrophages M1 et M2 afin d’élucider leur rôledans l’AAA et d’identifier de nouveaux biomarqueurs. Cinq protéines différentielles ont étéidentifiées impliquées dans la phagocytose ou la réponse au stress.L’étude des différents types cellulaires impliqués dans l’AAA pourrait nous permettre unemeilleure compréhension de ses mécanismes et l’identification de biomarqueurs potentiels de cettepathologie. / Abdominal aortic aneurysm (AAA) is a vascular disease consisting in a dilatation of the abdominalaorta. As AAA is asymptomatic, it is important to identify new biomarkers in order to improve theearly care management of patients.The aim of this work was to study specifically the cell types involved in the development of AAA.After collection of an AAA samples bank, we located these cell types among the tissue andhighlighted 3 interesting areas: the intraluminal thrombus contained neutrophils, T lymphocytes,mast cells and M2 macrophages. M1 macrophages were located in the adventitia along with Blymphocytes and other mast cells. The media contained smooth muscle cells (SMC) with a phenotypevarying from a healthy state, to an aneurysmal one and to an apoptotic one.These results suggest a different role of the two subtypes of macrophages and an evolution ofSMCs during AAA. Thus, we chose to isolate these subtypes from the aneurysmal tissue with lasermicrodissection in order to study the proteins differentially expressed between them that could bepotential biomarkers. We showed that the plasmatic increase in peroxiredoxin-1 levels in AAApatients came from pro-inflammatory M1 macrophages and the increase in ADAMTS5 levels camefrom aneurysmal elongated SMCs.We then compared the whole proteome of M1 and M2 macrophages in order to elucidate theirrole in AAA and identify new biomarkers. Five differentially expressed proteins were identified,implicated in phagocytosis and stress response.The study of the different cell types involved in AAA could enable us a better understanding of itsmechanisms and allow the identification of new potential biomarkers for this pathology

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.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

Cells in the body respond to mechanical loads in ways that are crucial to normal and disease physiology. Understanding these processes is difficult due to the complex mechanical environment in vivo. In this research, we have developed several cell-stretching devices capable of subjecting cell cultures to non-uniform stretch distributions in order to investigate pathological responses of vascular smooth muscle cells to physiologic stretches. 10T1/2 cells were cyclically stretched with these devices for 24 hours upon silicone membranes, PDMS tubes, and within 3D PEGDA hydrogels. After stretching, altered cell behaviors were measured, including orientation, proliferation (quantified by BrdU incorporation), and gene expression (quantified by real-time, RT-PCR). Cells demonstrated marked changes in orientation, proliferation, and mRNA expression, which all varied with cellular location in the non-uniform environment. More specifically, increased orientation, increased proliferation, and more dramatically altered mRNA expression were found in regions of high, uniaxial stretch, relative to regions of low, near-equibiaxial stretch. These findings demonstrate the capabilities of graded stretch distributions to produce graded cell responses, indicating potentially localized smooth muscle cell behavior in a diseased artery. The novel devices employed herein will hopefully improve our understanding of these complicated cellular pathways, ultimately allowing for improved treatment or prevention of vascular disease.

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.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

During the development of atherosclerosis, contractile vascular smooth muscle cells (VSMCs) change to cells capable of migrating and proliferating to mediate repair, where the responses may be adaptive or mal-adaptive in effect. Cyclic adenosine monophosphate (cAMP)-elevating agents have been shown to inhibit migration of VSMC. cAMP activity within the cell is known to be ubiquitous and dynamic, requiring control through signal termination mechanisms for cellular homeostasis. Phosphodiesterase (PDE) enzymes are central to this critical regulatory process catalyzing the hydrolysis of cAMP. A great deal of insight into the role of PDEs in defining compartmentalization of cAMP signaling has arisen predominately from recent studies on the cAMP-specific PDE4 family. Compartmentalization of PDE4 is mediated by their unique N-terminal domains, which have been proposed to provide the “postcodes/zipcodes” for cellular localization. PDE4D isoforms vary widely, yet their conservation over evolutionary time suggests important non-redundant roles in distinct cellular processes. To study the potential role of individual PDE4D isoforms we seek to utilize the unique N-terminal targeting domains that are proposed to be responsible for their protein-protein interactions and site-directed localization. Herein, we report on the expression, targeting and localization of five “long” PDE4D isoforms and the impact on cell morphology of certain amino-terminal domains of individual PDE4D constructs expressing green fluorescent protein (NT-PDE4D/GFP) in human aortic smooth muscle cells (HASMCs). Through the development of engineered NT-PDE4D/GFP expression plasmids, we were able to study the cell biological impacts associated with the overexpression of individual PDE4D amino-terminal variants in HASMCs. We show that NT-PDE4D5/GFP and NT-PDE4D7/GFP expressing cells exhibited an elongated cell morphology, where this effect was much more marked in NT-PDE4D7/GFP expressing cells, exhibiting multiple leading edge structures and highly elongated “tails”. We identify a potential role for PDE4D7 targeting in the regulation of cell polarity and migration. Our results suggest the novel idea that PDE4D7, rather than the four other long PDE4D isoforms (PDE4D3, PDE4D5, PDE4D8, or PDE4D9), represents the dominant PDE4D variant involved in controlling cAMP-mediated effects on cell tail retraction dynamics. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2014-03-13 13:00:31.684 / Video I: Time-lapse video of GFP-expressing cell migration in HASMC. GFP expressing cells did not differ in cell migration or morphology compared to non-injected control cells. HASMCs were microinjected with GFP construct. Representative images of micoinjected GFP cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X) / Video II: Time-lapse video of NT-PDE4D7/GFP-expressing cell migration in HASMC. NT-PDE4D7/GFP expressing cells exhibit elongated tail and decrease in cell migration compared to non-injected control cells. HASMCs were microinjected with NT-PDE4D7/GFP construct. Particle tracking of NT-PDE4D7 cells showed cleaving and full detachment of elongated tail. Representative images of micoinjected NT-PDE4D7 cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X)

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.