Atherosclerotic cardiovascular diseases (CVD) are the leading cause of death and often develop due to obesity-induced complications including hyperlipidemia, elevated blood pressure (BP), inflammation, and oxidative stress. Epidemiological, animal model, and cell culture studies indicate that citrus, and grapefruit specifically, exert cardiovascular health benefits, likely due to the high flavonoid content in citrus fruits. Grapefruit and/or isolated grapefruit flavonoids elicit cardiovascular benefits via improvements in lipid metabolism and endothelial reactivity, and by antioxidant and anti-inflammatory actions. The aim of this work was to determine the role of six-week daily consumption of grapefruit on weight, lipid, and BP control as well as inflammatory and oxidative stress markers in overweight/obese adults. Further, we sought to evaluate the acute, postprandial effects of grapefruit consumption on metabolic, inflammatory, and oxidative stress markers in response to a high fat, high calorie (HFHC) double meal challenge. Participants were randomized to either a grapefruit group (n=42) in which they consumed 1.5 grapefruit/day for six weeks or to a control condition (n=32). Ten participants who completed the feeding trial also participated in the postprandial study. On two test days participants consumed a HFHC meal for breakfast and again for lunch. A ruby red grapefruit was consumed with breakfast on the first test day. Blood samples were collected at baseline and for the subsequent eight hours on each day. In the feeding trial, grapefruit consumption resulted in reductions in waist circumference (p<0.001), systolic BP (p=0.03), total cholesterol (p=0.001), and LDL-cholesterol (p=0.021) compared to baseline values. F2-isoprostanes and hsCRP values were nonsignificantly lower in the grapefruit vs. control arm following the intervention (p=0.063 and p=0.073, respectively). In the postprandial evaluation, insulin concentrations were significantly higher 30 minutes (p=0.007) and 2 hours (p=0.025) post HFHC + grapefruit meal consumption vs. HFHC alone. HFHC + grapefruit intake resulted in lower IL-6 concentrations after two hours (p=0.017) and lower F2-isoprostanes after 5 hours (p=0.0125). These findings suggest that regular grapefruit consumption may reduce CVD risk by targeting many of the risk factors and pathogenic factors involved in endothelial dysfunction. However, this dietary change alone is unlikely to result in significant CVD risk reduction.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

Urotensin-II (U-II) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study, we hypothesized that selective overexpression of UT in an SMC-specific fashion would increase atherosclerotic lesion formation in a hypercholesterolemic mouse model. The objectives were to demonstrate the role of UT in this mouse model of atherosclerosis, and to elucidate some of the mechanism involved in the process. We used four strains of mice; wildtype (WT), UT+ (a transgenic strain expressing human UT driven by the alpha-SM22 promoter), ApoE knockout (ko), and UT+/ApoE ko. All animals were fed a high-fat diet for 12 weeks. Western blot analysis revealed a significant increase in UT expression in UT+ and ApoE ko mice (P&lt;0.05). Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT+/ApoE ko but not in UT + mice when compared to wild type mice (P&lt;0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT +, ApoE ko and UT+/ApoE ko compared to WT mice (P&lt;0.05). Oral administration of the UT receptor antagonist SB-657510A for 10 weeks in a group of ApoE ko mice fed a high fat diet resulted in a significant reduction of lesion (P&lt;0.001). Immunohistochemistry revealed the presence of strong expression of UT and U-II proteins in the atheroma of UT+, ApoE ko and UT+/ApoE ko mice, particularly in foam cells. SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P&lt;0.05). The present findings suggest that the use of UT receptor antagonists may reduce lesion formation through reduced foam cell formation and lipid uptake, demonstrating an important role for UT in the pathogenesis of atherosclerosis.

Atherosclerosis is a complex disease which develops silently over decades and can lead to acute myocardial infarction or stroke, the main cause of death worldwide. Apoliporotein E (apo E) is a glycoprotein known for its major role in lipid metabolism and its pro-atherogenic effects. Swine make a unique and viable research model as it shares most of the anatomic and physiologic characteristics with humans, notably for the the cardiovascular system. In addition, it is the only animal species, other than nonhuman primates, that develops atherosclerosis spontaneously. In this study we examined the feasibility for creating an apo E-deficient pig model of atherosclerosis using RNA interference (RNAi) and somatic cell nuclear transfer (SCNT). The knockdown efficiency was tested in porcine granulosa cells. It varied from 45% to 82% compared to control cells, as revealed by real-time PCR analysis. Accordingly, short hairpin RNA-expressing vectors were constructed and used to transfect porcine fetal fibroblast cells. Cell lines with stable chromosomal integration were established and used to produce embryos by SCNT. Development of SCNT embryos to the blastocyst stage (33%) was comparable to non-transgenic embryos. The integration of the shRNA into the genome of GFP-expressing embryos was revealed by PCR and gel electrophoresis. These findings indicate that porcine embryos harboring shRNA-specific to apo E created by SCNT may lead to the production of apo E-deficient pigs. These pigs would be a promising new animal model for advancing atherosclerosis research.

Coronary atherosclerosis is one of the leading cause of mortality in developed countries, and is increasingly diagnosed via X-ray computed tomography. Due to the large resulting volume of data, recent research has been directed towards developing automated methods of screening CT scans for coronary atherosclerosis. This task typically consists of lumen extraction, plaque detection, plaque quantification, and material discrimination. In this paper, we describe a novel set of techniques for accomplishing the first three steps, which aim to provide higher precision than previous efforts. We also discuss how such a high-precision detection and quantification system could be used to significantly improve on the state of the art in material discrimination. Our methods extract lumen for 71.2% of centreline points, detect plaque with a detection sensitivity of 67% on CTA reference data, and quantify plaque with a linear weighted kappa coefficient of 0.08.

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-22 19:24:04.594

The effect of waveform shape and extension of negative flowrate are studied at two Reynolds numbers (Re = 80, Re = 200) on stented channels by looking at the kinematics and dynamics of the flow. The waveforms are reconstructed by Fourier decomposition of a waveform corresponding to left anterior descending (LAD) of coronary artery. The stents are modeled by an immersed boundary method. Two stent geometries are created which are the idealizations of two clinical ones. The first geometry is an idealization of XIENCE V stent which is called Lambda stent and the second one is the idealization of Endeavor stent and is called X stent. The former has larger inter-strut spacing and smaller thickness than the latter one. The use of immersed boundary method for creating the stents is validated, and the spatial resolution requirements are determined. The shape of the waveform is changed by systematically filtering out the higher modes of Fourier decomposition and the negative flowrate extension is reduced by shifting the waveform to the positive flowrate. The presence of the stent causes vortical structures to be created between stent struts. These vortices are migrating to the centre of the channel and disappear. It is observed that the confined geometric feature of X stent and its larger thickness, leads to larger areas of flow recirculation which causes smaller wall-shear-stress parameters with respect to Lambda stent and more deviation of the flow from a healthy vessel. The importance of the convective terms of the Navier-Stokes equations was studied at the two Reynolds numbers for both stents. It is observed that at high Reynolds number (Re = 200), the convective terms play significant role throughout the waveform cycle while at low Reynolds number (Re = 80), the effect of convective terms become negligible during negative flowrate. Moreover the convective terms become more significant for flow in a channel with Lambda stent than X stent due to the specific shape and size of the stents. The kinematics of the flow corresponds to the study of vortex timing. It was found that this timing is mainly affected by the waveform and Reynolds number rather than the stent geometry. The time at which vortex creation occurs is coincident with the time at which wall shear stress changes its sign in an unstented channel. Therefore the analytical solution of unsteady channel flow can be used as a tool for analysing the kinematics of the flow, / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2012-06-07 17:41:52.501

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

An important part of repair processes activated by vascular injury is the recruitment of vascular smooth muscle cells (SMC) from the existing contractile coat. Phenotypic modulation of SMCs enables these cells to proliferate and migrate into the vessel intima. Despite its importance in vessel repair, this plasticity of SMCs can also promote both the pathogenesis of atherosclerosis as well as neointimal formation following revascularization- induced injury. Vascular growth factors are major contributors to the migratory and proliferative responses to injury. IGF-1 is one such growth factor that elicits a response via its receptor, the IGF-1R, a classical tyrosine kinase receptor. However, it has been suggested that the IGF-1R may also be coupled to a heterotrimeric G protein and can thus initiate cellular responses via this alternate pathway. The objective of this study was to investigate the structural aspects of IGR-1R coupling to a heterotrimeric G protein in SMCs, as well as the contribution of this pathway to the cellular responses. In a porcine primary SMC culture model, IGF-1R co-precipitated with both the α- and β-subunits of a G protein, with the latter demonstrating activation dependent precipitation. The specific Gα class activated by IGF-1R was Gαi, in a manner that was independent of the activity of the tyrosine kinase. Both Gαi1 and Gαi2 directly interacted with the receptor. Gβγ mediated the activation of MAPK and its inhibition was sufficient to attenuate both the proliferation and migration of SMCs in vitro. In contrast, the contribution of Gαi was related to regulation of protein translation and histone modification. The data supports the conclusion that IGF-1 regulates the phenotype of vascular SMCs at least partially via a non-classical G protein-coupled receptor. Investigation into the individual subunits of the G protein complex led to the elucidation of a model in which both components play an integral role in the IGF-1 response, independent of the receptor tyrosine kinase activity. In one case, an interplay of specific Gαi-subunits leads to modulation of the VSMC translational and transcriptional responses, while in the other, release of the Gβγ-subunit activated the MAPK response in a manner that significantly contributes to both the migration and proliferation of SMCs.

Atherogenesis is the leading cause of death in the developed world, and is putting considerable monetary pressure on health systems the world over. Although the risk factors are well understood, unfortunately, the initiation and development of this disease still remains relatively poorly understood, but it is becoming increasingly identifiable as a dysfunction of the endothelial cells that line the walls of arteries. The prevailing haemodynamic environment plays an important role in the focal nature of atherosclerosis to very specific regions of the human vasculature. Disturbed haemodynamics lead to very low wall shear stress, and inhibit the transport of important blood borne chemicals. The present study models, both computationally and mathematically, the transport and hydrolysis of important blood borne adneosine nucleotides in physiologically relevant arterial geometries. In depth analysis into the factors that affect the transport of these low diffusion coefficient species is undertaken. A mathematical model of the complex underlying endothelial cell dynamics is utilised to model production of key intracellular molecules that have been implicated into the complex initiation processes of atherosclerosis; hence regions of the vasculature can be identified as being 'hot spots' for atherogenesis. This model is linked into CFD software allowing for the assessment of how 3D low yields and mass transfer affect the underlying cell signalling. Three studies are undertaken to further understand nucleotide variations at the endothelium and to understand factors involved in determining the underlying cell dynamics. The major focus of the first two studies is geometric variations. This is primarily due to the plethora of evidence implicating the geometry of the human vasculature, hence the haemodynamics, as an influential factor in atherosclerosis initiation. The final model looks at a physiologically realistic geometry to provide a more realistic reproduction of the in vivo environment.