Atherosclerosis is a slow-developing disease of large and medium sized arteries, and is the premier cardiovascular disease that underlies myocardial and cerebral infarction, aneurysm, stroke and gangrene of the extremities. At least 17 million people die of atherosclerotic complications each year worldwide, with another 15 million surviving unstable events. Despite therapeutic advances such as drug-eluting stents and statins, which reduce cardiovascular events by around 25%, there is an urgent need for additional strategies to complement these treatments and further reduce morbidity and mortality. Inflammation plays a fundamental role in mediating all stages of atherogenesis. The innate immune response has long been implicated in atherogenesis, and activation of the complement system has been associated with all stages of disease. In particular, C5b-9 (membrane attack complex) has been detected in human plaques and may be pathogenic. Since C5b-9 is produced in plaques then the complement activation product 5a (C5a) must also be generated. However, very little is known about the role of C5a in atherogenesis. Indeed, elevated levels of serum C5a have been detected in patients with advanced atherosclerosis and recently the classical C5a receptor, CD88, has been detected on most of the cells found in human atherosclerotic plaques. To date, no studies examining specific C5a receptor antagonism in an animal model of atherosclerosis have been performed. This thesis explored the potential therapeutic benefits of inhibiting C5a, using the C5a receptor antagonist, PMX53, in the ApoE knockout (ApoE-/-) mouse model of atherosclerosis. In Chapter 2, expression of both receptors to C5a, CD88 and C5L2, in aortae of ApoE -/- mice was explored. CD88 and C5L2 mRNA expression was detected in the aorta of ApoE -/- mice at 3, 5, 9,12, 17 and 25 weeks of age. CD88 expression in ApoE -/- mice increased with time, and with macrophage accumulation within the plaque, as indicated by an increase in expression of the macrophage marker, F4/80. Expresssion of CD88 was significantly increased at 17 and 26 weeks of age, compared with age-matched wild-type controls. C5L2 was also expressed albeit at much lower levels compared with wild-type controls. Having established the presence of C5a receptors in ApoE -/- mice, in Chapter 3, the effects PMX53-treatment on ApoE -/- mice on a normal chow diet was examined. PMX53 treatment (3 mg/kg; tri-weekly s.c., plus ~1mg/kg/day p.o. for 20 weeks) resulted in a significant reduction in neointimal area and therefore the intima:media ratio in the brachiocephalic artery compared to untreated controls (P < 0.05; n = 6-8). PMX53 treatment also reduced collagen content and outward remodelling of the brachiocephalic artery. In Chapter 4, studies exploring the effects of PMX53-treatment in the more inflammatory environment created by a high fat (or Western-type diet) were explored. Male ApoE -/- mice were treated with PMX53 from 5 – 25 weeks of age (3 mg/kg; tri-weekly s.c., plus v ~1mg/kg/day p.o.). Mice were placed on a high fat diet from 10 weeks of age. While PMX53- treated did not affect neo-intimal area, it did result in a significant increase in cell density (P<0.01; n=12) and a significant reduction in buried caps (P < 0.05; n = 12) in the brachiocephalic artery compared with untreated animals. Interestingly, PMX53-treated mice also had significantly reduced total cholesterol compared with untreated controls (P < 0.05; n = 12). These results provide the first evidence for a role for C5a in plaque destabilisation and cholesterol metabolism. Finally, Chapter 5 described the expression of CD88 and C5L2 in cultured primary rat vascular SMC was explored. Expression of CD88 and C5L2 was detected by Western blot; immunocytochemical analysis demonstrated intracellular expression of both C5L2 and CD88. Conversely, radioligand binding experiments suggested the presence of ~25000 cell surface receptors with a high affinity to C5a (KD = 0.3 nM). After establishing the presence of receptors to C5a, experiments were conducted to determine whether C5a has any functional effects on these cells. C5a induced a moderate increase in TNF-α release after 4 hours of treatment (P < 0.05, n = 3), but did not affect SMC proliferation (n = 3). In summary, this study is the first to demonstrate the benefits of specifically inhibiting C5a in a mouse model of atherosclerosis. These findings suggest that C5a plays a role in atherogenesis in ApoE -/- mice and that the C5a receptor antagonist PMX53 may have therapeutic potential in human atherosclerotic disease.

<p>Atherosclerosis is a serious threat to public health and a major cause of morbidity and mortality. In this doctoral research, the feasibility of using whole-body magnetic resonance angiography (WBMRA) was studied as a principal aim both in patients and in an epidemiological setting. Secondary aims were to create a score for assessment of the degree of atherosclerosis with the use of WBMRA and to investigate the correlation between this score and various cardiovascular (CV) risk factors. </p><p>WBMRA was found feasible both in atherosclerotic patients and in an elderly population from the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS). All subjects except one completed the examination without any adverse events. A large proportion (93-99%) of the vessel segments could be evaluated and the results of a smaller comparison between WBMRA and conventional invasive x-ray angiography were reasonable regarding the assessed degree of maximum stenosis or occlusion. This indicates the safety and robustness of the WBMRA method.</p><p>Unsuspected significant vascular abnormalities were found in patients with atherosclerotic symptoms and significant vascular abnormalities were present in elderly subjects without any self-reported vascular disease. The prevalence rates of vascular abnormalities in the carotid, renal, and inflow and runoff arteries of the lower limbs were estimated in an elderly population. A total atherosclerotic score (TAS) reflecting the degree of luminal narrowing was created for the WBMRA method and was significantly related to Framingham risk score (FRS) and to the amount of abdominal visceral adipose tissue, interleukin-6, and leptin and was inversely significantly related to adiponectin. </p><p>Studies with outcome data of the PIVUS cohort are needed for further validation of the WBMRA method and to determine whether TAS can be used as an adjunct for CV risk assessment. Meanwhile, the correlation with FRS indicates that TAS could be of value for this purpose.</p>

Ph.D. / Biomedical Engineering / The cardiovascular disease atherosclerosis is directly linked to the functions of the endothelium, the monolayer of endothelial cells (ECs) that line the lumen of all blood vessels. EC functions are affected by fluid shear stress (FSS), the tangential force exerted by flowing blood. In vivo FSS is determined by vascular geometry with relatively straight vessels producing high, unidirectional FSS and vessel branch points and curvatures producing low, oscillatory FSS. While these distinct FSS conditions differentially regulate EC functions, they also dramatically affect EC shape and cytoskeletal structure. High and unidirectional FSS induces EC elongation and cytoskeletal alignment, while concurrently promoting EC functions that are atheroprotective. In contrast, low and oscillatory FSS induces cobblestone-shaped ECs with randomly oriented cytoskeletal features, while simultaneously promoting EC functions that create an athero-prone vascular environment. Whether these distinct EC shapes and cytoskeletal structures influence EC functions, independent of FSS, is largely unknown. The overall hypothesis of this study is that cell shape and cytoskeletal structure regulate EC functions through mechanisms that are independent of FSS. Due to advances in surface engineering in the field of micropatterning, EC shape can be controlled independent of external forces by creating spatially localized surface cues. In this research, lanes of protein were micropatterned on glass surfaces to induce EC elongated shape in the absence of FSS. In Aim 1, micropattern-elongated EC (MPEC) shape and cytoskeletal structure were fully characterized and determined to be comparable to FSS-elongated ECs. Thus, inducing EC elongation on micropatterned lanes provides a platform for studying the functional consequences of EC shape, independent of FSS. Using this model, the following important markers of EC functions related to atherosclerosis were evaluated to determine the influence of EC shape and cytoskeletal alignment: extracellular matrix deposition (Aim 2), inflammatory function(Aim 3), and thrombotic potential (Aim 4). The results indicate that EC-elongated shape and cytoskeletal alignment participate in promoting selected EC functions that are protective against atherosclerosis, independent of FSS. Since EC shape is governed by the cytoskeleton, this data suggests that the cytoskeleton plays an active role in the regulation of EC functions that promote cardiovascular health.

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.

Introduction : Linflammation intervient à tous les stades dans la pathogénie de l'athérosclérose. Initialement, la maladie est caractérisée par une dysfonction endothéliale favorisée par les facteurs de risque cardiovasculaires classiques, des forces hémodynamiques anormales, et des substances vasoactives. A la faveur de cette dysfonction, des processus inflammatoires participent à la formation de la plaque dathérosclérose (1).Les lipoprotéines de basse densité pénètrent dans l'intima artérielle et subissent des modifications chimiques oxydatives responsables de réactions inflammatoires secondaires via la transcription nucléaire du facteur B. Des monocytes circulants activés adhèrent ensuite à la surface endothéliale, la traversent et se différencient en macrophages (activation via l'expression de molécules d'adhésion à la surface endothéliale : VCAM-1 et ICAM-1, qui lient des ligands de la famille des intégrines présents sur la membrane des leucocytes ; sélectines E et P). Cette migration subendothéliale est favorisée par la présence de chémoattractants. Les macrophages se transforment en cellules spumeuses en phagocytant les LDL oxydées via des récepteurs "scavengers". Les lipides d'abord intracellulaires vont devenir extracellulaires et se regrouper pour former un amas appelé le cur lipidique. Des lymphocytes T activés sont aussi présents et interagissent avec les macrophages, entretenant une réaction inflammatoire chronique qui favorise la croissance de la plaque. La plaque adulte est composée d'une chape fibro-musculaire (cellules musculaires lisses, protéines de matrice extracellulaire) séparant le coeur lipidique de la lumière artérielle. Au début, la croissance de la plaque nimplique pas obligatoirement une réduction de la lumière, laugmentation du volume de la plaque dans la paroi étant compensée par une majoration de la taille du vaisseau (remodelage positif). Des cytokines pro-inflammatoires (TNF-, interleukines 1, ...) induisent l'expression de métalloprotéinases par les cellules de la plaque ce qui conduit à sa fragilisation. Dautres cytokines ont des propriétés anti-inflammatoires et l'activité des métalloprotéinases peut être neutralisée par des inhibiteurs spécifiques naturels (TIMP-1, TIMP-2). La stabilité de la plaque résulte donc déquilibres complexes encore imparfaitement compris. La rupture de la plaque est le point de départ de l'évolution thrombotique dont l'importance est variable mais il est vraisemblable que certaines érosions ou ruptures n'entraînent pas de thrombose, favorisant par contre la croissance de la plaque. Activation inflammatoire induite par l'angioplastie dans les syndromes coronariens stables et instables, influence des dommages myocardiques : La CRP ultrasensible (CRPus) possède un pouvoir prédictif de complications chez les patients atteints d'infarctus aigu du myocarde, d'angor stable ou instable. L'origine de cette inflammation est incertaine : réaction inflammatoire au sein même de la plaque ? embolisation a partir d'une plaque friable avec nécrose infra-clinique ? Nous avons donc étudié l'influence pronostique des marqueurs inflammatoires mesurés avant et après l'angioplastie (ACP) et leur relation avec les marqueurs spécifiques de dommage myocardique (troponines). 200 patients (39 à 85 ans) traités par ACP avec ou sans stent entre 01/97 et 05/99 furent étudiés et suivis afin de dépister la survenue de complications. 20% des patients présentaient avant lACP une CRP us > à 6 mg/l. Après ACP, les taux de CRP us > à 6 mg/l et à 12 mg/l étaient respectivement de 40,8 % et 26,4%. Avant lACP, tous les patients présentaient une valeur de troponine I normale alors qu'après lACP, 10,6 % avaient une troponine I > à 5µg/l. Durant le suivi moyen de 38 mois, nous avons recensé 52 événements cardiovasculaires majeurs : 9 décès, 12 infarctus, 5 pontages aorto-coronaire, 26 resténoses, 13 ACP sur une autre lésion que celle traitée initialement. En analyse multivariée, étaient prédictifs de complication : la troponine I après ACP, la CRP us après ACP, les antécédents d'hypertension artérielle et dACP préalable (p<0.05). Nous avons évalué une association entre entre la CRP us après ACP et l'élévation de troponine. Le test t de Student montrait une différence moyenne entre les taux de CRP us pré- et post-ACP de 3,75 mg/l chez les patients avec troponine < à 5µg/l après ACP et de 6,85 mg/l chez ceux avec troponine >5µg/l (p < 0,02). Dans cette étude, l'inflammation et lembolisation après ACP sont prédictives d'événements cardiovasculaires à moyen terme le. L'association entre la CRP us et la troponine après ACP suggère un lien entre ces 2 marqueurs, les patients présentant une inflammation accrue courant un risque supérieur dembolisations péri-procédurales, ce qui pourrait expliquer leur pronostic moins favorable (2). Impact pronostique à long terme de l'activation inflammatoire dans les syndromes coronariens stables Limportance pronostique de la CRP us dans l'angor stable n'est pas établie; la plupart des études préalables évaluaient le court terme or les événements plus tardifs reflètent plus la progression de l'athérosclérose qu'une complication de l'angioplastie à proprement parler. Nous avons donc analysé l'impact pronostique à long terme de la CRP us avant et après ACP et l'influence de l'amplitude de lélévation de ce marqueur sur le devenir à long terme des patients dilatés pour un angor stable. Entre 08/98 et 05/99, 89 patients traités pour un angor stable furent inclus prospectivement et suivis pendant 6 ans. La CRP us moyenne était de 3,35 mg/l avant ACP et de 8,69 mg/l après. La plupart des patients présentait une élévation de ce paramètre ( CRP moyen = 5,34 mg/l ; ≥ à 3 mg/l chez 43 patients). Nous n'avons pas retrouvé de corrélation entre une élévation de CRP > à 3 mg/l et une élévation de troponine T >0,01µg/l. Durant le suivi, 38 patients ont présenté une complication cardiovasculaire (3 décès, 6 infarctus, 6 pontages, 12 ACP sur une autre lésion que celle traitée initialement, 11 resténoses). On notait un taux supérieur d'événements chez les patients présentant une  CRP > à 3 mg/l à la fois à un an de suivi (18 vs 2,2 %; p < 0,001) et à 6,6 ans (39,3 vs 3,4 %; p < 0,001).En analyse multivariée, les paramètres prédictifs indépendants étaient un antécédent d'infarctus du myocarde préalable, une  troponine T > à 0,01µg/l et une  CRP > à 3 mg/l (p = 0, 004, p = 0,013 et p = 0,004 respectivement).L'élévation de CRP us > 3 mg/l après ACP présentait une valeur prédictive d'événements cardiovasculaires supérieure à celle de la CRP us, considérée isolément avant ou après ACP. Donc, bien que le niveau inflammatoire basal soit important, lindice prédictif de complication le plus puissant est l'augmentation de ce marqueur induit par langioplastie. Une élévation importante de CRP ultrasensible après ACP chez certains patients pourrait refléter l'hyperréactivité de leur système inflammatoire aux agressions vasculaires. Une telle hyperréactivité pourrait être un marqueur de progression de l'athérosclérose et/ou d'instabilité clinique future (3). Activation neutrophilique dans les syndromes coronariens instables Nous avons évalué l'existence dune activation des polynucléaires secondaires à une angioplastie avec pose simultanée dune endoprothèse (stenting direct) au niveau d'une lésion responsable d'un syndrome coronarien instable. Nous avons aussi mesuré, la libération de cytokines associée. L'activation des polynucléaires a été évaluée par la mesure des taux de myéloperoxydase (MPO), de lactoferrine (LCF) et d'élastase.Vingt patients en angor instable furent étudiés (19 hommes; âge moyen : 53,9 ± 12,1 ans). Ils furent séparés en 2 groupes : groupe A : 15 patients traités par stenting direct de la lésion responsable ; groupe B : 5 patients n'ayant pas subi de revascularisation. Des prélèvements biologiques étaient réalisés chez les patients après l'insertion de l'introducteur fémoral (T0), 30après lACP ou après la procédure diagnostique (T1), puis 30 minutes, 3, 6, 12 et 24 heures après (T2 à T6). Les deux groupes étaient comparables du point de vue clinique et angiographique. Dans le groupe contrôle, aucune variation ne fut observée pour les dosages de MPO et de LCF. Dans le groupe A, une augmentation plasmatique du niveau de MPO fut observée immédiatement après l'angioplastie (p = 0,0009 à T1 vs T0; p < 0,0001 à T2 vs T0; p = 0,008 pour T3 vs T0). Pour la LCF, l'élévation était similaire (p = 0,004 pour T1 vs T0 et pour T2 vs T0). Des différences significatives furent observées à T1, T2 et T3 entre les groupes A et B pour la MPO (p = 0,015; p = 0,005; p = 0,042 respectivement) et pour la LCF (p = 0,0001; p = 0,0005; p = 0,003 respectivement). Le test ANOVA démontrait une valeur de p < à 0,0001 pour la MPO et la LCF dans le groupe A. Pour l'élastase et le TNF-, nous n'avons pas observé de différence significative au sein des groupes et entre les groupes. Nous avons observé une élévation plasmatique de l'IL-6 dans les 2 groupes (test ANOVA : p= 0,0065 pour le groupe B et p< 0,0001 pour le groupe A). Pour la première fois, nous rapportons une élévation rapide et intense d'enzymes neutrophiliques spécifiques (MPO et LCF) après pose dun stent coronarien (4). Cette libération pourrait en partie être responsable de la libération secondaire de cytokines (IL-6, IL-12, IL-8, CRP us). Il reste à déterminer si ces phénomènes peuvent influencer le pronostic clinique ou représenter une cible thérapeutique. Activation neutrophilique dans les syndromes coronariens stables; comparaison avec les tableaux cliniques instables L'activation des polynucléaires neutrophiles est donc impliquée dans la physiopathologie des syndromes coronariens instables et est aussi observée après ACP. Les leucocytes activés relâchent de la MPO, un agent oxydant puissant dont l'activité entraîne la production de dérivés d'oxygène réactif. Cette enzyme est augmentée chez les patients présentant un syndrome coronarien aigu, plus particulièrement chez ceux qui vont présenter une évolution clinique défavorable. Actuellement, on ignore si la MPO intervient dans la rupture de plaque ou contribue directement aux lésions tissulaires. Elle favorise l'athérogenèse via la production de molécules pro-athérogènes (dérivés oxydés de l'oxyde nitrique par exemple) et en utilisant comme substrat l'oxyde nitrique qui lui est athéroprotecteur. Nous avons comparé l'expression des cytokines inflammatoires et l'activation leucocytaire induites par un direct stenting chez des patients en angor stable par rapport aux instables. Quinze patients (groupe A) en angor instable furent comparés à 11 patients (groupe B) en angor stable. Tous présentaient une seule lésion sévère cible, traitable par langioplastie et pose simultanée dun stent. Les échantillons sanguins furent prélevés comme décrit dans létude précédente. A l'exception d'un âge moyen supérieur dans le groupe B, tous les paramètres cliniques et biologiques étaient similaires dans les deux groupes. Dans le groupe A, une augmentation du niveau plasmatique de MPO fut observée immédiatement après la pose du stent avec une élévation parallèle de la LCF. Dans le groupe B, nous avons également observé une élévation de MPO et de LCF après l'intervention qui était néanmoins inférieure que dans le groupe A (ANOVA test : p < 0,001 à la fois pour la MPO et la LCF dans les deux groupes). Pour l'élastase, aucune différence significative ne fut observée entre les groupes. Les niveaux plasmatiques d'IL-8 et dIL-12 ne furent pas significativement affectés dans chacun des groupes mais leurs valeurs moyennes étaient supérieures chez les patients stables, bien que restant dans les limites de la normale (< 30pg/ml chez les patients normaux; n = 34). Nous avons également observé une élévation significative de lIL-6, 12 heures après l'intervention dans les 2 groupes. Le pic sérique de ces cytokines survenait néanmoins plus tard par rapport à la libération aiguë de MPO et de LCF. Cette observation souligne l'importante activation des neutrophiles après pose de stent, démontrant que la rupture mécanique d'une plaque d'athérosclérose par un stent, est rapidement suivie d'une libération denzymes spécifiques. Lactivation des polynucléaires neutrophiles est plus intense dans les syndromes coronariens aigus que dans les situations cliniques stables. Donc, parallèlement aux modifications des taux de MPO trans-cardiaque observées dans l'angor instable (reflétant l'inflammation généralisée de l'arbre coronaire), nous rapportons pour la première fois que le stenting coronarien a un effet direct sur lélévation de la MPO.(5) Conclusions : Nos travaux montre que : 1) Langioplastie coronaire est associée à une élévation des paramètres inflammatoires systémiques, limportance de ces manifestations inflammatoires possédant une signification pronostique. 2) Le niveau basal et lincrément secondaire des paramètres inflammatoires sont hautement prédictifs des évènements cardio-vasculaires futurs suggérant que la réactivité individuelle est déterminante. 3) Les neutrophiles par leurs enzymes spécifiques interviennent précocement dans la cascade inflammatoire induite. 4) Lintensité de la réponse est différente en fonction de la présentation clinique (Hyper-réactivité diffuse? Composition de la plaque?). Une meilleure compréhension de la pathogénie de linsuffisance coronarienne devrait permettre une meilleure stratification du risque impliquant une prise en charge thérapeutique maximalisée et le développement de stratégies thérapeutiques alternatives plus efficaces tant en prévention primaire que secondaire. Références : 1. Gach O, Piérard L, Legrand V. Inflammation and atherosclerosis. State of the Art in 2004-2005. Rev Med Liège 2005; 60: 235-241. 2. Gach O, Louis O, Martinez C, Legrand V. Impact of markers of myocardial damage and inflammation after PCI on late outcome. Heart 2002; 87. 3. Gach O, Legrand V, Biessaux Y, Chapelle JP, Vanbelle S, Pierard L. Long term prognostic significance of High-Sensitivity C-Reactive Protein before and after coronary angioplasty in patients with stable angina pectoris. Am J Cardiol, 2007, 99:31-35. 4. Gach O, Biemar C, Nys M , Deby-Dupont G, Chapelle JP, Deby C, Lamy M, Pierard LA, Legrand V. Early release of neutrophil markers of activation after direct stenting in patients with unstable angina. Coron Artery Dis 2005; 16: 59-65. 5. Gach O, Nys M, Deby-Dupont G, Chapelle JP, Lamy M, Piérard LA, Legrand V. Acute neutrophil activation in direct stenting: comparison of stable and unstable angina patients. Int J Cardiol. 2006; 112(1): 59-65.

Atherosclerosis is a serious threat to public health and a major cause of morbidity and mortality. In this doctoral research, the feasibility of using whole-body magnetic resonance angiography (WBMRA) was studied as a principal aim both in patients and in an epidemiological setting. Secondary aims were to create a score for assessment of the degree of atherosclerosis with the use of WBMRA and to investigate the correlation between this score and various cardiovascular (CV) risk factors. WBMRA was found feasible both in atherosclerotic patients and in an elderly population from the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS). All subjects except one completed the examination without any adverse events. A large proportion (93-99%) of the vessel segments could be evaluated and the results of a smaller comparison between WBMRA and conventional invasive x-ray angiography were reasonable regarding the assessed degree of maximum stenosis or occlusion. This indicates the safety and robustness of the WBMRA method. Unsuspected significant vascular abnormalities were found in patients with atherosclerotic symptoms and significant vascular abnormalities were present in elderly subjects without any self-reported vascular disease. The prevalence rates of vascular abnormalities in the carotid, renal, and inflow and runoff arteries of the lower limbs were estimated in an elderly population. A total atherosclerotic score (TAS) reflecting the degree of luminal narrowing was created for the WBMRA method and was significantly related to Framingham risk score (FRS) and to the amount of abdominal visceral adipose tissue, interleukin-6, and leptin and was inversely significantly related to adiponectin. Studies with outcome data of the PIVUS cohort are needed for further validation of the WBMRA method and to determine whether TAS can be used as an adjunct for CV risk assessment. Meanwhile, the correlation with FRS indicates that TAS could be of value for this purpose.

The overall goals of this project were to develop microstructurally based constitutive models to characterize the mechanical behavior of arteries and to investigate the effects of HIV proteins and antiretroviral drugs on the microstructure and mechanical behavior. To this end we created several constitutive models in aim 1 using a rule of mixtures approach, investigated the role of viral proteins in aim 2 through the use a transgenic mouse model, and studied the effects of the antiretroviral drug AZT administered to mice in aim 3. It is well known that the local mechanical environment which cells experience mediates growth and remodeling and that subsequent growth and remodeling can change that mechanical environment. This remodeling includes changes in the content and organization of the constituents of arteries (collagen, elastin, and smooth muscle cells). The first aim thus created models that incorporated the content and organization of these constituents using a rule-of-mixtures approach. The models we developed were able to capture the mechanical behavior of the arteries as well as previously developed phenomenological models while providing more physical meaning to the parameters, some which can be measured experimentally for incorporation into future models. Aims 2 and 3 investigated the mechanical and microstructural changes to murine arteries in response to HIV proteins or the drug AZT. While the development of antiretroviral therapy has greatly increased the life expectancy of patients with HIV, a number of other complications and co-morbidities including cardiovascular disease have become apparent. While clinical data has implicated both the virus and the antiretroviral drugs as playing roles, this work addressed the need of investigating these effects in a controlled manner. Specifically we used mouse models and focused on the two subclinical markers of increased intima-media thickness and arterial stiffening. Aim 2 used a transgenic mouse that expressed most of the human HIV proteins. We observed both intima-media thickening and arterial stiffening in alignment with clinical data. Other changes that also support a proatherogenic phenotype included decreased elastin content and changes in cathepsin activity. Aim 3 administered the antiretroviral drug AZT to healthy mice and we also observed the same subclinical markers of atherosclerosis including intima-media thickening and arterial stiffening as well as the other proatherogenic changes of decreased elastin and changes in cathepsin activity. Several other parameters including axial behavior, opening angles, collagen content, and collagen fiber angles were also quantified. These were important to fully characterize the vessel and may also be incorporated in the future into the constitutive models developed in aim1. In conclusion, in aim 1 we developed a microstructurally based constitutive model of arteries that effectively captures the mechanical behavior and includes parameters that have more physical meaning and some of which are experimentally tractable. Aims 2 and 3 both observed several subclinical markers of atherosclerosis in mice that express HIV proteins or were given AZT, providing a good model for future work and suggesting that both the HIV virus and antiretroviral drugs may play roles in the development of atherosclerosis in HIV.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

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.