Dissertations / Theses on the topic 'Atherosclerotic calcification'

PET imaging, using the bone tracer Na18F, allows the non-invasive location of atherosclerotic plaques that are at risk of rupture. However, the spatial resolution of PET is only 4-5 mm, limiting the mechanistic information this technique can provide. In this thesis, the use of fluorescence and Raman imaging to elucidate the mechanism of micro-calcification within atherosclerotic plaques has been investigated. A number of fluorescent probes to detect fluoride and calcium have been synthesised. One of the fluoride probes has been shown to be selective for fluoride however, the concentration of fluoride required to activate the probe is order of magnitudes higher than the amount of Na18F used for PET imaging making it problematic to use for future studies. On the other hand, a calcium probe has been shown to: selectively bind to hydroxyapatite (HAP); permit visualisation and quantification of HAP in both vascular and bone cell models; and effectively stain cultured aortic sections and whole mouse aorta for OPT imaging. Building on these preliminary data, fluorescence imaging and immunohistochemistry (IHC) imaging of both healthy and atherosclerotic tissue that were previously subjected to PET imaging, were successfully carried out showing the ability of the probe to detect HAP in human vascular tissue. IHC staining for Osteoprotegerin (OPG) and Osteopontin (OPN), two bone proteins recently detected in vascular tissue, showed the co-localization of OPG with the probe. Conversely, the OPN was shown to localize in areas surrounding high OPG and probe signal. To determine the exact composition of vascular calcification, Raman spectroscopy was also used. It is believed that the biosynthetic pathway to HAP passes through a series of transitional states; each of these has different structural characteristics which can be studied using Raman spectroscopy. In particular, HAP has a strong characteristic Raman peak at 960 cm-1. An increase in HAP concentration has been detected by Raman in both calcified cell models and aortic sections. When human vascular tissue was analysed, an additional peak at 973 cm-1 was present suggesting the presence of whitlockite (WTK) in this tissue as well as HAP.

Introduction: Atherosclerosis is a chronic inflammatory disease characterized by the buildup of cholesterol, fat and other debris within arterial walls. Atherosclerotic lesions undergo a calcification process with similarities to bone remodeling. In mice, the type-2 cannabinoid receptor (CB2) is known to regulate bone remodeling processes and has also been shown to alter atherosclerotic lesion characteristics. However, the role of CB2 in lesion calcification is still unclear. CB2 modulates bone remodeling by affecting differentiation of osteogenic precursor cells; thus we hypothesize that CB2 alters lesion calcification by altering osteoblastogenesis and osteoclastogenesis of precursor cells of vascular origin. To test this hypothesis, we studied the role of CB2 receptor in mediating osteoclastogenesis and osteoblastogenesis from murine monocyte/macrophage and smooth muscle cell lines in vitro. Methods: RAW264.7 cells are a murine monocyte/macrophage cell line known to undergo osteoclastogenesis in response to receptor activator of nuclear factor kappa B ligand (RANKL). RAW264.7 cells were cultured in media containing RANKL and supplemented with either CB2 agonists or antagonists. Effects on RANKL-induced osteoclastogenesis were then evaluated by measuring the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP) activity and further verified by microscopic quantitation of multi-nucleate TRAP-positive osteoclasts. MOVAS-1 cells are a murine vascular aortic smooth muscle cell line known to differentiation into osteoblasts when cultured in osteogenic media. MOVAS-1 cells were cultured in osteogenic media supplemented with CB2 agonists or antagonists. Effects on osteoblastogenesis were evaluated by measuring marker enzyme activity. Alizarin red staining was performed to visualize and quantitate effects on calcium deposition. Results: RAW264.7 cells treated with Win55, 212-2, a nonselective CB agonist, or HU-308, a selective CB2 agonist, displayed a dose-dependent decrease in RANKL-induced TRAP activity. Co-administration of a CB2-selective antagonist (SR144528), but not a CB1-selective antagonist (AM251), blocked this effect. Visual quantitation of multinucleated TRAP-positive cells confirmed Win55,212-2 treatment reduced osteoclastogenesis in RANKL-treated RAW264.7 cells. Induction of osteoblastic differentiation of MOVAS-1 cells, as determined by ALP activity, was enhanced by supplementation with Win55, 212-2 or 2-archidonyl glycerol. Co-administration of SR144528, but not AM251, reduced the induction of ALP activity in MOVAS-1 cells by Win55,212-2 and 2-AG. Alizarin red staining revealed increased calcium deposition in cultures of MOVAS-1 cells treated with Win55,212-2 compared to those cultured in osteogenic medium without Win55,212-2. Conclusions: These results demonstrate that CB2 activation can affect osteogenic differentiation of vascular cells in vitro. These results support the hypothesis that CB2 signaling promotes lesion calcification by altering the balance of osteoclastic and osteoblastic differentiation of vascular precursors.

Background: Calcification of atherosclerotic plaques is associated with vulnerability to rupture and increased risk of myocardial infarction. The mechanism of plaque calcification is unclear, but has been shown to be a cell-mediated process involving complex signaling pathways affecting the osteogenic transcription factor Runt-related transcription factor 2 (Runx2). The type-2 cannabinoid receptor (CB2) modulates processes involved in bone remodeling and our prior studies determined that CB2 alters the composition of early lesions in hyperlipidemic Ldlr-/- mice; however, the function of CB2 in plaque calcification is unknown. Therefore, we tested the hypothesis that CB2 modulates plaque calcification by evaluating the effects of systemic CB2 gene deletion on lesion calcification and aortic expression of Runx2 in Ldlr-/- mice. Results: Groups (n≥8) of 8-week old CB2+/+Ldlr-/- (WT) and CB2-/- Ldlr-/- (CB2-/-) mice were fed a high fat diet (HFD) for up to 24 weeks. Standard blood plasma analysis showed no difference in HFD-induced hyperlipidemia between WT and CB2-/- mice. Aortic levels of endocannabinoids, anandamide and 2-archidonylglycerol, were significantly elevated after 12 weeks of HFD feeding as determined by LC-MS/MS. En face analysis revealed the extent of atherosclerosis in the aortic arch and thoracic aorta did not differ between WT and CB2-/- mice, but was ~1.9-fold greater in the abdominal aortas of CB2-/- mice (17.0±1.3% vs 9.0±1.3%, p=0.002). Calcification of aortic root lesions was ~2.3 fold greater in CB2-/- mice compared to WT mice (12.9±1.1% vs 5.6±1.2%, p=0.002) as revealed by von Kossa staining. Western blot analysis showed significantly increased expression of Runx2 in aortas of WT mice compared to CB2-/- after 20 weeks of HFD (2.55±0.25 fold, p Conclusion: Systemic CB2 deficiency enhances lesion calcification and is associated with altered aortic expression of Runx2. These results provide novel mechanistic insights into the function of CB2 signaling in the pathogenesis of atherosclerosis and vascular calcification that may lead to the development of therapies aimed at stabilizing calcified plaque.

The objectives of this thesis are: a) To examine racial/ethnic differences in coronary artery calcification (CAC) and CAD, between symptomatic South Asians and Caucasians, matched for age, gender and conventional cardiovascular risk factors, b) To assess, using a meta-analysis model, the natural history of and stability of measurements of coronary artery calcium scoring (CACs) based on data collected from two large published trials: St Francis and EBEAT, c) To investigate the prevalence of coronary artery calcification in individuals with CT evidence for AVC, mitral valve calcification (MAC) or of both of them (AVC+MAC), d) To assess any potential association between premature CAD (<55 years in first-degree male relatives and <65 years in first-degree female relatives) and CAC in a large cohort of asymptomatic individuals. We found that coronary artery calcification is more extensive and diffuse in symptomatic patients of South Asian ethnic origin as compared to Caucasians, despite similar conventional risk factors for CAD. This is more evident in those >50 years of age, suggesting potential genetic or other risk factors yet to be determined. The natural history of coronary artery calcification was overtime progression in the majority of subjects, irrespective of gender. The higher variability in RCA measurements could be related to the low baseline CACs or exaggerated movement of the right side atrioventricular ring, whereas those for LCA brances are influenced by the branch allocation of the CACs. Valve calcification is not isolated but involve also and the coronary arteries. The presence of calcification in the aortic valve or combined aortic and mitral valves predicted coronary artery calcification. Additionally patients in whom both valves have become calcified tend to have severe coronary artery calcification. And finally, there is no relationship between the prevalence and extent of coronary artery calcification and the presence of family history of coronary heart disease in asymptomatic individuals with none of the conventional risk factors for atherosclerosis.

Objective: In the aortic wall calcification occurs as a pattern o f micro and macrocalcification changing significantly aortic wall biomechanical properties. The aim o f the w ork is biom echanical properties studying of aortic wall in the conditions of macro and microcalcification.

Introduction Coronary artery disease (CAD) is the most common cause of death in Europe and North America and early detection of atherosclerosis is a clinical priority. Diagnosis of CAD remains conventional angiography, although recent technology has introduced non-invasive imaging of coronary arteries using computed tomographic coronary angiography (CTCA), which enables the detection and quantification of coronary artery calcification (CAC). CAC forms within the arterial wall and is usually found in or adjacent to atherosclerotic plaques and is consequently known as sub-clinical atherosclerosis.  The conventional cardiovascular (CV) risk factors used to quantify the estimated 10-year coronary event risk comprise dyslipidaemia, hypertension, diabetes mellitus, obesity, smoking and family history of CAD. Nevertheless, their relationship with significant (>50%) stenosis, their interaction with the CAC score and their predictive ability for CAC presence and extent has not been fully determined in symptomatic patients.   Methods   For Papers 1-4 we took patients from the Euro-CCAD cohort, an international study established in 2009 in Umeå, Sweden. The study data gave us the CAC score and the CV risk factor profile in 6309 patients, together with angiography results for a reduced cohort of 5515 patients. In Papers 1 and 2 we assessed the risk factors for significant stenosis, including CAC as a risk factor. Paper 2 carried out this analysis by geographical region: Europe vs USA and northern vs southern Europe. Paper 3 investigated the CV risk factors for CAC presence, stratified by age and gender, while Paper 4 assessed the CV risk factors for CAC extent, stratified by gender.  In paper 5 we carried out a systematic review and meta-analysis of all studies of the risk factor predictors of CAC presence, extent and progression in symptomatic patients. From a total of 884 studies, we identified 10 which fitted our inclusion criteria, providing us with a total of 15,769 symptomatic patients. All 10 were entered in the systematic review and 7 were also eligible for the meta-analysis.   Results Paper 1:           Among risk factors alone, the most powerful predictors of significant coronary stenosis were male gender followed by diabetes, smoking, hypercholesterolaemia, hypertension, family history of CAD and age; only obesity was not predictive. When including the log transformed CAC score as a risk factor, this proved the most powerful predictor of >50% stenosis, but hypercholesterolaemia and hypertension lost their predictive ability. The conventional risk factors alone were 70% accurate in predicting significant stenosis, the log transformed CAC score alone was 82% accurate but the combination was 84% accurate and improved both sensitivity and specificity.  Paper 2:           Despite some striking differences in profiles between Europe and the USA, the most important risk factors for >50% stenosis in both groups were male gender followed by diabetes. When the log CAC score was included as a risk factor, it became by far the most important predictor of >50% stenosis in both continents, followed by male gender. In the northern vs southern Europe comparison the result was similar, with the log CAC score being the most important predictor of >50% stenosis in both regions, followed by male gender.  Paper 3:           Independent predictors of CAC presence in males and females were age, dyslipidaemia, hypertension, diabetes and smoking, with the addition of family history of CAD in males; obesity was not predictive in either gender. The most important predictors of CAC presence in males were dyslipidaemia and diabetes, while among females the most important predictors of CAC presence were diabetes followed by smoking. When analysed by age groups, in both males and females aged <70 years, diabetes, hypertension and dyslipidaemia were predictive, with diabetes being the strongest; in females aged <70 years, smoking was also predictive. Among those aged ≥70 years, the results are completely different, with only dyslipidaemia being predictive in males but smoking and diabetes were predictive in females.  Paper 4:           In the total cohort, age, male gender, diabetes, obesity, family history of CAD and number of risk factors predicted an increasing CAC score, with the most important being male gender and diabetes. In males, hypertension and dyslipidaemia were also predictive, although diabetes was the most important predictor. Diabetes was similarly the most important risk factor in females, followed by age and number of risk factors. Among patients with CAC, hypertension, dyslipidaemia and diabetes predicted CAC extent in both males and females, with diabetes being the strongest predictor in males followed by dyslipidaemia, while diabetes was also the strongest predictor in females, followed by hypertension. Quantile regression confirmed the consistent predictive ability of diabetes.  Paper 5:           In the systematic review, age was strongly predictive of both CAC presence and extent but not of CAC progression. The results for CAC presence were overwhelmed by data from one study of almost 10,000 patients, which found that white ethnicity, diabetes, hypertension and obesity were predictive of CAC presence but not male gender, dyslipidaemia, family history or smoking. With respect to CAC extent, only male gender and hypertension were clearly predictive, while in the one study of CAC progression, only diabetes and hypertension were predictive. In the meta-analysis, hypertension followed by male gender, diabetes and age were predictive of CAC presence, while for CAC extent mild-moderate CAC was predicted by hypertension alone, whereas severe CAC was predicted by hypertension followed by diabetes.   Conclusion Our investigation of the Euro-CCAD cohort showed that the CAC score is far more predictive of significant stenosis than risk factors alone, followed by male gender and diabetes, and there was little benefit to risk factor assessment over and above the CAC score for >50% stenosis prediction. Regional variations made little difference to this result. Independent predictors of CAC presence were dyslipidaemia and diabetes in males and diabetes followed by smoking in females. The risk factor predictors alter at age 70. The most important risk factor predictors of CAC extent were male gender and diabetes; when analysed by gender, diabetes was the most important in both males and females. Our studies have consistently shown the strong predictive ability of male gender in the total cohort and diabetes in males and females and this is reflected in the meta-analysis, which also found hypertension to be independently predictive. Interestingly, dyslipidaemia does not appear to be a strong risk factor.

The presence of aortic calcification causes severe tissue overstretching. Neutrophils and macrophages promote experimental abdominal aortic aneurysm formation. Aim: to compare the number of neutrophils andmacrophages in the tissue of the atherosclerotic aorta with calcification and without it.

Les pathologies cardiovasculaires sont le plus souvent l’aboutissement des processus liés à l’athérosclérose. Elles représentent la première cause de morbi-mortalité dans le monde et leur incidence s’accroit avec le vieillissement de la population et l’expansion de facteurs de risques comme le diabète ou l’obésité. La sténose valvulaire aortique (SVA) est la valvulopathie la plus fréquente dans les pays développés présentant de nombreux points communs avec l’athérosclérose vasculaire. En plus des facteurs de risque, les lésions valvulaires et les lésions vasculaires partagent des similitudes dans les processus physiopathologiques impliqués comme l’inflammation, la fibrose, l’angiogenèse et la calcification. Ce dernier processus apparait dans les stades avancés des pathologies liées à l’athérosclérose et joue un rôle critique via son implication dans la stabilité de la plaque ou l’épaississement des cuspides valvulaires aortiques. Les macrophages, cellules issues de la différenciation des monocytes infiltrés, jouent un rôle prépondérant dans ces lésions via les phénotypes classiques (M1) et alternatifs (M2). Néanmoins cette dichotomie ne reflète pas complètement la variété de leur plasticité et les différents phénotypes induits notamment par le microenvironnement des monocytes/macrophages (zones riches en lipides, zones riches en fer ou zones riches en calcification). Dans la valve aortique, les cellules interstitielles de valve (VIC) forment la population cellulaire la plus présente au sein de la valve aortique. Ces cellules jouent un rôle déterminant dans le maintien du tissu valvulaire, mais également dans les processus de calcification menant à la SVA. Dans un premier temps, cette thèse a pour but d’étudier la capacité des macrophages à former des ostéoclastes, cellules responsables de la dégradation de la matrice osseuse, au sein des plaques d’athérosclérose. Dans un second temps, ce travail se focalisera sur les processus de calcification de la valve aortique via l’étude du rôle de la leptine dans les calcifications valvulaires (étude a priori) puis dans une étude transcriptomique sans a priori de VIC issues de valve sténosées et non-sténosées. Nos résultats sur les macrophages montrent ex vivo que les cellules en bordure des calcifications vasculaires sont des macrophages alternatifs de type M2. In vitro, ces cellules sont incapables de se différencier en ostéoclastes et de résorber une matrice osseuse. Pour l’étude de l’effet de la leptine sur les VIC, nous montrons que la leptine sérique est plus élevée chez des patients présentant une SVA, nous confirmons que la leptine et son récepteur sont exprimés au sein des valves aortiques et que la leptine favorise la différenciation ostéoblastique des VIC de manière dépendante des voies Akt et ERK. Enfin, l’étude transcriptomique a permis de mettre en évidence une nouvelle voie métabolique dérégulée dans les VIC. Cette enzyme est sous exprimée dans les VIC issues de valves pathologiques et dans les zones calcifiées des valves aortiques sténosées. Par ailleurs, le traitement des VIC par le produit de cette enzyme en milieu procalcifiant inhibe la calcification. Cette thèse met en avant de nouveaux indices sur les processus de calcification observés dans les plaques d’athérosclérose et les valves aortiques sténosées. Ces résultats décrivent l’impossibilité des M2 à former des ostéoclastes capables de résorber les calcifications. Il sera intéressant d’étudier le phénotype des macrophages en bordure des calcifications des valves aortiques sténosées. D’autre part, il sera intéressant d’étudier l’origine de la leptine dans la valve et son mécanisme d’action sur les VIC. Enfin, ce travail a mis à jour une nouvelle voie métabolique, impliquée dans le développement des calcifications valvulaires, qui pourrait constituer une voie thérapeutique innovante dans le traitement médicamenteux de la SVA
Cardiovascular diseases (CVD) are the most often outcome of atherosclerosis processes. CVD are the first leading cause of death rate with an increasing incidence due to ageing populations and expansion of risk factors such as diabetes mellitus or obesity. Aortic valve stenosis (AVS) is the most frequent valvulopathy in developed countries sharing common points with vascular atherosclerosis. More than only risk factors, valvular and vascular lesions share common pathophysiological processes implicated in the development of the disease such as inflammation, fibrosis, angiogenesis and calcification. This last process appears in late stages of atherosclerosis diseases and play critical roles via implication in plaque stability or thickening of the aortic valve. Macrophages are cells deriving from infiltrated monocytes, playing an important role in the inflammatory state of lesions via classical (M1) or alternative phenotypes (M2) phenotypes. Nevertheless, this dichotomy does not reflect completely the variety of their plasticity and different phenotypes induced by the microenvironment of monocytes/macrophages (lipid riche zone, iron riche zone or calcium rich zone). In the aortic valve, valvular interstitial cells (VIC) are the most prominent cell type found in the aortic valve. These cells play a major role not only in the valve tissue homeostasis but also in the calcification processes leading to AVS. In a first part, the aim of this thesis is to elucidate the ability of macrophages to differentiate into osteoclasts, cell type responsible for bone matrix remodeling, inside atherosclerosis plaques. In a second part, this work will focus on the calcification processes occurring in the aortic valve via the study of the role of leptin in valvular calcification (association study) and then in a transcriptomic analysis of VIC isolated from calcified versus non calcified aortic valves (genome-wide expression study). Our results about macrophages show that ex vivo cell surrounding vascular calcification are alternative M2 macrophages. In vitro, these cells are no able to differentiate into true osteoclasts nor to resorb calcium deposits. Concerning the role of leptin on VIC, the results show that serum leptin is higher in patients with AVS, leptin and its receptors are expressed in the aortic valves and leptin enhances the osteoblast différenciation of VIC in an Akt and ERK dependant manner. Finally, the transcriptomic analysis allowed to highlight a new pathway deregulated in VIC. This enzyme is underexpressed in VIC isolated from calcified aortic valves and in the calcified zonesAbstract4of stenosed aortic valves. Otherwise, treating VIC with the product of this enzyme in a procalcifying medium inhibits calcification processes.This thesis highlights new insights into the calcification processes occurring in atherosclerosis lesions and calcified aortic valves. These results describe that M2 macrophages cannot differentiate into osteoclasts and reverse calcification formation inside atherosclerosis plaques. In parallel, it would be interesting to study the macrophages phenotypes surrounding calcium deposits in stenosed aortic valves. Then, it will be interesting to decipher the origin of leptin and its precise mechanism of action on VIC. Finally this work points out a new metabolic pathway implicated in the development of valvular calcification which could be a medical treatment of SVA

Calcifications in carotid atheromas can be detected in a panoramic radiograph (PR) of the jaws. A carotid artery calcification (CAC) can indicate presence of significant (≥ 50%) carotid stenosis (SCS). The aim of this thesis was to (1) determine the prevalence of SCS and burden of atherosclerotic disease among patients revealing CACs in PRs, (2) determine the prevalence of CACs in PRs among patients with SCS, (3) analyze whether the amount of calcium and/or (4) the radiographic appearance of the CACs, can improve the positive predictive value (PPV) for SCS detection among patients with CACs in PRs. The thesis is based on four cross-sectional studies. Two patient groups were prospectively and consecutively studied. Group A represented a general adult patient population in dentistry examined with PR presenting incidental findings of CACs. These patients were examined with carotid ultrasound for presence or absence of SCS and their medical background regarding atherosclerotic related diseases and risk factors was reviewed. An age and gender matched reference group was included for comparisons. Group B comprised patients with ultrasound verified SCS, examined with PR prior to carotid endarterectomy. The PRs were analysed regarding presence of CACs. The extirpated plaques were collected and examined with cone-beam computed tomography (CBCT) to determine the amount of calcium. The radiographic appearance of CACs in PRs from Group A and B were evaluated for possible association with presence of SCS. In Group A, 8/117 (7%) of patients with CAC in PRs revealed SCS in the ultrasound examination, all were found in men (8/64 (12%)). Patients with CACs in PRs revealed a higher burden of atherosclerotic disease compared to participants in the reference group (p <0.001). In Group B, where all patients had SCS, 84% revealed CACs in PRs and 99% of the extirpated plaques revealed calcification. CACs with volumes varying between 1 and 509 mm3 were detected in the PRs. The variation in volume did not correlate to degree of carotid stenosis. The radiographic appearance that was most frequently seen in neck sides with SCS (65%) was also frequently found in neck sides without SCS (47%) and therefore the PPV did not improve compared to the PPV solely based on presence of CACs. CACs in PRs are more associated with SCS in men than in a general population and patients with CACs in PRs have a higher burden of atherosclerotic disease. The majority of patients with SCS show CACs in PRs and the majority of extirpated carotid plaques reveal calcification. The volume of CAC and specified radiographic appearance does not increase the PPV for SCS in patients with CACs in PRs. In conclusion patients with CACs in PRs, and without previous record of cardiovascular disease, should be advised to seek medical attention for screening of cardiovascular risk factors.
Bakgrund Inom ramen för specialist- och allmäntandvård utförs panoramaröntgen-undersökningar dagligen på såväl barn som vuxna. En panoramaröntgenbild (PB) är en översiktsbild som är specifikt anpassad till att återge området för tänder och käkar. Utöver det, avbildas även delar av halsen och som bifynd ibland förkalkningar belägna i området för halspulsådern (karotiskärlet). Dessa förkalkningar kallas för karotisförkalkningar och är ett tecken på åderförkalkning. Åderförkalkning består i huvudsak av en fettrik plackansamling i kärlväggen. Placket kan med tiden förkalkas till varierande grad. Det är dessa förkalkningar vi kan se i PB. När en åderförkalkning ökar i volym kan den utgöra en förträngning i kärlet. Då förträngningen av kärldiametern är ≥ 50% benämns åderförkalkningar belägna i karotiskärlet för ”signifikanta karotisstenoser” (SKS). Graden av förträngning bedöms som regel med ultraljudsundersökning av halskärlen. Bitar av SKS kan lossna varvid det bildas små blodproppar. Eftersom halspulsådern försörjer främre hjärnhalvan med blod så kan dessa bitar täppa till ett av hjärnans blodförsörjande kärl och leda till stroke (slaganfall). För att minska risken att drabbas av stroke kan man ibland operera bort SKS (karotisplacket). Syfte Syftet med denna avhandling var att ta reda på (1) hur många av de patienter som blir undersökta med PB inom tandvården som uppvisar karotisförkalkningar, hur stor andel som har SKS samt utreda om patienter med förkalkningar i PB i större utsträckning är drabbade av hjärtkärlsjukdomar/risk faktorer, (2) hur ofta utopererade karotisplack innehåller kalk och hur ofta patienter med känd SKS uppvisar karotisförkalkningar i PB, (3) huruvida förkalkningsmängden i utopererade karotisplack är korrelerad till förträngningsgrad, och (4) huruvida det finns något specifikt radiografiskt utseende på karotisförkalkningar i PB som kan användas för att identifiera en större andel patienter med SKS bland patienter som uppvisar karotisförkalkningar i PB, det vill säga minska risken för att skicka patienter utan SKS på ultraljudsundersökning. Material och metoder Materialet bestod av två huvudgrupper av patienter. Grupp A bestod av patienter undersökta inom tandvården med PB som uppvisat karotisförkalkningar. Alla dessa patienter undersöktes med ultraljud för att bedöma förekomst av SKS. Den medicinska journalen granskades avseende tidigare förekomst av åderförkalkningsrelaterade sjukdomar och risk faktorer. En köns- och åldersmatchad kontrollgrupp utan karotisförkalkningar i PB analyserades på motsvarande sätt för jämförelse. Grupp B bestod av patienter med känd SKS som före operativt avlägsnande av karotisplack undersöktes med PB. PB granskades avseende förekomst av karotisförkalkning och utopererade karotisplack avseende kalkinnehåll. Förkalkningsmängden i de utopererade karotisplacken korrelerades dels till möjlighet att identifiera karotisförkalkning i PB samt till förträngningsgraden i kärlen. Karotisförkalkningarnas utseende delades in i grupper för att utvärdera om vissa utseenden i större utsträckning kunde associeras till förekomst av SKS. Resultat I Grupp A uppvisade 8/117 (7%) patienter SKS, alla var män, 8/64 (12%). Patienter med karotisförkalkningar i PB hade oftare åderförkalkningsrelaterade sjukdomar och risk faktorer (p < 0,001). I Grupp B hade 84% av patienterna med SKS karotisförkalkning i PB. Bland de utopererade karotisplacken innehöll 99% förkalkningar och förkalkningsvolymen varierade från 1-509 mm3. Möjligheten att upptäcka karotisförkalkning i PB var oberoende av om förkalkningsvolymen var stor eller liten. Förkalkningsvolymen var heller inte korrelerad till hur stor förträngning av kärlet en SKS (≥ 50%) orsakat. Ett radiografiskt utseende på karotisförkalkningar i PB noterades i 65% av de halssidor som hade en SKS. Detta specifika radiografiska utseende återfanns dock även i 47% av halssidor utan SKS. Andelen falskt positiva patienter var således fortsatt hög. Slutsats Vi fann att 12% män med karotisförkalkningar i PB, undersökta i en generell population inom tandvården, uppvisar SKS. Patienter med karotisförkalkningar i PB uppvisar fler riskfaktorer och är oftare drabbade av hjärt-kärlsjukdomar än patienter utan karotisförkalkningar i PB. Majoriteten av patienter med SKS uppvisar karotisförkalkningar i PB och nära 100% av utopererade karotisplack innehåller kalk. Förkalkningsmängden påverkar inte möjligheten att upptäcka karotisförkalkning i PB. Förkalkningsmängd och specificerade radiografiska utseenden hos karotisförkalkningar i PB förutsäger inte SKS bättre än definitionen ”förkalkning ja eller nej”. Dessa parametrar kan således inte användas till att förfina urvalet bland patienter som uppvisar karotisförkalkning i PB mot högre andel patienter med SKS. Individer med karotisförkalkningar i PB bör uppmanas konsultera vården för undersökning av eventuella risk faktorer för hjärt-kärlsjukdom.

Atherosclerosis is a chronic inflammatory disorder of medium and large vessels. Immune signaling and dyslipidemia are two of several processes which influence lesion development in atherosclerosis. Cannabinoids, such as those found in marijuana, exert their effects through two cannabinoid receptors, CB1 and CB2. Recent studies using CB2 knockout mice and CB2-selective ligands have shed light on a protective role of CB2 in early stages of atherosclerosis. However, the role of CB2 in advanced stages of atherosclerosis remains unclear. To determine if CB2 plays a role in advanced atherosclerotic lesion composition and progression, we investigated the effects of systemic CB2 gene deletion on advanced atherogenesis in Ldlr-null mice fed an atherogenic high fat diet (HFD) for 20-24 weeks. CB2 deficiency did not significantly affect aortic root lesion area, however, CB2-/- mice had a significant increase (~1.9 fold) in the percentage of abdominal aorta surface occupied by lesion. CB2-/- mice also displayed increased lesional macrophage content (~2.3 fold) and an unstable phenotype characterized by significantly reduced smooth muscle cell/macrophage ratio and increased matrix metalloproteinase-9 activity and mineralization. These results suggest that although CB2 does not affect the size of atherosclerotic lesions, it does modulate the cellular and extracellular matrix composition and promotes a stable phenotype. CB2+/+ and CB2-/- mice were also subjected to treatments with either CB2-selective agonist, JWH-015, or antagonist, SR144528, over the last four weeks of a 24 week atherogenic diet to identify the effects of CB2 stimulation on calcification of advanced lesions. No change was observed in body weight or cholesterol in response to either treatment. SR144528 reduced triglycerides and mineralization of aortic root lesions in CB2+/+ mice only. Aortic Runx2 and osteopontin were increased in response to JWH-015 by a CB2-dependent mechanism. Administration of synthetic cannabinoids in an ex vivo organ culture of CB2+/+ aortas revealed increased vascular calcification in response to CB2 blockade and decreased vascular calcification in response to CB2 activation. All together, these results support a protective role for CB2 in late stages of atherosclerosis and suggests that drugs targeting CB2 may be beneficial in the treatment of advanced atherosclerosis by affecting osteogenic mechanisms implicated in the mineralization of lesions.

Les complications cardio-vasculaires sont fréquentes et souvent associées à une augmentation de la mortalité chez les patients en insuffisance rénale chronique (IRC). L’électrocoagulation du cortex d’un rein et la néphrectomie controlatérale chez les souris invalidées pour l’apolipoproteine E (apoE-/-) nous ont permis de développer un modèle caractérisé par une IRC modérée et stable pendant plusieurs semaines. Dans ce modèle, nous avons montré l’apparition plus rapide de lésions athéromateuses et de calcifications vasculaires de souris apoE-/- après réduction néphronique que chez les souris non urémiques. Dans deux études interventionnelles, nous avons utilisé ce modèle pour évaluer l’efficacité de l’antioxydant N-acétylcystéine et du sevelamer. Nous avons observé que les deux traitements diminuaient la progression de l’athéromateuse des souris apoE-/- urémiques par comparaison avec les souris apoE-/- urémiques témoins. De plus, le sevelamer retardait la progression des calcifications vasculaires chez la souris apoE-/- urémique. Ces effets étaient associés à une baisse de l’expression de la nitrotyrosine (marqueur du stress oxydant).

Background: Atherosclerosis is an important cause of stroke. Ultrasound offers the convenience of real-time and detailed assessment of carotid plaque features as well as arterial wall thickening and composition. Evaluation of these features is important for determining patients’ risk of suffering vascular events and also contributes to selecting the best treatment strategy. Methods: Using ultrasound data analysis we have determined plaque features in the bifurcation and internal carotid artery (ICA), including: surface plaque irregularities, calcification, echogenicity (grey scale median-GSM) and other textural plaque features (Juxtaluminal black area, entropy, coarseness). In addition, intima media thickens (IMT) and its grey scale median (IM-GSM) was measured in common carotid artery (CCA). Using Cone Beam CT (CBCT) we have quantified calcification volume of the carotid plaques extracted after carotid endarterectomy procedure. For the meta-analysis we have used comprehensive meta-analysis software version 3. Study I: We have included 39 patients and we compared carotid plaque features of the contralateral arteries with those located ipsilateral to symptomatic side and arteries of asymptomatic patients. Study II: The accuracy of US to detect atherosclerosis calcification was assessed against CBCT in 88 patients. Study III: Based on the previous vascular events in coronary, carotid and lower extremity arterial system, 87 patients were divided into three groups: asymptomatic, symptoms in one vascular system and symptoms in more that one vascular system. IMT, IM-GSM and plaque features were compared between groups. Study IV: We have meta-analyzed ten cohort prospective studies evaluating carotid plaque echogenicity for cerebrovascular symptoms prediction. Results: Study I. Plaques of the contralateral to symptomatic arteries had similar features to those in symptomatic and more vulnerable than asymptomatic arteries. Study II. Carotid ultrasound was accurate in detecting calcification volumes of ≥8mm3 with very high sensitivity but it was less accurate in detecting lower calcification volumes (<8mm3). Carotid calcification was not different between symptomatic and asymptomatic patients. Study III. Echogenicity of the intima-media complex (IM-GSM), but not its thickness (IMT), was significantly decreased with increasing number of arterial systems affected by atherosclerosis. IM-GSM was lower in patients with prior myocardial infarction and stroke. Study IV. Carotid plaque echogenicity evaluated by US could predict future cerebrovascular events in patients with asymptomatic, relative risk RR 2.72 (95% CI, 1.86 to 3.96), and recurrent symptoms in symptomatic patients, RR 2.97 (95% CI, 1.85-4.78). Conclusion: Plaques located in the contralateral to symptomatic arteries have similar features as symptomatic side and more vulnerable than asymptomatic arteries. Carotid ultrasound could accurately detect larger but not smaller carotid plaque calcification volumes (<8 mm3). Low IM- GSM could identify patients with multi-system atherosclerosis disease, suggesting a better marker for determining systemic atherosclerosis disease burden compared to conventional IMT. Finally, carotid plaque echogenicity predicts future cerebrovascular events in patients with symptomatic and asymptomatic carotid stenosis.

Vascular calcification is strongly correlated with the clinical manifestations of atherosclerosis, heart attacks and strokes. The calcification process resembles bone formation and involves the osteogenic trans-differentiation of smooth muscles cells within the arterial wall. Cannabinoid receptors are known to modulate bone formation and are present in atherosclerotic vessels, suggesting they may also play a role in modulating calcification. Therefore, we evaluated the effects of cannabinoids on the expression of osteogenic proteins by vascular smooth muscle cells undergoing calcification.

L’athérosclérose est l’une des premières causes des maladies cardiovasculaires. Le développement de complications de plaque telles que la calcification et l’angiogénèse intra-plaque influence grandement la stabilité de la plaque et donc le pronostic vital du patient. Certains facteurs de risque sont connus pour expliquer le développement de ces complications comme le diabète ainsi que la maladie rénale chronique. Cependant, tous les mécanismes impliqués ne sont à ce jour pas élucidés. Il est donc essentiel de comprendre quels sont les mécanismes moléculaires mis en jeu dans la progression de ces lésions. Dans une étude préliminaire et dans la littérature, il a été montré que la LRG1 (leucine-rich α-2 glycoprotéine 1) s’accumulait dans le plasma de patients atteints de diabète de type II mais également dans un contexte de maladie rénale chronique. En premier lieu, nous avons démontré une élévation plasmatique de la LRG1 dans un modèle d’athérosclérose sévère chez la souris (souris ApoE KO nourries avec un régime riche en gras). Nous avons observé la présence de la LRG1 au sein même des plaques d’athérome compliquées et qu’elle se localisait préférentiellement au niveau des régions calcifiées à la fois dans le modèle murin mais aussi dans des plaques carotidiennes humaines. Nous avons montré in vitro que l’expression de la LRG1 était augmentée dans des cellules endothéliales à la suite d’une stimulation pro-inflammatoire avec du TNF-α. De manière intéressante, il a été décrit que la LRG1 pouvait se fixer à un récepteur accessoire de la famille du TGF-β. Sachant que cette voie est fortement impliquée dans la modulation de la calcification dans les cellules musculaires lisses vasculaires (CMLV), nous avons donc étudié le rôle de la LRG1 dans ces cellules. Nous avons identifié que la LRG1 était capable d’induire la calcification des CMLV in vitro en augmentant le contenu calcique et en modulant l’expression de gènes impliqués dans la transition ostéochondrogénique. Pour conclure, nos résultats montrent pour la première fois le rôle de la LRG1 dans la calcification vasculaire ce qui suggère un rôle de cette protéine dans le développement de complications de plaque d’athérome
Atherosclerosis is the primary cause of cardiovascular disease. The development of plaque complications such as calcification and neo-angiogenesis strongly impacts plaque stability and is a good predictor of mortality in patients with atherosclerosis. Despite some known risk factors of plaque complications like diabetes mellitus and chronic kidney disease, the mechanisms involved are still not fully understood. We and others have identified that the concentration of circulating leucine-rich α-2 glycoprotein 1 (LRG1) was increased in diabetic and chronic kidney disease patients. Using Apolipoprotein E knockout (ApoE KO) mice fed with high fat diet, we showed that plasma LRG1 was also increased in a mouse model of atherosclerosis. We also observed that LRG1 was present in complicated calcified plaque in vivo and that LRG1 was preferentially localised in calcification areas in mice. This result was also confirmed in human carotid endarterectomy specimens. In vitro we showed that LRG1 expression was enhanced in endothelial cells by inflammatory mediators such as Tumor necrosis factor-alpha (TNF-α). Interestingly, LRG1 is known to bind a co-receptor of the Transforming growth factor beta (TGF-β) family, a pathway largely described to control the calcification process in vascular smooth muscle cells (VSMC). Accordingly, we researched the role of LRG1 on VSMC calcification and we identified that LRG1 was able to induce VSMC calcification in vitro by increasing calcium content and the expression of osteoblastic markers. In conclusion, our results identified for the first time a role of LRG1 on vascular calcification and suggest a role of this molecule in the development of plaque complications in patients with atherosclerosis

Les maladies cardiovasculaires sont la première cause de mortalité dans le monde. Une meilleure compréhension des mécanismes physiopathologiques est nécessaire. Dans ce travail de thèse nous nous sommes intéressés à deux pathologies cardiovasculaires : l’athérosclérose et l’insuffisance cardiaque. Récemment, nous avons identifié le récepteur LRP1 et la protéine adaptatrice ShcA comme étant deux protéines impliquées dans deux de ces pathologies cardiovasculaires. Nousavons montré que ShcA joue un rôle protecteur dans l’insuffisance cardiaque. Chez les souris déficientes en ShcA au niveau cardiaque, nous observons une cardiomyopathie caractérisée par une dilatation du ventricule gauche associée à une perte de la contractilité. Nous avons montré que ShcA est essentiel à l’organisation des sarcomères et ceci très tôt durant l’embryogenèse. Dans une deuxième partie nous avons montré qu’en l’absence de PPARgamma, LRP1 était nécessaire à la calcification vasculaire en activant la voie prochondrogénique de Wnt5a. Nous avons montré que PPARgamma protège de la calcification vasculaire en induisant l’expression de Sfrp2 qui agit comme un antagoniste de Wnt5a
Cardiovascular disease is the number one cause of death worldwide. A better understanding of the pathophysiological mechanisms is necessary. In this thesis we are focused on two cardiovascular diseases: atherosclerosis and heart failure. Recently, we identified the LRP1 receptor and the adapter protein ShcA as two proteins involved in two of these cardiovascular diseases. We have shown that ShcA exerts a protective role against heart failure. Mutant mice lacking ShcA in the heart exhibit a dilated cardiomyopathy with reduced cardiac contractility. Myocyte ultrastructure analysis shows that Shc A is essential to maintain sarcomeric intégrity in early embryonic heart development. in last part we have shown vascular calcification in the absence of PPARgamma requires expression of LRP1 in vascular smooth muscle cells. LRP1 promotes a Wnt5a-dependent prochondrogenic pathway. We show that PPARgamma protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2 (Sfrp2, wich functions as a Wnt5a antagonist

L'athérosclérose consiste en l'accumulation de lipoprotéines de faible densité (LDL) dans la paroi vasculaire qui provoque l'infiltration de leucocytes, principalement des monocytes. Ces cellules se différencient alors en macrophages pour éliminer les LDL infiltrées en favorisant le transport inverse du cholestérol vers les lipoprotéines de haute densité.Cependant, lorsque l'efflux de cholestérol devient inefficace, les macrophages s’engorgent delipides, forment des cellules spumeuses qui meurent par apoptose ou nécrose. Ils déversent alorsleur contenu lipidique dans la matrice extracellulaire, formant ainsi le coeur nécrotique. Descristaux de cholestérol peuvent se former dans l’intima et sont phagocytés par les macrophages.Ils provoquent alors des dommages lysosomiaux qui activent le complexe inflammasomeNLRP3. Ce complexe entraîne la maturation de la caspase 1 qui clive les pro-cytokines en IL1Bet IL18 actives. Ces interleukines sont alors libérées et stimulent la sécrétion d'interféron γ(IFNγ) par les lymphocytes T CD4+, provoquant la prolifération et la migration des CellulesMusculaires Lisses (CML) vers le coeur nécrotique pour former la chape fibreuse. En plus desnombreux facteurs de risque des maladies cardiovasculaires comme le diabète ou le tabagisme,la perturbation des rythmes nycthéméraux comme chez les travailleurs postés, augmentel’incidence de ces maladies. Le récepteur nucléaire Rev-erb-α est un composant central del’horloge moléculaire. Il est en outre un régulateur important du métabolisme des lipides et dela réponse inflammatoire. Il représente donc une cible thérapeutique de choix dans le traitementde ces maladies multifactorielles liées à un dérèglement de l’horloge. Mes travaux montrentque l’absence de Rev-erbα dans un modèle murin pro-athérogène LDLr-/- entraînel’augmentation des taux plasmatiques de lipides et accélère le processus d'athérogenèse. Deplus, Rev-erb-α régule l'expression circadienne des composants de la voie NLRP3 et lasécrétion d'IL1B et IL18 dans les macrophages. Au niveau moléculaire, Rev-erb-α réprimedirectement la voie NLRP3, tandis que la déficience en Rev-erbα augmente l'activation de cettevoie. Ce mécanisme régulerait la susceptibilité aux stimuli inflammatoires dans des modèlesd'inflammation aiguë comme la péritonite ou l'hépatite fulminante en fonction du moment dela journée auquel les souris ont été stimulées. L'absence de Rev-erbα induit égalementl’expression de Nlrp3, Il1β et Il18 dans des modèles d'inflammation chronique commel'athérosclérose ainsi que l’expression de NLRP3 dans les macrophages des lésions d'athérome.De plus, IL1B et IL18 stimulent la différenciation des CML en cellules ostéoblastiques quiforment de la calcification intimale. Il est intéressant de noter que l'absence de Rev-erbα dansle fond génétique LDLr-/- entraîne une augmentation de la calcification intimale des lésions par rapport aux souris LDLr-/-Rev-erbα+/+, tandis que l'absence de Rev-erb-α dans les CMLprimaires augmente leur différenciation en ostéoblastes et la formation d'un dépôt calcique invitro. De manière intéressante, cet effet de la délétion de Rev-erbα observé sur le dépôt calciquein vitro est exacerbé lorsque les CML primaires sont stimulées en culture avec de l’IL1B. Reverb-α ne jouerait donc pas seulement un rôle dans la production d’IL1B par les macrophagesmais également dans la réception et l’interprétation de ce signal par les CML. L’activation deRev-erb-α par son ligand inhibe ces effets. Ces travaux mettent en évidence l’importance deRev-erb-α dans la régulation circadienne de la réponse inflammatoire et les phénomènesassociés au développement des maladies cardiovasculaires, identifiant Rev-erb-α comme unenouvelle cible thérapeutique qui permettrait de cibler avec une seule molécule plusieurs aspectsde la pathologie
Atherosclerosis is chronic inflammatory disease of the vascular wall, which consists inthe accumulation of Low Density Lipoproteins (LDL) into the vascular wall triggering theinternalization of leucocytes. Recruited monocytes differentiate into macrophages to removeLDL through the so-called reverse cholesterol transport pathway. However, when flux isimpaired, lipid-laden macrophages become foam cells leading to their apoptosis or necrosis.Their lipid content and cellular debris are then released in the extracellular matrix, thus formingthe necrotic core. In addition, laden-cholesterol crystals trigger lysosomal damage inmacrophage, which activates the NLRP3 inflammasome complex. NLRP3 inflammasome isinvolved in the maturation of the pro-caspase 1, which subsequently cleaves the pro- IL1B andpro-IL18 into mature IL1B and IL18. These interleukins are then released and stimulate thesecretion of interferon γ by CD4+ T cells, leading to the proliferation and the migration ofSmooth Muscle Cells (SMC) toward the necrotic core to form the fibrous cap. In addition tothe numerous risk factors of cardiovascular diseases such as diabetes or smoking, disruption ofdaily rhythms, as seen in shiftwork, increases the incidence of these diseases. Beyond its majorrole in metabolism and inflammation control, the nuclear receptor Rev-erb-α is also a corecomponent of the molecular clock. Altogether, it thus represents a putative therapeutic target inthe treatment of such multifactorial diseases related to clock impairment. My work shows thatthe deletion of Rev-erbα in a murine model of atherosclerosis (LDLr-/-) leads to an increase inplasma lipid levels and accelerates atherogenesis. In addition, Rev-erb-α regulates the circadianexpression of the NLRP3 pathway components as well as the subsequent secretion of IL1B andIL18 in primary human and mouse macrophages. At the molecular level, Rev-erb-α directlyrepresses the NLRP3 pathway, whereas Rev-erbα deficiency enhances its activation. Thismechanism may regulate the circadian susceptibility to inflammatory stimuli depending on thetime of challenge in acute inflammation models such as fulminant hepatitis or peritonitis. Theabsence of Rev-erbα also induces the expression of Nlrp3, Il1β and Il18 in models of chronicinflammation such as atherosclerosis as well as the expression of NLRP3 in lesionalmacrophages. In addition, IL1B and IL18 stimulate the differentiation of SMCs into osteoblastlikecells that form intimal calcification. It is noteworthy that Rev-erbα deletion in LDLr-/- miceis associated to an increase in lesion calcification in vivo, while the absence of Rev-erb-α inprimary SMCs increases their differentiation into osteoblasts and the formation of calciumdeposit in vitro. Interestingly, this differential effect observed on calcium deposition in vitro isexacerbated when primary SMCs are stimulated in culture with IL1B. Therefore, Rev-erb-α may be involved not only in macrophage-mediated IL1B production but also in the sensing ofsuch signal by SMCs. Conversely, treatment with Rev-erb ligands inhibits these effects. Thiswork emphasizes the key role of Rev-erb-α in the circadian regulation of the inflammatoryresponse and in the development of cardiovascular diseases, thus identifying Rev-erb-α as anew therapeutic target that act on several aspects of the pathology

A gordura pericárdica (GP), um componente do tecido adiposo visceral, tem sido consistentemente relacionada com aterosclerose coronária na população geral. Este estudo avaliou a associação entre GP e a calcificação arterial coronária (CAC) em pacientes com doença renal crônica (DRC) não dialítica. Este é um estudo transversal post-hoc da linha de base de coorte prospectiva de 117 pacientes com DRC em seguimento ambulatorial sem doença coronária manifesta (idade, 56,8 ± 11 anos; 64% do sexo masculino; 95,1% hipertensos; 25,2% diabéticos; 15,5% com história prévia de tabagismo; e estágios 2 a 5 da DRC e ritmo de filtração glomerular estimado de 36,8 ± 18,1 ml/min). O escore de CAC, volume de GP e gordura visceral abdominal (GVA) foram medidos por tomografia computadorizada. A associação da GP, como variável contínua, com a presença de CAC foi analisada por regressão logística multivariada. CAC (escore de cálcio>0) esteve presente em 59,2% dos pacientes. Na comparação com os pacientes sem CAC, aqueles com CAC eram 10 anos mais velhos, apresentaram maior proporção de homens (78,7% versus 42,9%, p < 0.001), tiveram maior circunferência de abdominal (95,9 ± 10,7 versus 90,2 ± 13,2 centímetros, p=0,02), maior volume de GP (224,8 ± 107,6 versus 139,1 ± 85,0 cm³, p < 0,01), e maior área de GVA (109,2 ± 81,5 versus 70,2 ± 62,9 cm², p=0,01). Em análise multivariada ajustada para idade, sexo, diabetes, história de tabagismo, história de tabagismo, e hipertrofia ventricular concêntrica; GP esteve significantemente associada com a presença de CAC (OR: 1,88 95% IC: 1,03-3,43 por desvio padrão, p=0,04). GP permaneceu associada com CAC mesmo após ajuste adicional para ritmo de filtração glomerular e fósforo sérico (OR: 1,85 95% IC: 1,00 - 3,42, p=0,05). A GP está independentemente associada com CAC em pacientes com DRC não dialítica.
Pericardial fat (PF), a component of visceral adipose tissue has been consistently related to coronary atherosclerosis in the general population. This study evaluated the association between PF and coronary artery calcification (CAC) in non-dialysis dependent chronic kidney disease (CKD) patients. This is a post-hoc cross sectional analysis of the baseline of a prospective cohort of 117 outward CKD patients without manifest coronary artery disease (age, 56.9 ± 11.0 years, 64,1% males, 95.1% hypertensive, 25.2% diabetics, 15.5% ever smokers, CKD stage 2 to 5 with estimated glomerular filtration rate 36.8 ± 18.1 ml/min). CAC scores, PF volume and abdominal visceral fat (AVF) areas were measured by computed tomography. The association of PF as a continuous variable with the presence of CAC was analyzed by multivariate logistic regression. CAC (calcium score >0) was present in 59.2% patients. On the comparison with patients with no CAC, those with CAC were 10 years older on average, had a higher proportion of male gender (78.7% vs. 42.9%, p < 0.001), and had higher values of waist circumference (95.9 ± 10.7 versus 90.2 ± 13.2 cm, p=0.02), PF volumes (224.8±107.6 versus 139.1±85.0 cm³, p < 0.01) and AVF areas (109.2 ± 81.5 versus 70.2 ± 62.9 cm², p=0.01). In the multivariate analysis, adjusting for age, gender, diabetes, smoking and, left ventricular concentric hypertrophy, PF was significantly associated with the presence of CAC (OR: 1.88 95% CI: 1.03-3.43 per standard deviation, p=0.04). PF remained associated with CAC even after additional adjustments for estimated glomerular filtration rate or serum phosphorus (OR: 1.85 95% CI: 1.00-3.42, p=0.05). PF is independently associated with CAC in non-dialysis dependent CKD patients

Objetivo: Avaliar a relevância de fatores de risco tradicionais para doença cardiovascular (FRC), fatores relacionados ao lúpus e densidade mineral óssea (DMO) na calcificação prematura de artérias coronárias (CAC) em mulheres jovens com lúpus eritematoso sistêmico (LES). Métodos: Noventa e quatro pacientes lúpicas do sexo feminino com duração de doença 5 anos e idade menor que 45 anos foram selecionadas consecutivamente para este estudo. Os fatores de risco cardiovascular analisados foram: diabetes mellitus, hipertensão arterial sistêmica, dislipoproteinemia, fumo, índice de massa corpórea (IMC), insuficiência ovariana e renal. Fatores de risco relacionados ao LES estudados foram: duração de doença, critérios ACR, SLICC/ACR modificado (excluindo escores relacionados à aterosclerose), SLEDAI, tratamento com glicocorticóide e ciclofosfamida. A densidade mineral óssea de corpo inteiro, coluna lombar e colo do fêmur foram realizadas por densitometria de dupla emissão de fontes de raios-X (DXA). Calcificação de artérias coronárias foi determinada usando tomografia computadorizada com 16 multidetectores. Resultados: Calcificação prematura de artérias coronárias foi identificada em 12 (12,7%) dos pacientes, havendo associação com maior freqüência de pacientes com FRC (p=0,008), maior número de FCR (p=0,003), idade (p=0,025), duração de doença (p=0,011) e SLICC (p=0,011). A análise individual dos FRC demonstrou que a presença de menopausa (p=0,036), dislipidemia (p=0,003) e hipertensão (p=0,006) foram significativamente associados com calcificação coronariana. Análise de regressão logística múltipla usando FRC, idade, duração de doença, SLICC e DMO de corpo inteiro revelou que apenas duração de doença (p=0,042) e DMO de corpo inteiro (p=0,023) permaneceram fatores significantes para calcificação coronariana. Conclusão: Identificamos que duração de doença e DMO reduzida são preditores independentes para calcificação coronariana prematura em mulheres jovens com LES, sugerindo um mecanismo subjacente comum
Objective: To evaluate the relevance of traditional cardiovascular risk factors (CVR), disease-related risk factors and bone mineral density (BMD) for premature coronary artery calcification (CAC) in young female systemic lupus erythematosus (SLE). Methods: Ninety-four female SLE patients 5 years disease duration and age <45 years were consecutively selected for this study. Cardiovascular risks (CVR) analyzed were: diabetes mellitus, arterial hypertension, dyslipoproteinemia, smoking, body mass index (BMI), ovarian and renal insufficiency. SLE-related risk factors evaluated were: disease duration, ACR criteria, modified SLICC/ACR (excluding atherosclerosis-related scores), SLEDAI, glucocorticoid and cyclophosphamide treatment. Bone mineral density (BMD) in whole body, lumbar spine and femoral neck was assessed by dual X ray absorptiometry (DXA). Coronary artery calcification was determined using the 16-slice multidetector computed tomography. Results: Premature coronary artery calcification was identified in 12 (12.7%) patients and was associated with a higher frequency of patients with CVR (p=0.008), a higher mean number of CVR (p=0.003), mean age (p= 0.025), mean disease duration (p=0.011) and mean SLICC (p=0.011). Individual analysis of CVR demonstrated that the presence of menopause (p= 0.036), dyslipidemia (p= 0.003) and hypertension (p=0.006) were significantly associated with coronary calcification. Additionally, premature calcification was associated with a lower whole body BMD (p=0.013). Multiple logistic regression analysis using CVR, age, disease duration, SLICC and whole body BMD revealed that only disease duration (p=0.042) and whole body BMD (p=0.023) remained significant factors for coronary calcification. Conclusion: We have identified that disease duration and decreased BMD are independent predictors for premature coronary calcification in young women with SLE, suggesting a common underlying mechanism

O HDL-c é um fator de risco cardiovascular negativo e sua concentração plasmática apresenta relação inversa com a incidência de eventos cardiovasculares. Entretanto, as evidências relativas ao grupo de indivíduos com níveis de HDL-c acima do percentil 95 da população geral ainda são escassas e o impacto da hiperalfalipoproteinemia (HALP) sobre o risco cardiovascular continua representando motivo de controvérsia na literatura médica. Alguns estudos em populações específicas associam a HALP a aumento do risco cardiovascular. Ao mesmo tempo, outros estudos identificaram populações de indivíduos hipoalfalipoproteinêmicos com marcada longevidade. Assim, demonstrou-se aparente dissociação entre níveis de HDL-c e risco cardiovascular em determinadas populações, reconduzível a aspectos disfuncionais da HDL. O objetivo do presente estudo foi verificar o papel da HALP na determinação do risco cardiovascular; comparar a prevalência de doença cardiovascular subclínica, avaliada por meio da quantificação ultrassonográfica da Espessura Íntimo-Medial Carotídea (EIMC), entre portadores de HDL-c >= 90mg/dL (grupo HALP) e portadores de concentrações de HDL-c atualmente consideradas normais (entre 40 e 50mg/dL para os homens e entre 50 e 60mg/dL para as mulheres); e avaliar características e função da HDL em portadores de HALP por meio do estudo de sua composição, de sua capacidade de efluxo de colesterol, e de sua atividade anti-inflamatória e antioxidante, correlacionando estas características com a presença de doença cardiovascular subclínica avaliada por meio da determinação da EIMC, da Velocidade de Onda de Pulso (VOP) e da presença de Calcificação Arterial Coronariana (CAC) avaliada pela TCMD. Para responder estas perguntas, o presente estudo foi articulado em dois braços: Braço 1: Análise da coorte do estudo ELSA com o objetivo de determinar a prevalência de HALP em uma população geral; definir o perfil demográfico, antropométrico e metabólico dos portadores de HALP; e comparar a prevalência de doença vascular subclínica deste grupo com controles da mesma coorte com níveis normais de HDL-colesterol. Braço 2: Recrutamento de 80 voluntários hígidos e portadores de HALP para avaliação da correlação entre presença de doença vascular subclínica, e aspectos estruturais e funcionais da HDL. Em seus dois braços, o estudo levou a quatro conclusões principais: 1) Níveis marcadamente elevados de HDL-c estão associados a menor espessura íntimo-medial carotídea quando comparados a níveis de HDL-c considerados normais pelas diretrizes vigentes. Embora portadores do fenótipo HALP apresentem, como grupo, um perfil metabólico mais favorável que o encontrado em indivíduos com HDL-c normal, a associação entre EIMC e HALP foi independente dos fatores de risco tradicionais, indicando que a menor prevalência destes últimos em portadores de HDL-c marcadamente elevado justifica apenas parcialmente a menor prevalência de doença vascular subclínica neste grupo; 2) Embora a HALP se apresente como um fenótipo ateroprotetor, há indivíduos com níveis marcadamente elevados de HDL-c que evoluem com doença cardiovascular, clínica ou subclínica. Neste contexto, nossos resultados indicam correlação entre os três métodos avaliados para estudar doença vascular subclínica em portadores de HALP: EIMC, VOP e CAC; 3) Os fatores de risco tradicionais continuam exercendo seu peso na determinação do risco cardiovascular em portadores de HALP. Idade, tabagismo, hipertensão arterial, hipertrigliceridemia e altos níveis de LDL-c apresentaram associação estatisticamente significativa com a presença de doença vascular subclínica no grupo estudado; 4) A avaliação da composição e da função da HDL em portadores de HALP pode permitir identificar indivíduos especificamente mais suscetíveis à aterosclerose. Nossos resultados indicam que, em particular, a atividade anti-inflamatória da HDL, avaliada pela capacidade de inibição da produção de IL-6; o efluxo de colesterol e a capacidade de transferência de triglicérides apresentaram associação independente com menor espessura íntimo-medial carotídea em portadores de HALP, enquanto níveis mais altos de Apo A-IV se associaram a maior grau de doença cardiovascular subclínica
HDL-c is a negative cardiovascular risk factor and its plasma concentration is inversely related to the incidence of cardiovascular events. However, evidence of benefit among subjects with HDL-c levels above the 95th percentile of the general population is still scarce and the impact of hyperalphalipoproteinemia (HALP) on cardiovascular risk continues to represent matter of debate in the medical literature. Some studies with specific populations indicated an increased cardiovascular risk associated with HALP. In addition, other reports identified groups of patients with marked hypoalphalipoproteinemia and longevity. Hence, there could be a dissociation between HDL-c levels and cardiovascular risk in certain populations, possibly due to dysfunctional HDL particles. The aim of this study was to investigate the role of HALP phenotype in determining cardiovascular risk; to compare the prevalence of subclinical cardiovascular disease, assessed by ultrasound measurement of Carotid Intima-Media Thickness (CIMT) among patients with HDL-c >= 90mg/dL (HALP group) and patients with HDL-c currently considered normal (40-50mg/dL for men and 50-60mg/dL for women); and to evaluate HDL functionality in patients with HALP through the study of its composition, its cholesterol efflux capacity, and its anti-inflammatory and antioxidant activity; correlating those characteristics with the presence of subclinical cardiovascular disease assessed by CIMT, Pulse Wave Velocity (PWV) and Coronary Artery Calcification (CAC). To answer these questions, the present study was articulated into two arms: Arm 1: ELSA-Brasil study cohort analysis in order to assess HALP prevalence in a general population, defining demographic, anthropometric and metabolic profile of HALP individuals; and comparing the prevalence of subclinical vascular disease among HALP subjects with controls with normal HDL-c. Arm 2: Recruitment of 80 healthy volunteers with HALP to study the correlation between subclinical vascular disease and HDL functionality in this group. Our study led to four main conclusions: 1) markedly elevated HDL-c is associated with lower CIMT compared to the control group with normal HDL-c levels. Although individuals with HALP display a more favorable metabolic profile than subjects with normal HDL-c, the association between CIMT and HALP was independent of traditional risk factors, indicating that the lower prevalence of subclinical vascular disease in this group is only partially justified by the lower prevalence of cardiovascular risk factors; 2) Although HALP can be regarded as an atheroprotective phenotype, there are individuals with markedly elevated levels of HDL-c who develop cardiovascular disease. Our results indicate good correlation of the three methods here adopted to study subclinical vascular disease among HALP patients: CIMT, VOP and CAC; 3) Traditional risk factors continue to exert their weight in determining cardiovascular risk in patients with HALP. Age, smoking, hypertension, hypertriglyceridemia and high levels of LDL-c were significantly associated with the presence of subclinical vascular disease among HALP individuals; 4) the assessment of the HDL composition and functionality in patients with HALP may allow to identify individuals specifically more susceptible to atherosclerosis. Our results indicate that, in particular, cholesterol efflux capacity, the anti-inflammatory activity of HDL, and triglyceride transfer capacity were independently associated with lower CIMT in HALP individuals, while higher levels of Apo A-IV were associated with a greater burden of subclinical cardiovascular disease

Introdução: Doenças tireoidianas subclínicas incluem hipotireoidismo e hipertireoidismo subclínicos. A associação entre doença tireoidiana subclínica e morbimortalidade cardiovascular é controversa e os dados sobre a relação entre essas condições clínicas e aterosclerose subclínica são escassos. Objetivos: Este estudo objetiva avaliar a associação entre doença tireoidiana subclínica, calcificação arterial coronariana (CAC), doença arterial coronariana (DAC), índice de espessura de médio-íntima carotídea média (IMT) e velocidade de onda de pulso carotídeo-femoral (cf-VOP) no Estudo Longitudinal de Saúde do Adulto (ELSA-Brasil). Métodos: Incluímos sujeitos eutireóideos, definidos como tendo TSH entre 0,4 e 4,0 mUI/L e T4L entre 0,8 e 1,9ng/dL, indivíduos com hipotireoidismo subclínico, definido como TSH > 4,0 mUI/L e T4L normal, e hipertireoidismo subclínico, definido como TSH < 0,4 mUI/L e T4L normal. Excluímos os indivíduos com as demais disfunções tireoidianas, em uso de medicação que altera a função tireoidiana, e com doença cardiovascular prévia. Na análise de angiotomografia, excluímos também os sujeitos com hipertireoidismo subclínico pelo pequeno número que impedia a análise e, na análise de cf-VOP, doença renal crônica, indivíduos em uso de diuréticos e de anti-hipertensivos. As associações entre quintis de TSH, CAC > 100 e DAC foram avaliadas por regressão logística e as associações entre IMT, VOP (como variáveis contínuas ou categorizadas com ponto de corte no percentil 75 amostral) e níveis de TSH ou doenças tireoidianas subclínicas foram avaliadas por regressões logísticas e lineares multivariadas. Todos os modelos foram ajustados por variáveis demográficas e fatores de risco cardiovasculares. Resultados: A análise de CAC incluiu 3.836 sujeitos, mediana de idade de 49 anos (IQR=44-56), 1.999 (52,1%) mulheres. CAC > 100 associou-se independentemente com o primeiro quintil (OR ajustado=1,57, IC 95%=1,05-2,35, P=0,027), usando o terceiro como referência. Na análise de angiotomografia, foram incluídos 796 sujeitos, mediana de idade de 55 anos (IQR=48-60 anos), 406 (51%) mulheres. O primeiro quintil associou-se independentemente com CAC (OR ajustado=1,76, IC 95%=1,09-2,82, P= 0,02), DAC (OR ajustado=1,73, IC 95%=1,08-2,79, P=0,023), mas não com extensão de doença. Na análise de IMT, foram incluídos 8.623 sujeitos, mediana de idade de 50 anos (IQR=45-57 anos), 4.624 (53,6%) mulheres, na subanálise de hipotireoidismo subclínico, e 8.193, com mediana de idade de 50 anos (IQR=44-57 anos), 4.382 (53,5%) mulheres, na subanálise de hipertireoidismo subclínico. Hipotireoidismo subclínico, mas não hipertireoidismo subclínico, associou-se ao IMT como variável contínua (beta=0,010, IC 95%=0,0004-0,019, P=0,041) e categorizado no percentil 75 ajustado para sexo, idade e raça (OR ajustado=1,30, IC95%=1,07-1,61, P=0,010). Na análise de cf-VOP, foram incluídos 8.341 sujeitos, mediana de idade de 50 anos (IQR=44-56 anos), 4.383 (52,5%) mulheres, na subanálise de hipotireoidismo subclínico, e 7.790, mediana de idade de 50 anos (IQR=44-57 anos), 4.191 (53,8%) mulheres, na subanálise de hipertireoidismo subclínico. Cf-VOP não se associou com doença tireoidiana subclínica. Conclusões: Em análises diferentes, CAC e DAC associaram-se com primeiro quintil de TSH usando-se o terceiro como referência. O IMT associou-se com hipotireoidismo subclínico e a cf-VOP não se associou com disfunção tireoidiana subclínica
Introduction: Subclinical thyroid disease includes subclinical hypothyroidism and subclinical hyperthyroidism. Association between subclinical thyroid disease and cardiovascular morbidity and mortality is controversial and data about the relationship between those clinical conditions and subclinical atherosclerosis is scarce. Objectives: This study aims to evaluate the association between subclinical thyroid disease, coronary artery calcification (CAC), coronary artery disease (CAD), mean common carotid intima-media thickness (IMT) and carotid-femoral pulse wave velocity (cf-PWV) in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). Methods: We included euthyroid subjects, defined as TSH between 0.4 and 4.0 mIU/l and FT4 between 0.8 and 1.9 ng/dL, and individuals with subclinical hypothyroidism, defined as TSH > 4.0 mIU/l and normal FT4, and subclinical hyperthyroidism, defined as TSH < 0.4 mIU/L and normal FT4. We excluded individuals with other thyroid disorders, subjects who used medication that altered thyroid function, subjects with past of cardiovascular disease. In computed angiotomography analysis, we have excluded subjects with subclinical hyperthyroidism because of the small sample, and in cf-PWV analysis, we have excluded individuals with chronic kidney disease, use of anti-hypertensive and diuretics. The association between TSH quintiles was evaluated in logistic regression models for CAC and CAD, and the association between IMT, cf-PWV (as continuous variables or as factor, categorized at 75th sample\'s percentile) and TSH levels or subclinical thyroid diseases was evaluated by multivariate logistic and linear regression models. All models were adjusted for demographic variables and cardiovascular risk factors. Results: CAC analysis included 3,836 subjects, median of age 49 years (IQR=44-56), 1,999 (52.1%) women. CAC > 100 was independently associated with first quintile of TSH, using the third quintile as the reference (adjusted OR=1.57, 95% CI=1.05-2.35, P=0.027). Computed angiotomography analysis included 796 subjects, median of age 55 years (IQR=48-60), 406 (51%) women. CAD and CAC > 0 was independently associated with first quintile in comparison with third quintile (adjusted OR=1.73, 95% CI=1.08-2.79, P=0.023 and adjusted OR=1.76, 95% CI=1.09-2.82, P= 0.02, respectively), but not with burden of disease. In IMT analysis, 8,623 subjects were included, median of age 50 years (IQR=45-57 years), 4,624 (53.6%) women in the subclinical hypothyroidism subanalysis, and 8,193, median age 50 years (IQR = 44-57 years), 4,382 (53.5%) women, in the subclinical hyperthyroidism subanalysis. Subclinical hypothyroidism, but not subclinical hyperthyroidism, was independently associated with IMT as continuous variable (beta=0.010, IC 95%=0.0004-0.019, P=0.041) or as factor categorized at 75th percentile adjusted for age, sex and race (adjusted OR=1.30, 95% CI=1.07-1.61, P=0.010). In cf-PWV analysis, 8,341 subjects were included, median of age 50 years (IQR=44-56 years), 4,383 (52.5%) women in the subclinical hypothyroidism subanalysis, and 7,790, median age 50 years (IQR = 44-57 years), 4,191 (53.8%) women in subclinical hyperthyroidism subanalysis. Cf- PWV was not associated with subclinical thyroid disease. Conclusion: In separated analysis, CAC and CAD was independently associated with first quintile of TSH using the third as the reference; IMT was independently associated with subclinical hypothyroidism, and cf-PWV was not associated with subclinical thyroid diseases

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.

Chan Fat-Yiu.
Thesis (M.Sc.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 85-93).
Abstracts in English and Chinese.
ACKNOWLEDGEMENTS --- p.4
SUMMARY --- p.5
ABBREVIATIONS --- p.9
LIST OF TABLES --- p.11
LIST OF FIGURES --- p.13
Chapter CHAPTER I --- INTRODUCTION --- p.14
Chapter 1.1 --- The Historical Aspects of C-Reaction Protein --- p.15
Chapter 1.2 --- Biochemistry of CRP --- p.16
Chapter 1.3 --- Physiology of CRP --- p.18
Chapter 1.4 --- Current Clinical Applications of Serum CRP Assay --- p.19
Chapter 1.5 --- Recent Findings of CRP --- p.21
Chapter 1.5.1 --- Pathophysiology of atherosclerosis --- p.22
Chapter 1.5.2 --- A nother atherogenic risk factor: hs- CRP --- p.26
Chapter 1.5.3 --- Can hs-CRP replace other risk factors? --- p.30
Chapter 1.5.4 --- Altering hs-CRP result in medication --- p.32
Chapter 1.6 --- Methods of Measurement of CRP Concentration --- p.33
Chapter 1.7 --- Analytical Considerations in the Measurement of hs-CRP --- p.34
Chapter CHAPTER II --- OBJECTIVES AND SIGNIFICANCE --- p.36
Chapter 2.1 --- Objectives --- p.37
Chapter 2.2 --- Issues and Problems --- p.37
Chapter 2.3 --- Significance and Value of this Study --- p.38
Chapter CHAPTER III --- MA TERIALS AND METHODS I Setting up the serum hs-CRP assay on the Hitachi 911 Analyzer --- p.39
Chapter 3.1 --- Materials --- p.40
Chapter 3.1.1 --- Reagents from Roche Diagnostics --- p.40
Chapter 3.1.2 --- Reagents for the Beckman Coulter Array ® Analyzer --- p.40
Chapter 3.1.3 --- In-house reagents --- p.41
Chapter 3.2. --- Apparatus and Equipment --- p.41
Chapter 3.2.1 --- Hitachi 911 Analyzer --- p.41
Chapter 3.2.2 --- Beckman Coulter Array ® 360 Analyzer --- p.42
Chapter 3.3 --- The Tina-quant a C-Reactive Protein (Latex) Ultrasensitive Assay --- p.42
Chapter 3.3.1 --- Priniciple of the Dual-Radius Enhanced Latex (DuREL´ёØ) technology --- p.42
Chapter 3.3.2 --- Assessment of Analytical Performance --- p.45
Chapter CHAPTER IV --- MA TERIALS AND METHODS II Serum hs-CRP in Chinese chronic-renal-failure patients with atherosclerotic vascular disease or cardiac valve calcification --- p.48
Chapter 4.1 --- Patient Recruitment --- p.49
Chapter 4.2. --- Blood Specimens --- p.49
Chapter 4.3 --- Assay Methods --- p.50
Chapter 4.3.1 --- hs-CRP --- p.50
Chapter 4.3.2 --- TC --- p.50
Chapter 4.3.3 --- TG --- p.51
Chapter 4.3.4 --- HDL-C --- p.51
Chapter 4.3.5 --- LDL-C --- p.52
Chapter 4.3.6 --- Apo A-1 --- p.52
Chapter 4.3.7 --- Apo B --- p.53
Chapter 4.3.8 --- Lp(a) --- p.53
Chapter 4.4 --- Ultrasound measurement of carotid artery inter-media thickness --- p.53
Chapter 4.5 --- Statistical analysis --- p.54
Chapter CHAPTER V --- RESUTLSI Setting up the serum hs-CRP assay on the Hitachi 911 Analyzer --- p.55
Chapter 5.1 --- Imprecision --- p.56
Chapter 5.2 --- Linearity --- p.56
Chapter 5.3 --- Recovery --- p.56
Chapter 5.4 --- Detection Limit --- p.57
Chapter 5.5 --- Carry-over --- p.57
Chapter CHAPTER VI --- RESULTS II Serum hs-CRP in Chinese chronic-renal-failure patients with atherosclerotic vascular disease or cardiac valve calcification --- p.63
Chapter 6.1 --- Patient Recruitment --- p.64
Chapter 6.2 --- Chinese chronic-renal-failure patients with AVD --- p.64
Chapter 6.3 --- Chinese chronic-renal-failure patients with CVC --- p.65
Chapter CHAPTER VII --- DISCUSSION I Performance of the serum hs-CRP assay on the Hitachi 911 Analyzer --- p.75
Chapter 7.1 --- "Imprecision, Detection Limit, Linearity, and Recovery of hs-CRP Assay" --- p.76
Chapter 7.1.1 --- Imprecision --- p.76
Chapter 7.1.2 --- Detection Limit --- p.76
Chapter 7.1.3 --- Linearity --- p.76
Chapter 7.1.4 --- Recovery --- p.77
Chapter 7.2 --- Overall Performance --- p.77
Chapter CHAPTER VIII --- DISCUSSION II Serum hs-CRP in Chinese chronic-renal-failure patients with atherosclerotic vascular disease or cardiac valve calcification --- p.79
Chapter 8.1 --- CAPD Patients --- p.80
Chapter 8.2 --- Serum hs-CRP Concentration of AVD and CVC Patients --- p.81
Chapter 8.3 --- Other risk factors in AVD and CVC Patients --- p.82
Chapter 8.4 --- Conclusion --- p.83
REFERENCES --- p.85

博士
國防醫學院
生命科學研究所
105
Abnormal activation of components of the innate immune system, such as Toll-like receptor (TLR), has been implicated in the pathological progression of metabolic inflammatory diseases including atherosclerosis and vascular calcification. Functional phenotype of vascular smooth muscle cells (VSMCs) is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of TLR2 and TLR4 in the progression of vascular diseases, their roles in the regulation of VSMC function remains unclear. The goal of the present study was to elucidate the mechanism by which TLR2 and TLR4 regulate VSMC function and phenotypic switching. Migration assay revealed that activation of TLR2 and TLR4 by their specific agonists induced VSMCs migration. Inhibition experiments indicated that IL-6 secretion and VSMC migration induced by agonists of TLR2 and TLR4 were mediated through the activation of p38 mitogen-associated protein kinase (p38 MAPK) and extracellular signal–regulated kinase (ERK) 1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR2 and TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited agonists of TLR2 and TLR4-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed IL-6 production and VSMC migration-induced by TLR2 and TLR4. Rac-1 inhibitor suppressed TLR2 and 4-driven VSMC migration but not IL-6 production. Further experiments with inhibitors and siRNA found that TLR4 activates PI3K/ATK signaling to induce F-spondin expression, subsequently controls CREB-mediated IL-6 production to promote VSMC migration. These findings provide mechanismistic insights into the essential role of F-spondin in VSMC function and atherosclerosis. In addition to VSMC migration, agonists of TLR2 and TLR4 also promoted VSMC chondrogenesis and calcification as demonstrated by Alcian blue and Alizarin red S (ARS) staining. Neutralizing anti-IL-6 antibodies attenuated TLR2 and TLR4-mediated VSMC chondrogenesis, but did not affect VSMC calcification. Interestingly, TLR2 and TLR4 activated c-Jun N-terminal kinases (JNKs) - and ERK1/2 signaling to downregulate calcification inhibitor, osteoprotegerin (OPG) levels, which in turn not only promoted VSMC chondrogenesis but also trigged VSMCs calcification. Importantly, the lower OPG level and increased atherogenic calcification were clearly detected in high-fat diet (HFD)-fed ApoE-/- mice compared with HFD-fed ApoE-/-Tlr2-/- mice. Based on our findings, we concluded that, upon specific ligand binding, both TLR2 and TLR4 activate MAPKs to trigger VSMC migration and VSMC chondrogenesis, which in turn progressively triggers atherogenesis and atherogenic calcification.

Vascular calcification (VC) is a feature of atherosclerotic plaques and is associated with co-morbidities such as diabetes and chronic kidney disease. While its presence is used by cardiologists to identify higher risk patients, increasing data suggests that the morphology of calcification is also important to the stability of a plaque, whereby smaller growths, particularly in the fibrous cap, increase likelihood of rupture and larger sheets decrease likelihood. Although lipoproteins, namely high (HDL), low (LDL), very low (VLDL) density lipoproteins and lipoprotein (a) (Lp(a)), have been previously studied in their relationship to general plaque growth and progression, their roles in influencing plaque calcification are less understood due to experimental variations within the literature. This thesis therefore aims to study the roles of HDL, LDL, VLDL and their pro-atherogenic oxidised forms (ox) on atherosclerotic VC in vitro, in vivo and in human plasma samples ex vivo, and aims to assess these using standardised models. Using in vitro calcification assays developed in chapter 3, we observed that both oxVLDL and oxLDL increase vascular smooth muscle cell (VSMC) calcification, whereas native HDL reduces mineralisation. This effect of HDL however is not observed in reconstituted or oxidised species, showing a pro-calcification effect for oxidised lipoprotein species. PCR and western blot techniques identified raised Runx2, RANKL, RANK and lowered OPG as potential calcification molecules that may be influenced by lipoproteins in these cells, however further studies are needed to elucidate the full mechanisms and solidify these results. In vivo, modifying VLDL into a pro-atherogenic phenotype via apo CIII deletion on an Apo E knock out background had only minor effects on plaque characteristics when challenged with an atherogenic diet. Interestingly, triglyceride levels positively correlated with calcification markers both in vivo and ex vivo, suggesting a role for triglyceride rich lipoproteins in the stabilisation of plaques. Furthermore, the results for triglyceride involvement in VC carried into in vivo studies using pro-atherogenic Apo E knockout and pro-atherogenic/pro-calcification Apo E x OPG double knock out mice fed an atherogenic diet for 40 weeks and infused with HDL during the final 4 weeks of the study. Alongside the in vivo results, the triglyceride level in human plasma correlated with calcification markers in ex vivo human studies. While we expected to see changes in correlation with Lp(a) levels, none were detected in this thesis, prompting more precise investigation in the future when examining this particle. This thesis therefore shows roles for several lipoprotein forms in vascular calcification in standardised experiments, providing a starting point for comparison and further experimentation in the future. In continuing this research, a broader spectrum of markers will need to be analysed and further mechanistic studies would provide insight into molecules which may be therapeutically targeted for late-stage and culprit plaque stabilisation.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021

Yes
Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of highrisk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using 18F-sodium fluoride (18F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of 18F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse 18F-NaF binding. We show that 18F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. 18F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of 18F-NaF may foster new approaches to developing treatments for vascular calcification.