No description available.

La sténose aortique (SA) est la maladie valvulaire la plus fréquente dans notre société. Elle est caractérisée par un remodelage fibrocalcique conduisant à une obstruction progressive du flux sanguin. La lipoprotéine(a) (Lp[a]), une lipoprotéine similaire à la lipoprotéine de faible densité, est un facteur de risque génétique fortement associé à la SA. Malheureusement, les concentrations plasmatiques de Lp(a) sont très peu influencées par des facteurs extrinsèques, tels qu’un régime alimentaire ou une médication hypolipidémiante. Des études suggèrent que la Lp(a) serait associée aux processus de calcification dans le développement de la SA. La tomographie par émission de positons couplée à la tomographie axiale permet de détecter le processus précoce lié à calcification de la valve aortique. En effet, cette technique d’imagerie nucléaire permet d’identifier et de quantifier la microcalcification au niveau de la valve aortique, un marqueur fortement lié au développement futur de calcium. L’impact de la Lp(a) sur la microcalcification de la valve aortique n’a jamais été évalué. La mesure de la microcalcification chez des individus sans SA ayant des concentrations plus ou moins élevées de Lp(a) a été effectuée. Notre hypothèse était que les individus ayant des concentrations élevées de Lp(a) ont une microcalcification plus élevée, lorsque comparée aux individus ayant des concentrations plus faibles de Lp(a). Les résultats de cette étude ont révélé que les individus sans SA mais ayant des concentrations élevées de Lp(a) présentent une microcalcification plus importante que les individus ayant de plus faibles concentrations de Lp(a). La réalisation de ce projet de recherche nous a permis d’observer cliniquement un processus actif de calcification chez des individus avec des concentrations élevées de Lp(a), et ce, malgré l’absence clinique de la maladie, illustrant l’importance de cette lipoprotéine dans le développement de la SA. / Aortic stenosis (AS) is the most common valve disease in our society. It is characterized by fibrocalcific remodelling leading to progressive obstruction of blood flow. Lipoprotein(a) (Lp[a]), a lipoprotein similar to low-density lipoprotein, is a genetic risk factor strongly associated with AS. Plasma concentrations of Lp(a) are very little influenced by extrinsic factors, such as diet or lipid-lowering medication. Studies suggest that Lp(a) would be associated with calcification processes in the development of AS. Positron emission tomography coupled with computed tomography allows the early process related to calcification of the aortic valve to be detected. This nuclear imaging technique identifies and quantifies microcalcification at the aortic valve, a marker strongly linked to the future development of calcium. The impact of Lp(a) on aortic valve microcalcification has never been evaluated. Microcalcification measurements in individuals without AS with high or low concentrations of Lp(a) were performed. Our hypothesis was that individuals with high concentrations of Lp(a) have higher microcalcification when compared to individuals with lower concentrations of Lp(a). The results of this study revealed that individuals without AS but with high concentrations of Lp(a) have a higher microcalcification than individuals with lower concentrations of Lp(a). The completion of this research project allowed us to observe clinically an active calcification process in individuals with high concentrations of Lp(a) despite the clinical absence of the disease, illustrating the importance of this lipoprotein in the development of AS.

Most global calcification is carried out by organisms which are also photosynthetic. In this study, the coccolithophore Emiliania huxleyi (Lohmann) Hay and Mohler and two species of hermatypic coral were used to: examine the effect of dissolved inorganic carbon (DIC) and light on photosynthesis and calcification; and determine the extent to which these two processes interact. A novel method of producing coccolith-less (non-calcifying) cells from calcifying cells of the same strain of E huxley! was developed thus allowing photosynthesis and calcification to be studied separately. The kinetics of photosynthesis in both types of cell, and of calcification in coccolith-bearing cells, were shown to be biphasic with respect to DIC concentration. The hiatus in all three cases was located at 1 mM DIC. This unusual pattern was shown to be the product of two carbon uptake mechanisms: an anion exchanger working at all DIC concentrations and an external carbonic anhydrase active only at low DIC concentrations. In contrast to the commonly-held view, this study demonstrated that calcification did not promote photosynthesis in E. huxleyi. Nevertheless, there was clearly strong biological control of calcification in this alga since DIC uptake was mediated by an anion transporter and a dehydroxylating enzyme. This work also showed that in E huxleyi, DIC addition enhanced photosynthesis at both limiting and saturating photon flux densities and that bicarbonate affected photochemical processes directly. Photosystem II activity was stimulated and non-photochemical quenching was reduced, possibly protecting the photosynthetic apparatus from damage by light. In the two corals; Porites porites and Acropora sp., strong biological control of calcium carbonate precipitation was also evident. Again, calcification did not stimulate photosynthesis. Calcification rates of Acropora sp. were monitored in the dark and although these were lower than in the light, they still increased dramatically with bicarbonate addition. This showed that high concentrations of the bicarbonate ion can compensate for the lack of light. Hence, it seems that in hermatypic corals, light-dependence of calcification may be facultative and not obligate. It is therefore clear from the results of this study that calcification and photosynthesis are not as closely coupled as has been previously thought. In neither E. huxleyi, nor in the hermatypic corals, were photosynthetic and calcification rates saturated at the present ambient DIC concentration of seawater.

Ce mémoire porte sur l’étude de pyrophosphates de calcium hydratés (CPP : Ca2P2O7•nH2O), composés rencontrés dans des micro-calcifications pathologiques associées à l’arthrose et, dans certains cas, responsables d’arthropathie destructive. Ces cristaux, présents dans les articulations de patients arthritiques, possèdent un fort potentiel inflammatoire susceptible d’engendrer une dégradation aigüe du cartilage. Cependant, les mécanismes de formation des phases de CPP et d’activation de leur potentiel inflammatoire n’ont pas encore été entièrement décrits. Nous nous sommes intéressés à l’étude des conditions de formation des différentes phases de pyrophosphates de calcium hydratés in vitro ainsi qu’à la caractérisation fine de chacune des phases d’intérêt biologique avec des outils de laboratoire et de grands instruments afin de mieux comprendre leurs propriétés physico-chimiques et d’améliorer leur identification in vitro et in vivo. Dans un premier temps, un protocole de synthèse a été établi permettant la synthèse de quantités importantes de chacune des phases pures de CPP (CPP amorphe, CPP dihydratés monoclinique et triclinique et CPP tétrahydraté). Les conditions de synthèse associées à la formation de chacune de ces phases, pH et température notamment, ont été explorées. Des échantillons purs ont été utilisés comme références pour les différentes études physico-chimiques et structurales qui ont ensuite été menées. Les échantillons de référence ont été caractérisés finement, d’un point de vue structural avec notamment la résolution de plusieurs structures cristallines (diffraction des rayons X et des neutrons, sur poudre et monocristal) mais aussi au travers de différentes analyses spectroscopiques (spectroscopies FTIR et Raman, RMN du solide) et d’analyses de la morphologie des cristaux (microscopies électroniques à balayage et en transmission, diffraction électronique). Chacune de ces analyses complémentaires, couplées à des modélisations ab initio, a permis de préciser les hypothèses suggérant un rôle de la surface des cristaux dans le potentiel inflammatoire de ces phases. Une troisième partie est consacrée à l’exploration de différentes techniques de synthèse mettant en œuvre différents milieux (cristallisation en solution et en gel). Ces expériences ont permis d’établir des comparaisons avec les processus de formation observés in vivo et d’évolution in vitro à haute température des phases de CPP. Finalement des études ex vivo de cartilages calcifiés seront présentées, mettant en évidence les avantages de ces techniques de caractérisation de laboratoire comme outils de diagnostic. Ce travail permet ainsi de préciser les mécanismes physico-chimiques liés aux différentes phases de CPP in vitro et in vivo afin de mieux comprendre la formation de ces phases et leur potentiel inflammatoire associé, tout en améliorant les possibilités de diagnostic des arthropathies microcristallines. / The present work concerns the study of hydrated calcium pyrophosphates (CPP: Ca2P2O7•nH2O), a group of phases detected in pathological microcalcifications and associated with arthritis. These crystals are frequently observed in the synovial fluid of arthritic patients and they were described as having a high inflammatory potential which could induce a severe degradation of cartilage. However, the mechanisms involved in the formation of the CPP crystals and the activation of their inflammatory potential are not fully understood. This work is focused on the study of the synthesis conditions of CPP in vitro and on the fine characterization of CPP phases of biological interest using laboratory equipments and large-scale facilities. The aim of this work was to describe the physico-chemical properties of these materials, including inflammatory potential, and to improve their identification in vivo and in vitro. First, a synthesis protocol was designed for the production of significant amounts of pure samples for each of the CPP phases (amorphous CPP, monoclinic and triclinic dihydrated CPP and tetrahydrated CPP). Different conditions, including pH and temperature, were studied to achieve the synthesis of reference materials. These samples were precisely characterized using complementary techniques to determine their crystalline structures (powder and single crystal X-ray diffraction and neutron diffraction) as well as using spectroscopic (FTIR and Raman spectroscopies, MAS-NMR) and morphologic analyses (SEM, TEM and electron diffraction). These analyses, combined with ab initio modeling, clarified the hypotheses concerning the role of the crystal surface on the adsorption properties of CPP crystals and their inflammatory potential. The third part of this thesis is focused on the study of CPP synthesis conditions, by using different experimental setups to study crystallization in solution and in gel. A comparison with in vivo formation processes and in vitro high temperature evolution phenomena of these phases was established based on the results of these experiments. Finally, ex vivo analyses of pathological cartilage are presented, highlighting the advantages of different laboratory characterizations as medical diagnostic tools. This work contributes to clarify the physico-chemical characteristics of CPP phases in vitro and in vivo, to improve the knowledge on the formation and the evolution of these phases, their properties including inflammatory potential, and to facilitate their identification in vivo.

Vascular calcification (VC) is accelerated in patients with chronic kidney disease (CKD), resulting in increased risk of cardiovascular disease and mortality. Although the consequences of VC are associated with elevated pulse wave velocity (PWV) and left ventricular hypertrophy (LVH), the temporal impact on blood pressure changes is unknown. Mineral imbalance in CKD greatly contributes to the development of VC, and elevated serum phosphate is a major risk factor. Magnesium, which plays an important role in bone regulation, has been recently shown to be a modifier of VC, but whether magnesium inhibits calcification in CKD is unknown. A modified adenine model of CKD was developed in rats, characterized by mineral imbalance and progressive VC. During the development of VC, pulse pressure increased, which was driven by a drop in diastolic blood pressure, rather than systolic hypertension. Continuous pressure recordings in conscious rats using radiotelemetry revealed a significant increase in systolic variability associated with development of VC. Regional VC was associated with regional changes in the hemodynamic profile of the CKD rats. For example, only thoracic aortic calcification was associated with elevated PWV and pulse pressure. In contrast, the presence of abdominal and thoracic calcification differentially affected proximal and distal arterial pressure wave forms. CKD animals exhibited LVH, which was further increased by the presence of VC. In addition, fibroblast growth factor 23, which regulates renal excretion of phosphate, was elevated in CKD animals at every time point and was associated with LVH independently from VC. Development of VC was characterized in an in vitro organ model. Phosphate elevation in vitro caused VC in aortas. In vitro, magnesium supplementation inhibited initiation and progression of VC. CKD animals given a magnesium diet also demonstrated attenuated development of VC. In patients with stage 3-5 CKD (excluding dialysis), dietary phosphate was associated with the progression of coronary artery calcification even after adjusting for use of phosphate binders, total dietary energy and total dietary protein. Given the serious negative outcomes associated with development of VC, these findings fill key gaps in knowledge regarding the detection, management, prevention and treatment of VC in CKD. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-12-01 15:12:54.388

Encapsulating peritoneal sclerosis (EPS) is a rare complication of long-term peritoneal dialysis (PD). EPS is associated with extensive thickening and fibrosis of the peritoneum resulting in the formation of a fibrous cocoon encapsulating the bowel leading to intestinal obstruction. The presence of peritoneal thickening, peritoneal calcification and bowel obstruction is considered to be diagnostic of EPS. The current understanding of the pathogenesis of EPS is through the 'two-hit' fibrosis model. This model, however, does not explain the development of peritoneal calcification in patients with EPS. This thesis addresses the hypothesis that altered bone mineral metabolism in ESRF patients together with the mechanical stress of PD influences mesothelial cells to differentiate into osteoblasts promoting calcification in peritoneal tissue. Peritoneal calcification leads to increased tissue stiffness causing progressive fibrosis and the development of EPS. We compared the temporal evolution of the levels of bone mineral markers during PD between patients who developed EPS and control patients on PD. We found that raised serum levels of calcium, phosphate and alkaline phosphatase during PD increased the risk of development of EPS. We compared peritoneum from patients with EPS with that of PD patients without EPS using histological techniques. We found that calcification, organised fibrillary collagen and elastic fibres were significantly more abundant in the EPS peritoneum. Peritoneal calcification was also generalised and distributed not only on the peritoneal surface but also in the sub-mesothelial zone of fibrosis. EPS peritoneum also exhibited osteocalcin, an osteogenic protein, suggesting a cellular mechanism of calcification. Atomic force microscopy of EPS peritoneum showed increased stiffness when compared to control PD peritoneum with the areas of calcification possibly contributing to the increase in tissue stiffness. Human omental cells (HOMCs) were isolated by protease digestion and characterised using a panel of mesothelial markers. HOMCs were cultured in phosphate rich media and phosphate and calcium rich media. HOMCs when cultured with high extracellular levels of calcium showed accelerated mineralisation with upregulation of osteogenic transcription factor runx-2 suggesting osteoblastic transformation. In summary, this thesis indicates that poorly controlled secondary hyperparathyroidism is a risk factor for the development of EPS. On a background of PD related simple sclerosis, uncontrolled secondary hyperparathyroidism can lead to the transformation of mesothelial cells to osteoblasts. This leads to increased matrix deposition and matrix mineralisation causing increased matrix stiffness. Increase in matrix stiffness leads to progressive fibrosis culminating in EPS. Peritoneal calcification can act as the second hit leading to progressive fibrosis and development of EPS.

Σκοπός της παρούσας μελέτης είναι να εκτιμηθεί η κλινική σημασία των ενδοπροστατικών ασβεστώσεων σε νεαρούς ενήλικες άνδρες και να συσχετιστούν με κλινικές ή εργαστηριακές παραμέτρους που αφορούν τον προστάτη, ή άλλες ασθένειες. Ένας πληθυσμός 1374 νεαρών ενηλίκων ηλικίας 20-50ετων ελέγχθηκε υπερηχοργαφικά και επελέγησαν 101 περιπτώσεις που είχαν προστατικούς λίθους. Έγινε συλλογή και αξιολόγηση παραμέτρων από το ιστορικό την φυσική εξέταση και την συμπτωματολογία. Ελέγχθησαν εργαστηριακές παράμετροι του αίματος, των ούρων και του προστατικού εκκρίματος. Οι συμμετέχοντες χωρίσθηκαν σε ομάδες ανάλογα με τα χαρακτηριστικά μεγέθους(τύπος Α ή Β), αριθμού και κατανομής λίθων. Η συχνότητα της προστατική λιθίασης στην μελέτη μας ήταν 7,35%. Ηλικία άνω των 40 ετών σχετίζονταν στενά με το λιθιασικό φορτίο (p=0,034). Υπήρχε αυξημένη επίπτωση προστατίτιδας (28,7%), νεφρολιθίασης (27,3%) μεταξύ των ατόμων με προστατική λιθίαση. Οι τύπου Β λίθοι σχετίζονταν συχνότερα με συμπτώματα (p=0,007), χρόνιες μορφές προστατίτιδας (p=0,018), και αυξημένου λόγου Ca/Κρεατινίνης που είναι υποδηλωτικός ασβεστιουρίας (p=0,008). Η κεντρική εντόπιση των λίθων συνδέονταν συχνότερα με αυξημένο λόγο Ca/Κρεατινίνης (p=0,048). Η πλειονότητα (89,6%) των ατόμων με λιθίαση που δεν έχουν προστατίτιδα είναι εντελώς ασυμπτωματικοί. Οι μικρές πολλαπλές ασβεστώσεις είναι ένα φυσιολογικό, συχνά τυχαίο υπερηχογραφικό εύρημα του προστάτη που αντιπροσωπεύει περισσότερο διεργασία γήρανσης παρά μια νοσολογική οντότητα. Ωστόσο μεγαλύτεροι ή/και κεντρικότεροι προστατικοί λίθοι πιθανότατα σχετίζονται με υποκείμενη παθολογική κατάσταση που ενδεχομένως να απαιτεί διερεύνηση και θεραπεία. / The aim of the current study is to investigate the clinical significance of intraprostatic calcifications in young adults and also to examine any possible correlation with clinical or laboratory parameters concerning prostate or other diseases. A population of 1374 young adults, age 20 to 50 years, was screened with ultrasound imaging of the prostate and 101 cases with prostatic lithiasis were selected. Evaluation included history, physical examination and recording of lower urinary tract symptoms. Serum blood, urine and expressed prostatic secretion were examined with laboratory assays. The participants were divided into groups according to the size (type A or B), the number and the distribution characteristics of prostatic calculi. The prevalence of prostatic lithiasis in our series was calculated at 7,35%. Age over 40 y was closely related to calculus burden (p=0,034). There was a high incidence of prostatitis (28,7%) and nephrolithiasis (27,3%) among men with prostatic lithiasis. Type B large calculi were more often associated with symptoms (p=0,007), chronic prostatitis syndromes (p=0,018), and high Ca/Creatinine ratio suggesting hypercalciuria (p=0,008). Central localization was correlated with high Ca/Creatinine ratio (p=0,048). The great majority (89,6%) of patients with prostatic lithiasis who don’t suffer from prostatitis are completely asymptomatic. Small, multiple calcifications are normal, often incidental ultrasonographic finding in the prostate and represent a result of age rather than a pathologic entity. However, larger and /or central prostatic calculi may be related to underlying pathologic entities and require further evaluation and possibly treatment.

No description available.

Vascular calcification is the formation of mineralised tissue within the walls of arteries. The pathology has many similarities to embryonic bone formation and involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), regulate embryonic bone formation. Ceramide can be generated by lysosomal acid sphingomyelinase (L-SMase) and neutral sphingomyelinase (N-SMase), and be converted to sphingosine by acid ceramidase (ACDase) and subsequently to S1P by sphingosine kinases (SK1 & SK2). This study tested the hypothesis that ceramide and S1P also regulate VSMC matrix mineralisation. VSMCs were cultured in the presence of 3 mM β-glycerophosphate (BGP) to induce osteogenic differentiation and matrix mineralisation. During VSMC mineralisation there were decreases in the activities of L-SMase and N-SMase and increases in the levels of C18 and C20 ceramide. S1P levels also increased during mineralisation as did SK1 and SK2 mRNA and SK activity. These results demonstrate that ceramide and S1P have the potential to regulate VSMC mineralisation. The exogenous addition of C2 ceramide decreased the rate of VSMC matrix mineralisation. Consistent with this, when VSMCs were cultured with 3 mM BGP and the joint L-SMase and ACDase inhibitor, desipramine, total ceramide levels increased and no matrix mineralisation was detected. These findings suggest that ceramide is an inhibitor of VSMCs matrix mineralisation. It was also noted in the presence of 3 mM BGP and desipramine that the mineralisation-associated increase in S1P was inhibited. In agreement with this, when exogenous S1P was added to the VSMCs an increase in matrix mineralisation was observed. Thus, S1P acts as a promoter of matrix mineralisation. To determine how S1P was promoting matrix mineralisation the signalling roles of the ezrin, radixin and moesin (ERM) proteins were investigated. The short-term stimulation of VSMCs with S1P led to the phosphorylation of the ERM proteins and over the mineralisation time-course, when S1P levels increased, the levels of ERM phosphorylation also increased. When VSMCs were cultured in the presence of 3 mM BGP and the inhibitor of ezrin phosphorylation, NSC668394, a decrease in matrix mineralisation was observed. No increases in ERM phosphorylation were seen in the presence of desipramine during the mineralisation time-course Therefore, S1P may be increasing matrix mineralisation through promoting the phosphorylation of the ERM proteins. This work has demonstrated that ceramide inhibits and S1P promotes VSMC matrix mineralisation in vitro. Additionally, this work identifies activation of ERM proteins, downstream of S1P, as a novel signalling pathway promoting matrix mineralisation. Characterisation of novel regulators of VSMC matrix mineralisation in vitro gives insight into the complex mechanisms contributing to vascular calcification in vivo and will aid in identification of novel therapeutic targets.

L’hypertension artérielle pulmonaire (HTAP) est une vasculopathie progressive des vaisseaux pulmonaires. Elle est caractérisée par une augmentation anormale des résistances vasculaires pulmonaires causée par une oblitération et un remodelage des artères distales qui mènent ultimement à la défaillance cardiaque et au décès prématuré. La tomodensitométrie thoracique est un examen d’imagerie non invasive utile pour l’investigation de ces patients. Elle a une valeur essentiellement diagnostique, permettant de préciser de quel type d’HTAP il s’agit, ainsi que les conditions pulmonaires associées. On y observe également certains changements de l’arbre vasculaire pulmonaire. Récemment, le facteur de transcription Runt-related transcription factor 2 (RUNX2) fut associé à une cascade de signalisation promouvant la trans différenciation des cellules musculaires lisses en un phénotype similaire à l’ostéoblaste. Via l’augmentation de la rigidité artérielle, il fut avancé que cette voie moléculaire contribuerait à la pathogénèse de la maladie. La calcification vasculaire résulte d’un processus de remodelage similaire avec l’expression de marqueurs ostéoblastiques et une biominéralisation artérielle. Celle-ci constitue un facteur de risque cardiovasculaire bien établi et potentiellement une cible thérapeutique en athérosclérose et en hypertension artérielle. Cette calcification est visible à la tomodensitométrie grâce à sa radiodensité élevée la distinguant de la paroi vasculaire saine. Considérant le lien établi entre le phénotype ostéogénique et la calcification des artères, nous nous sommes intéressés au remodelage et à la biominéralisation artérielle pulmonaire à la tomodensitométrie thoracique et à sa valeur pronostique en HTAP. Nous avons documenté une augmentation de la radiodensité de la paroi des artères pulmonaires proximales à la tomodensitométrie. La radiodensité des artères démontra une bonne corrélation avec la calcification in situ chez une cohorte de 8 patients. Nous avons également documenté que cette radiodensité prédisait la survie chez les patients atteints d’HTAP. Ceci était indépendant des marqueurs traditionnels de sévérité clinique en HTAP, ainsi que de la réponse au traitement.