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Vascular calcification is a prominent cardiovascular condition found worldwide. This condition is predominantly found in the elderly population, and patients who suffer from chronic kidney disease, due to an imbalance of serum phosphate and calcium levels. For many years, vascular calcification was believed to be a passive pathological process which develops with ageing and/or lifestyle. Little has been documented about the disease until the 20th century, when interest in cardiovascular research grew amongst scientists. Indeed, vascular calcification underpins severe clinical outcomes and cardiovascular diseases have been labelled the global leading cause of death. Calcific aortic valve diseases (CAVD) is a progressive degenerative condition characterised by the development of lipo-calcification around the aortic valve leaflets leading to severe aortic stenosis and aortic regurgitation, which may ultimately lead to heart failure. At present there are no pharmaceutical therapies that can stop its progression and its molecular mechanisms are not fully understood. Recent findings have suggested that vascular smooth muscle cell (VSMC) calcification shares many common features with physiological skeletogenesis via the release of matrix vesicles (MVs), which are specialised structures that initiate mineralisation during bone formation. The ability for MVs to nucleate calcium and phosphate highly depend on their protein composition, as this may vary depending on active cell signalling and the microenvironment. This mechanism involving MV-regulated calcification has yet to be examined in CAVD. In this study, examined whether calcium and/or phosphate regulate VIC-derived MVs to induce calcification in the aortic valve. I used a primary rat valve interstitial cell (VIC) model, coupled with stenotic human valve tissues to characterise and study the mechanisms underpinning CAVD. X-ray fluorescence and diffraction analysis showed the mineral found in calcified human aortic valves to be hydroxyapatite (HA), the main component in bone. Additional imaging studies employing transmission electron microscopy (TEM) revealed particles that were similar in size and morphology to skeletal MVs. To further characterise VIC-derived MVs in vitro, I harvested MVs from rat VICs, and subsequently studied their protein composition using Isobaric tag for relative and absolute quantitation (iTRAQ) mass spectrometry. The data obtained from the proteomics analysis was compared to previous published studies on MV proteins derived from osteoblasts and VSMCs. The results showed the upregulation of numerous calcification regulators in MVs isolated from all 3 cell types, in particular, the Annexin family, which are known calcium binding proteins. Further studies conducted with Annexin 6, an established calcium regulator in arterial calcification, revealed its colocalisation with MV-enriched areas in calcified human aortic valve tissue suggesting it may play an important role in calcium regulation during CAVD.

The most active form of Vitamin D, 1alpha,25(OH)2D3, modulates cells via receptor mediated mechanisms. While studies have elucidated the pathway via the classical nuclear Vitamin D Receptor (VDR), little is known about the membrane-associated Vitamin D Receptor (ERp60). Caveolae and its characteristic protein Caveolin-1 have been involved in many signaling pathways due to its specific structure and physical configuration. Other studies have shown that many components of the Vitamin D pathway have been found in caveolae. This study hypothesizes that caveolae and Caveolin-1 are important for the effects of 1,25 Vitamin D signaling via ERp60. Research up to date have shown that in rat and mouse growth zone chondrocytes, cells deprived of intact caveolae either through disruption through beta-Cyclodextrin or genetic knockout do not exhibit the characteristic responses to Vitamin D through ERp60 when compared to chondrocytes with functional caveolae. Studies using immunofluorescence co-localization and caveolae fractionation have shown that ERp60 is localized in the caveolae domains. Cellular fractionation was also performed to examine the localization of the ERp60 receptor in lipid rafts and caveolae. Histology and transmission electron microscopy were also used to examine the physiological importance of caveolae and Caveolin-1 in growth plate morphology and cellular characteristics.

Les vésicules matricielles sont des organites extracellulaires impliques dans les processus de minéralisation. Nous avons détermine le mode d’action du strontium, ion contenu dans le principe actif d’un médicament antiostéoporotique sur les vésicules matricielles. Nous avons montre que le strontium agit en fonction de la concentration aussi bien sur la formation de l’hydroxyapatite que sur les activités phosphomonoéstérase et phosphodiestérase de la phosphatase alcaline tissu non spécifique. Pour des faibles concentrations (comprises entre 25 et 100^M), le strontium augmente l’activité phosphodiestérase et inhibe partiellement l’activité phosphomonoestérase. La balance entre la production de PPi, inhibiteur de la minéralisation et la production de Pi, essentiel a la formation d’hydroxyapatité pourrait être affectée par le strontium. / The matrix vesicles are extracellular organelles implicated in the process of mineralization.We determined the mode of action of strontium, ion contained in the active principle of an antiosteoporotic drug on the matrix vesicles. We showed that the action of strontium is concentration dependent on the hydroxyapatite formation and on phosphomonoesterase and phosphodiesterase activities of the tissue non specific alkaline phosphatase. At low concentration (between 25 and 100^M), strontium increased phosphodiesterase activity and inhibited partly phosphomonoesterase activity. The balance between the production of PPi, inhibitor of mineralization and the production of Pi, essential in the formation of hydroxyapatite could be affected by the strontium.

Trois dérivés d’amino acides sont reconnus d’une manière stéréo sélective par l’albumine du sérum bovin. Cette propriété a été observée dans le cas de la phosphatase alcaline de tissu non spécifique, (TNAP). Des inhibiteurs agissant à trois niveaux distincts sur les processus de minéralisation ont été cherchés: 1) TNAP ; 2) Formation de l’hydroxyapatite (HA); 3) Vésicules maticielles (VMs). Nous avons trouvé que des dérivés de benzothiophènes et de tétramisoles, solubles dans l’eau, sont des inhibiteurs spécifiques de TNAP. Un modèle qui permet de produire du HA, a été développé et a confirmé que les nucléotides sont des inhibiteurs de formation de HA. Nous avons montré que le médicament anti-rhumatisme sinomenine, n’ayant aucun effet sur le TNAP, ainsi que la théophylline ralentissaient tous les deux la formation de HA induits par les VMs. Ces modèles de minéralisation présentent un grand potentiel lors du criblage de médicaments pour le traitement de l’ostéoarthrose / Three amino acid derivatives were stereoselectively recognized by bovine serum albumin. Such property was also observed in the case of tissue non-specific alkaline phosphatase (TNAP), a marker in mineral formation. Inhibitors acting at three distinct levels on mineral formation were searched: 1) TNAP; 2) Hydroxyapatite (HA) formation; 3) Matrix vesicle (MV). We found that benzothiophene derivative of tetramisole are water soluble inhibitors of TNAP. A model producing HA as MVs was developed and served to screen HA inhibitors, confirming that several nucleotides inhibited HA formation. We demonstrated that the anti-rheumatic Chinese medicine sinomenine, having no effect on TNAP and theophylline, slowed down HA induced by MVs. The mineralization models presented a great potential to screen putative drugs to cure ostoarthritis.