Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (208-224). Also available online through Digital Dissertations.

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.

Les dystrophies musculaires sont un groupe de pathologies génétiques qui cause une faiblesse et une perte progressive des muscles squelettiques. Parmi elles, la dystrophie des ceintures de type 2B (LGMD2B) est caractérisée par des mutations dans le gène de la dysferline, entrainant de sévères dysfonctionnements, dont un défaut de réparation membranaire. Les ruptures de la membrane plasmique sont des évènements physiologiques induits par des contraintes mécaniques, comme lors de la contraction des fibres musculaires. Les cellules eucaryotes possèdent donc une machinerie protéique assurant une réparation rapide de larges ruptures membranaires. La liste exhaustive des composants de la machinerie de réparation et leur mode d’action reste à établir.Les annexines (Anx) sont de petites protéines solubles, au nombre de 12 chez les mammifères, qui partagent la propriété de lier les membranes exposant des phospholipides chargés négativement en présence de Ca2+. De nombreuses études ont montré l’implication de certaines Anx (AnxA1, A2, A4, A5, A6 et A7) dans la réparation membranaire de différents types cellulaires (muscle, cancer, endothélium…) et dans différentes espèces (souris, poisson-zèbre, homme…). La présence des Anx dans le muscle squelettique, et la participation de plusieurs membres de cette famille dans la réparation membranaire, soulèvent la question d’un rôle collectif de ces protéines dans la protection et la réparation des ruptures du sarcolemme.Les objectifs de ce travail ont été 1) d’identifier les Anx impliquées dans la réparation membranaire des cellules musculaires squelettiques humaines, 2) développer une stratégie de microscopie corrélative pour étudier le site de rupture et la distribution subcellulaire des Anx à haute résolution, 3) élucider la fonction des Anx dans le mécanisme de réparation, et 4) analyser les Anx dans des cellules musculaires dystrophiques. Avec des approches en biologie cellulaire et moléculaire, et en microscopie de fluorescence et électronique, nous avons donc étudié le comportement des Anx lors d’un dommage du sarcolemme.Nous avons ainsi montré que les AnxA1, A2, A4, A5 et A6 sont exprimées dans les myoblastes et les myotubes humains, et sont recrutées au site de rupture quelques secondes après le dommage, en formant une structure dense à l’extérieur du myotube endommagé appelé domaine « cap ». De plus, nous avons pu déterminer l’ordre relatif de recrutement des Anx au site membranaire endommagé. Les premières Anx à être recrutées sont l’AnxA1, suivies des AnxA6 et A5, les moins sensibles au Ca2+. Les dernières Anx recrutées sont les plus sensibles au Ca2+, les AnxA4 puis A2, qui semblent se lier à des vésicules intracellulaires initialement éloignées du site de rupture. Nous avons également étudié l’ultrastructure du site de rupture à haute résolution. Nos résultats ont révélé que le domaine « cap » correspondait à une accumulation de matériel membranaire qui est associé au Anx. En s’appuyant sur nos résultats et la littérature, nous avons proposé un modèle de réparation membranaire, impliquant les AnxA1, A2, A4, A5 et A6, dans les cellules musculaires squelettiques humaines. Nous nous sommes également intéressés à l’expression des Anx dans des lignées de cellules musculaires dystrophiques issues de patients atteints de dystrophies musculaires des ceintures de type 2B (déficients en dysferline) et 1C (déficients en cavéoline-3). Nous avons ainsi montré que le contexte pathologique perturbait l’expression de certaines Anx, sans en modifier leur localisation subcellulaire.En conclusion, ce travail de thèse montre que plusieurs membres de la famille des Anx sont impliqués dans la réparation membranaire, et agissent de concert pour réparer un dommage de la membrane plasmique. L’implication des Anx dans d’autres pathologies, comme le cancer et la pré-éclampsie, renforce l’intérêt de leur étude dans les processus de réparation membranaire et en font une cible thérapeutique potentielle. / Muscular dystrophy encompasses a group of genetic disorders which cause progressive weakness and wasting of skeletal muscle. Among them, limb girdle muscular dystrophy type 2B (LGMD2B) is characterized by mutations in the dysferlin gene leading to several dysfunctions including a failure in cell membrane repair process. Cell membrane disruption is a physiological phenomenon induced by mechanical stress, such as contraction of muscle fibers. Thus, eukaryotic cells have a repair protein machinery ensuring a rapid resealing of large cell membrane ruptures. The exhaustive list of components of the repair machinery and their interplay remain to be established.The annexin (Anx) family consists of twelve soluble proteins in mammals and share the property of binding to membranes exposing negatively charged phospholipids in a Ca2+-dependent manner. Several studies have shown the involvement of Anx (AnxA1, A2, A4, A5, A6 and A7) in membrane repair of different cell types (muscle, cancer, endothelium…) in different species (mouse, zebrafish, human…). The presence of different Anx in skeletal muscle, together with the participation of several members of the Anx family in membrane repair processes, raise the question of a collective role of these proteins in the protection and repair of sarcolemma injuries.The PhD project aimed 1) at identifying Anx that are essential for membrane repair in human skeletal muscle cells, 2) developing a correlative light and electron microscopy to study the wounded site and the Anx distribution at high resolution, 3) elucidating the function of each Anx in this process and 4) analyzing Anx in dystrophic muscle cells. Using approaches including cellular and molecular biology, fluorescence microscopy and transmission electron microscopy, we studied the behavior of Anx during sarcolemma damage.We showed that AnxA1, A2, A4, A5 and A6 are expressed in human myoblasts and myotubes, and are recruited at the disruption site within seconds after the sarcolemmal damage, forming a dense structure outside the cell, named the “cap” domain. Furthermore, we determined the relative order of Anx recruitment at the disruption site. The first Anx recruited are AnxA1, followed by AnxA6 and A5, the less sensitive to Ca2+. The last Anx recruited are the most sensitive to Ca2+, AnxA4 and A2. AnxA2 and A4 are instead rapidly recruited to intracellular vesicles present deeper in the cytosol. We also studied the ultrastructure of the disruption site at high resolution. Our results revealed that the “cap” domain correspond to a disorganized membrane structure, associated with the Anx. Thanks to our results and the literature, we have proposed a model for membrane repair involving Anx in human skeletal muscle cells. We also looked at the expression of Anx in dystrophic muscle cell lines from patients with limb girdle muscular dystrophy type 2B (dysferline deficient) and 1C (deficient in cadaveoline-3). We have thus shown that the pathological context disrupts the expression of some Anx, without altering their subcellular location.In conclusion, this work shows that several members of the Anx family are involved in membrane repair and act together to repair plasma membrane damage. The implication of Anx in other pathologies, such as preeclampsia or cancer, reinforces the interest of their study in the process of membrane repair.

This study investigated proteomic changes in bronchoalveolar lavage fluid (BALF) of beef calves to identify alterations related to development of naturally occurring bovine respiratory disease. BALF was collected from 162 healthy beef calves soon after weaning and transportation. Two-dimensional gel electrophoresis and mass spectrometric analysis revealed calves that later developed pneumonia had significantly lower levels of anti-inflammatory proteins including annexin A1, RAGE-binding protein, apolipoprotein-A, heat shock protein beta-1 and thioredoxin, but higher levels of antioxidant and pro-inflammatory proteins such as immunoglobulin light chain variable region, cyclophilin A, serum albumin precursor and glutathione S-transferase P. Difference in gel electrophoresis-based analysis further showed lower levels of annexin A1, annexin A2, peroxiredoxin I, calycyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3 in the calves that later developed pneumonia. Differences in annexin levels were partially confirmed by Western blot analysis. In healthy calves, immunohistochemistry revealed cytoplasmic expression of annexin A1 in surface epithelium of large airways, tracheobronchial submucosal glands, and goblet cells, and to a lesser degree in small airways but not in alveolar epithelium. Flow cytometry and immunocytochemistry labeled annexin A1 in blood and bronchoalveolar lavage neutrophils, monocytes, macrophages and lymphocytes. Annexin A2 expression was detected in surface epithelium of small airways, some mucosal lymphocytes, and endothelium, with weak expression in large airways, tracheobronchial submucosal glands and alveolar epithelium. For both proteins, the level of expression was similar in tissues collected 5 days after intrabronchial challenge with M. haemolytica compared to that from sham-inoculated calves. A sandwich ELISA for annexin A1 was developed. For use with BALF, the working range was 0.3-317 ng/ml and the sensitivity was 0.8 ng/ml. The coefficient of variation of intra-assay and the between assays was less than 20%. Together, these findings reveal annexins A1 and A2 as promising biomarkers of susceptibility to BRD in healthy at-risk calves. Further, the anti-inflammatory and pro-resolving functions of these proteins suggest roles in the pathogenesis of bacterial pneumonia of feedlot cattle. / Natural Sciences and Engineering Council (NSERC), Ontario Cattlemen’s Association, Ontario Ministry of Agriculture and Food and Ontario Veterinary College Fellowship Program