Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Abstract Vasostatin, the N-terminal 180 amino acids domain of calreticulin, induces apoptosis in endothelial cells and inhibits angiogenesis. However, the mechanism underlying the apoptosis induce by vasostatin remains elusive. In the present study, we investigated the role of (1) Fas /FasL pathway, (2) oxidative stress, and (3) nitric oxide (NO) in the apoptotic mechanism of vasostatin in endothelial cells. Recombinant vasostatin was generated and shown to induce apoptosis of bovine aortic endothelial cells (BAEC) as demonstrated by flow cytometry analysis, nucleus staining, and DNA fragmentation assay. Vasostatin elevated the levels of Fas and its adaptor, FADD, in BAEC. Furthermore, vasostatin treatment increased the activities as well as the expression of active form of caspase-8 and caspase-3 in BAEC. However, pretreatment with either caspase-3 inhibitor or caspase-8 inhibitor alone was not sufficient to blockade the vasostatin-mediated apoptosis, suggesting the involvement of other pathways. Extensive screening using an array of caspase inhibitors further supported such notion. Oxidative stress is frequently involved in the apoptosis of endothelial cells. Previous studies indicated that vasostatin enhanced WST-1-derived formazan formation despite its cytotoxic effect, suggesting vasostatin treatment might enhance the production of superoxide. By measuring the level of superoxide anion in cultured media by cytochrome c reducing test, it was found that vasostatin treatment increased the production of superoxide anion in endothelial cells. Antioxidants such as NAC, GSH, BHA partially attenuated the vasostatin-mediated cytotoxicity and cell death in endothelial cells. Noteworthingly, adding allopurinol, inhibitor of xanthine oxidase, but not other oxidase inhibitors abrogated the cytotoxicity of vasostatin, indicating that xanthine oxidase could be the source of ROS produced by vasostatin relate with apoptosis. The elecctrophoretic mobility shift assays (EMSA) suggested that vasostatin treatment increased the NF£eB DNA binding activity. Western blot analysis indicated vasostatin increased the levels of NF£eB but decreased I£eB level, which seemed to coincide with the EMSA findings. NO plays an important role in endothelial function. To investigate the role of NO in the cytotoxicity by vasostatin, analyzed the levels of NO metabolites in cultured media of endothelial cells and found that vasostatin treatment increased NO release in time- dependent manners. The expression of eNOS, but not iNOS, in endothelial cells was upregulated by vasostatin. Besides, vasostatin treatment also increased the AP-1 binding activities. Moreover, NOS inhibitor, L-NAME, or NO scavenger, carboxy-PTIO, slightly attenuated the cytotoxic effects of vasostatin in endothelial cells. In addition to direct cytotoxicity, NO may react with superoxide (O2-) to form peroxynitrite (ONOO-), which attacked the intracellular protein and caused the cell damage. Indeed, we also detected a dose-dependent increment in the nitrotyrosination of cellular protein by vasostatin treatment. Taking together, these results indicate that vasostatin induces apoptosis in endothelial cells via multiple pathways. The interactions between these distinct pathways remain to be elucidated in the future.

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Endothelial cells separate the bloodstream from the underlying tissue and play a crucial role in vascular homeostasis. They also form an important barrier for vascular drug delivery. This thesis contains preliminary studies targeted at understanding the mechanisms of binding and transport across endothelial cells cultured in vitro. Specifically, the first study investigates how the recombinant source of Factor IX (FIX), a blood coagulant protein used in the treatment of Hemophilia B, impacts surface ligand binding (FIX to its specific receptors) to bovine aortic endothelial cells (BAECs). Competitive binding experiments between 125I-FIX and FIX were undertaken to quantify the interaction of recombinant and transgenic FIX with BAECs and human collagen IV and determine if there was a measurable difference in binding affinity. Results indicate limited specific binding of 125I-FIX to BAECs and no binding to human collagen IV. Concrete conclusions were not drawn from this data due to technical issues during the experimental process. The second study investigates insulin-like growth factor-I (IGF-I) transport across both BAEC and MAC-T cells, a mammary epithelial cell line, cultured on tissue culture inserts. IGF-I is a circulatory growth factor implicated in the regulation of cell division and tissue proliferation. Competitive binding experiments between 125I-IGF-I and unlabeled protein (IGF-I, Y60L-IGF-I, a mutant of IGF-I, and IGF Binding Protein-3 (IGFBP-3)) were undertaken to quantify the binding and transport of IGF-I under various experimental conditions. Results confirmed earlier work from the Williams' laboratory indicating that 125I-IGF-I transport was enhanced by incubation with its non-receptor-binding analog, Y60L-IGF-I, but cell surface associated 125I-IGF-I was decreased by its presence. Other studies were undertaken but conclusive results could not be drawn. / Master of Science