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

Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1β in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells. IL-1β significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1β prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1β significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1β inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1β impedes the process of angiogenesis in myocardial endothelial cells.

Η ανάγκη εξατομίκευσης της θεραπείας ασθενών που πάσχουν από καρκίνο κρίνεται επιτακτική, λόγω του στενού θεραπευτικού εύρους των αντινεοπλασματικών φαρμάκων και των παρατηρούμενων δια-ατομικών διαφορών στη φαρμακοκινητική και στην κλινική ανταπόκριση. Ένα πολύ χρήσιμο εργαλείο στην εξατομίκευση της θεραπείας θεωρείται ότι είναι η πληθυσμιακή φαρμακοκινητική-φαρμακοδυναμική μοντελοποίηση, καθώς μπορεί να περιγράψει τις σχέσεις δόσης-ανταπόκρισης, να εξηγήσει την παρατηρούμενη μεταβλητότητα στην έκθεση στο φάρμακο ή στην κλινική ανταπόκριση και να καθοδηγήσει την επιλογή της δόσης βάσει της βέλτιστης αναλογίας οφέλους-κινδύνου για τη δεδομένη θεραπεία. Η παρούσα διδακτορική διατριβή επικεντρώθηκε στην μπεβασιζουμάμπη, ένα σχετικά καινούριο φάρμακο στοχευμένης θεραπείας για το οποίο δεν υπάρχουν αρκετά διαθέσιμα στοιχεία που αφορούν στη φαρμακοκινητική και φαρμακοδυναμική συμπεριφορά του. Σκοπός της συγκεκριμένης μελέτης ήταν ο χαρακτηρισμός της αλληλεπίδρασης της μπεβασιζουμάμπης με τον μοριακό της στόχο, VEGF165, σε ενήλικες ασθενείς με μεταστατικό ορθοκολικό καρκίνο που λαμβάνουν το φάρμακο σε συνδυασμό με χημειοθεραπεία (FOLFIRI, FOLFOX ή CAPIRI). Για αυτόν τον λόγο, προσδιορίστηκαν αρχικά οι συγκεντρώσεις της ολικής μπεβασιζουμάμπης και του ελεύθερου VEGF165 σε διάφορους κύκλους θεραπείας και στη συνέχεια, εφαρμόζοντας τη μη γραμμική μικτών επιδράσεων μοντελοποίηση με το υπολογιστικό πρόγραμμα NONMEM 7.3, αναπτύχθηκε ένα φαρμακοκινητικό μοντέλο σύνδεσης της μπεβασιζουμάμπης με τον VEGF165. Επιπλέον, διερευνήθηκε η επίδραση των δημογραφικών δεδομένων και των VEGF μονονουκλεοτιδικών πολυμορφισμών στην αλληλεπίδραση μεταξύ της φαρμακοκινητικής της μπεβασιζουμάμπης και των συγκεντρώσεων του VEGF165. Είναι η πρώτη φορά που χρησιμοποιήθηκε η TMDD προσέγγιση για τον χαρακτηρισμό της in vivo αλληλεπίδρασης μπεβασιζουμάμπης-VEGF165. Σύμφωνα με αυτή την προσέγγιση, η φαρμακοκινητική ενός φαρμάκου επηρεάζεται από την υψηλής συγγένειας δέσμευση με τον μοριακό του στόχο και την επακόλουθη αποικοδόμηση του σχηματιζόμενου συμπλόκου μέσω ενδοκυττάρωσης. Το αναπτυχθέν μοντέλο επέτρεψε την ικανοποιητική περιγραφή της φαρμακοκινητικής της μπεβασιζουμάμπης και των ιδιοτήτων σύνδεσής της με τον VEGF165. Η κάθαρση της μπεβασιζουμάμπης βρέθηκε να είναι 0.18 L/day, η τιμή αναφοράς της συγκέντρωσης του ελεύθερου VEGF165 ήταν 212 ng/L, η σταθερά του ρυθμού απομάκρυνσης του ελεύθερου VEGF165 ήταν 0.401 day-1 και η Kss ήταν 267 nM. Η επίδραση του πραγματικού σωματικού βάρους συνυπολογίστηκε στην εκτίμηση όλων των φαρμακοκινητικών παραμέτρων του μοντέλου. Κάποιες στατιστικά μη σημαντικές συσχετίσεις παρατηρήθηκαν μεταξύ της συγγένειας δέσμευσης του φαρμάκου και των VEGF-2578C/A και VEGF-634G/C πολυμορφισμών. Το αναπτυχθέν μοντέλο θα μπορούσε να αποτελέσει ένα χρήσιμο εργαλείο στην εξατομίκευση της θεραπείας και στην αξιολόγηση της κλινικής ανταπόκρισης ασθενών που λαμβάνουν μπεβασιζουμάμπη σε συνδυασμό με χημειοθεραπεία. / The need for individualized treatment in cancer patients is considered crucial due to the narrow therapeutic range of antineoplastic drugs and the observed inter-individual differences in pharmacokinetics and clinical response. Population pharmacokinetic-pharmacodynamic modeling has been recognized as a beneficial tool for personalizing treatment, as it can describe the dose-response relationships, explain the observed variability in drug exposure or response and guide dose selection based on the optimal benefit-risk ratio for a given treatment. The current doctoral thesis was focused on bevacizumab, a relatively new targeted therapy drug for which no sufficient data are available regarding its pharmacokinetic and pharmacodynamic behavior. The aim of the present study was to characterize the interaction of bevacizumab with its molecular target, VEGF165, in adult patients with metastatic colorectal cancer who receive the drug in combination with chemotherapy (FOLFIRI, FOLFOX or CAPIRI). For this reason, the concentrations of total bevacizumab and free VEGF165 were first determined in different cycles of treatment and then, a pharmacokinetic model for bevacizumab binding to VEGF165 was developed by using nonlinear mixed-effects modeling implemented in NONMEM 7.3 software. Moreover, the effect of demographic data and VEGF single nucleotide polymorphisms on the interplay between bevacizumab pharmacokinetics and VEGF165 concen-trations was investigated. This is the first time the TMDD approach was applied to characterize the in vivo bevacizumab-VEGF165 interaction. According to this approach, the pharmacokinetics of a drug is affected by its high affinity binding to its molecular target and subsequent degradation of the formed complex via endocytosis. The developed model allowed an adequate description of bevacizumab pharmacokinetics and its binding properties to VEGF165. Bevacizumab clearance was found to be 0.18 L/day, the free VEGF165 concentration at baseline was 212 ng/L, the elimination rate constant of free VEGF165 was 0.401 day-1, and Kss was 267 nM. The effect of actual body weight was taken into account in the estimation of all pharmacokinetic model parameters. Correlations, which were not statistically significant, were noticed between the binding affinity of the drug and the VEGF-2578C/A and VEGF-634G/C polymorphisms. The developed model could become a useful tool for individualizing treatment and evaluating clinical response of patients receiving bevacizumab in combination with chemotherapy.

Endometrial cancer is one of the most significant gynaecological malignancies that affect women from New Zealand and the rest of the world. One of the critical stages in the development of a tumour is the onset of hypoxia. The malignancy responds by having raised levels of Hypoxia Inducible Factor (HIF) that in turn induces increased production of Vascular Endothelial Growth Factor (VEGF). VEGF is a potent angiogenic factor that will mediate vascular supply of nutrients and oxygen to the developing tumour. The aim of this study was to investigate whether two compounds found in extracts of plant materials, Resveratrol (Resveratrol) and Epigallocatechin gallate (EGCG), altered the levels of VEGF in the supernatant of cultured endometrial cancer cells. Resveratrol is a phytoalexin that is found in many foods, such as grapes, nuts and berries, as well as in high concentrations in some red wines. 100 µM of resveratrol was added to cell cultures for 24 hours. VEGF levels in the supernatant were then analysed using ELISA. Resveratrol was found to have significant inhibitory effects in both primary endometrial cancer cell cultures and immortalised endometrial cancer cell cultures. Resveratrol was also shown to reverse the increase in VEGF caused by the hypoxia mimic cobalt chloride (CoCl₂). Epigallocatechin gallate (EGCG) is an antioxidant catechin extracted from green tea. The effect of EGCG was analysed using the same method as for resveratrol. 100 µM of EGCG was also shown to have a significant inhibitory effect on the level of VEGF in the supernatant of cultured endometrial cancer cells, as well as reducing the effect of CoCl₂. These results suggest that selected food compounds, resveratrol and EGCG, can reduce VEGF levels by inhibiting HIF. Further investigation This may have anti-tumour effects in women with endometrial cancer.

Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.

Understanding the processes that control the formation of blood (haematopoiesis), and blood vessels (vasculogenesis and angiogenesis) in vivo has huge clinical importance. The complex three-dimensional architecture of blood vessels is dynamic and aberrant regulation of either the growth or function of the vascular system may potentiate the spread of tumours, resulting in failure of physiological processes such as implantation and placental development, leading to a range of angiogenesis associated disorders for example diabetic retinopathy. Both embryonic and adult haematopoiesis are also three-dimensional, dynamic processes in which deregulation may result in blood disorders or leukaemia. The experiments herein describe my contribution to investigations into the molecular mechanisms involved in haematopoiesis and angiogenesis over a period of approximately 15 years, taking advantage of technical advances as they became available and adapting them to specific cell models. For example, microarray technology has facilitated discovery of new pathways and transcripts implicated in normal and pathological angiogenesis; central to this mechanism is the role of vascular endothelial growth factor (VEGF), a mitogen specific to endothelial cells. Chromosome immunoprecipitation (ChIP) technology subsequently revealed pathways of early mesoderm formation and the importance of gastrulation in this process. Transcriptional targets of the T-box transcription factor Brachyury were subsequently determined. Throughout this work, the human female reproductive tract provided a unique resource, as one of the rare sites of physiological angiogenesis with which to investigate endothelial cell biology and haematopoiesis. Embryonic stem cell-derived embryoid bodies subsequently proved to be an excellent model for the study of early blood vessel development in three dimensions (2003-5), and to follow early mesoderm development (2006-2010). Targets of Brachyury revealed the close association between blood vessel development, haematopoiesis and early mesoderm formation via a common haemangioblast precursor for blood and endothelial cell lineages. Data gathered by myself, and colleagues, from gene expression and transcription factor analysis is now being used to create lineage codes or routemaps for differentiation of stem cells to mature cells in-vitro and it is now possible to produce mature megakaryocytes and erythrocytes in vitro. The current challenge is to produce fully functional human platelets and enucleated red blood cells. Combined with the use of autologous induced pluripotent stem cells (iPSCs) this makes patientspecific tailoring of cell-based therapies a real possibility.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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

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

This study determines effects of oxygen levels on morphology and VEGF expression of developing chicken lungs following incubation in normoxia (21% O2), hypoxia (15% O2) or hyperoxia (30% O2), until developmental days 16 or 18. Lung morphology was assessed using light microscopy, while VEGF expression was determined with ELISA. In hypoxia, the proportion of parabronchial tissue and parabronchi including lumina increased from day 16 to 18 (61 to 68% and 74.2 to 82.2%, respectively). Non-parabronchial tissue was higher in hypoxia than in hyperoxia on day 16 (26 to 20%). However, by day 18, there were no differences between groups. VEGF expression was 33% higher in hypoxia than in hyperoxia on day 16 (736 vs. 492 pg/ml). On day 18, VEGF expression was 43% higher in hyperoxia than in normoxia (673 to 381pg/ml), and remained elevated by 40% in hypoxia over normoxia (631 pg/ml). VEGF may be a mechanism by which parabronchial tissue is stimulated from day 16 to 18 following exposure to chronic hypoxia.