Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

Phytochemicals are widely noted for their role in chemoprevention. Potato (Solanum tuberosum L.) is the third most important food crop worldwide and is considered a significant source of antioxidants, providing an ideal delivery system for beneficial compounds. The anti-proliferative and pro-apoptotic properties of potato bioactive compounds have been reported in vitro on human prostate cancer cell lines. However, in vivo studies are limited, and more information is needed to determine the chemopreventive properties of potato in the diet. The objective of this study was to evaluate the effects of potato bioactives on prostate cancer in vivo using a mouse model. Athymic nude mice received xenografts of human prostate cancer cells (PC-3) and were administered extracts of potato bioactives from either the white flesh Solanum bulbocastanum (PI243510) or CO112F2-2P/P (purple-flesh Colorado selection), while control mice received water. Neither potato extract provided a significant reduction in tumor growth nor reduced levels of the pro-angiogenic protein VEGF, but the S. bulbocastanum extract reduced expression of metastasis associated protein 1 (MTA1) in tumors, and both potato extracts reduced MTA1 expression in lungs, suggesting the need for further research on the potential chemopreventive or chemotherapeutic properties of potato bioactives.

Preeclampsia results from incomplete trophoblast invasion of the spiral arteries during early pregnancy. Vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) are critical factors involved in angiogenesis, invasion and hemostasis at the maternal-fetal interface. Both factors are transcriptionally regulated by hypoxia inducible factor (HIF), a heterodimeric complex consisting of HIF-1[beta] and either HIF-1[alpha] or -2[alpha] whose specificity or redundancy in gene regulation is cell-type specific. This study uses siRNA technology to dissect the mechanisms of hypoxia-mediated regulation of PAI-1 and VEGF expression in first trimester trophoblasts. Immortalized first trimester human extravillous trophoblasts (HTR8/SVneo cells) were maintained in serum-free and serum-containing media for 4h (n=3-4), 8h (n=6), 24h (n=5) and 48h (n=5) under normoxic (21% O2) and hypoxic (1-2% O2) conditions to determine a time of maximum induction of both VEGF and PAI-1. Subsequently, cells were maintained for 48h in the presence or absence of siRNA for HIF-1[alpha], HIF-2[alpha], HIF-1[alpha] + -2[alpha], a non-targeting (NT) sequence or Cyclophilin B (CB). Media were then removed, cells lysed, and Western blotting used to assess HIF-[alpha] knockdown. VEGF and PAI-1 levels in the media were quantified by ELISA and results expressed as pg or ng/[micro]g protein. Results from 3 to 8 independent experiments were analyzed using unpaired t-tests. Under hypoxic conditions treatment of cells with HIF-1[alpha], HIF-2[alpha] or HIF -1[alpha] + -2[alpha] siRNA resulted in >90% HIF-Ñ protein knockdown as determined by Western blotting. 48h of hypoxic treatment caused a statistically significant increase in PAI-1 levels (p<0.01) and VEGF levels (p<0.001) compared to normoxic controls. Under hypoxic conditions, PAI-1 levels were 4.75 [plus-minus] 0.46 ng/[micro]g protein and VEGF levels were 7.27 [plus-minus] 1.08 pg/[micro]g protein. Treatment with siRNA to HIF-1[alpha], HIF-2[alpha] and HIF-1[alpha] + -2[alpha] significantly reduced PAI-1 levels to 3.3 [plus-minus] 0.35 (p<0.02), 3.1 [plus-minus] 0.38 (p<0.03) and 2.4 [plus-minus] 0.19 (p<0.003), respectively. No significant difference in PAI-1 reduction was noted between the three HIF siRNA conditions. Under hypoxic conditions, levels of VEGF in cells treated with siRNA to HIF-1[alpha] (5.79 [plus-minus] 0.55), HIF-2[alpha] (5.50 [plus-minus] 1.24) and HIF-1[alpha] + -2[alpha] (4.24 [plus-minus] 0.93) were reduced compared to the hypoxic control (7.27 [plus-minus] 1.08), yet these effects did not reach statistical significance. However, when compared with the levels observed in cells treated with NT siRNA (9.90 [plus-minus] .98), all HIF siRNA treatments promoted a significant reduction in VEGF expression (p<0.003, p<0.02 and p<0.003 for HIF-1[alpha], HIF-2[alpha] and HIF-1[alpha]+ -2[alpha], respectively). In conclusion, these results indicate that hypoxia-mediated changes in PAI-1 and VEGF expression in trophoblasts are regulated similarly by both HIF-1[alpha] and HIF-2[alpha]. This provides important insight into the molecular mechanisms regulating hemostasis and trophoblast invasion as well as their potential dysfunction in pregnancies complicated by preeclampsia

Σκοπός της μελέτης ήταν η ανάδειξη της νεοαγγειογένεσης και ο προσδιορισμός της έκφρασης των VEGF-A, FGF-2, COX-2, AR και BCL-2 στην καλοήθη υπερπλασία και το αδενοκαρκίνωμα του προστάτη. Το υλικό αφορούσε σε δείγματα προστατικού ιστού μονιμοποιημένα και εγκλεισμένα σε παραφίνη, από 24 περιστατικά καλοήθους υπερπλασίας και 139 περιστατικά προστατικού αδενοκαρκινώματος. Τα τελευταία χωρίστηκαν περαιτέρω σε 3 υποομάδες (Grade I, II και ΙΙΙ) ανάλογα με το βαθμό διαφοροποίησης του νεοπλάσματος κατά Gleason (2-4, 5-7 και 8-10 αντίστοιχα). Χρησιμοποιήθηκε ανοσοϊστοχημική μέθοδος Βιοτίνης-Στρεπταβιδίνης-Υπεροξειδάσης, και εφαρμόστηκε ημιποσοτική μέθοδος για την εκτίμηση της ανοσοϊστοχημικής χρώσης. Τα ευρήματά μας ανέδειξαν σαφή αύξηση της νεοαγγείωσης (MVD) στο αδενοκαρκίνωμα του προστάτη σε σχέση με την καλοήθη υπερπλασία, η οποία εμφάνισε στατιστικώς σημαντική θετική σχέση με το βαθμό κακοήθειας των προστατικών νεοπλασμάτων (ANOVA p<0.001) και με την έκφραση των VEGF-A και COX-2 (ANOVA p<0.001). Αναδείχθηκε αντίστροφη συσχέτιση της πυρηνικής έκφρασης του ανδρογονικού υποδοχέα (AR) με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.0001) και την έκφραση του VEGF-A στο προστατικό στρώμα (p<0.001 Spearman r = -0.312). Η έκφραση της BCL-2 παρουσιάστηκε αυξημένη στα προστατικά αδενοκαρκινώματα και σχετίστηκε με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.001) και την έκφραση των VEGF-A και COX-2 (p<0.001). Η έκφραση του VEGF-A σε όλα τα περιστατικά αδενοκαρκινώματος του προστάτη εμφάνισε στατιστικώς σημαντική σχέση με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.0001). Ωστόσο στα πτωχής διαφοροποίησης αδενοκαρκινώματα η μέση τιμή της έκφρασης του VEGF-A παρουσίασε πτώση. Αντίθετα, η σημασία της COX-2 στον προστατικό καρκίνο αναδείχθηκε με την έκφρασή της τόσο στην καλοήθη υπερπλασία όσο και στα αδενοκαρκινώματα του προστάτη. Η έκφραση παρουσίασε σημαντική σχέση με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.01). Η σαφώς αυξημένη έκφραση της COX-2 σε σχέση με τη μείωση της έκφρασης του VEGF-A στα πτωχής διαφοροποίησης νεοπλάσματα πιθανόν υποδηλώνει την ύπαρξη ενός αγγειογενετικού διακόπτη στα νεοπλάσματα αυτά όπου η COX-2 φαίνεται να παίζει σημαντικότερο ρόλο από τον VEGF-A. Η σημαντική αυτή πληροφορία θα μπορούσε να βρει πιθανή θεραπευτική εφαρμογή, καθώς σε πτωχής διαφοροποίησης αδενοκαρκινώματα του προστάτη ίσως η θεραπεία με COX-2 εκλεκτικούς αναστολείς να είχε πολύ καλύτερα αποτελέσματα από τις αντι-αγγειογενετικές θεραπείες με αναστολείς του VEGF. / The aim of this study was to immunohistochemically evaluate the expression of VEGF-A, FGF-2, COX-2, AR and BCL-2 in benign prostatic hyperplasia (BPH) and prostate carcinoma in relation to microvessel density (MVD) and the Gleason grade of the neoplasms. A total of 139 cases of primary prostate carcinoma and 24 cases of benign hyperplasia were included in the study. Tumors were graded according to the Gleason grading system and further divided into 3 subgroups (GRADE I, II and III). The immunostaining was performed according to the Streptavidin-Biotin Complex Peroxidase method, in formalin fixed paraffin-embedded tissue. Mean micro vessel density (MVD) was strongly related to tumor grade, VEGF-A and COX-2 histoscore (ANOVA, p<0.001). The androgen receptor was localized in the nuclei of prostate epithelial cells in 97% of cases. The comparison of AR staining with tumor grade revealed an inverse relationship between these two parameters (ANOVA, p<0,0001). An interesting finding was the inverse relationship of stromal AR expression in relation to VEGF-A immunoreactivity. BCL-2 expression was correlated with tumor grade in prostate carcinoma cases (p<0.001) and was strongly correlated with COX-2 and VEGF-A expression (p<0.001). These findings suggest that BCL-2 may play a dual role in tumorigenesis, possibly through an angiogenetic axis. VEGF-A expression was detected in only 17% of BPH cases but all prostate cancer specimens demonstrated some degree of immunoreactivity. COX-2 immunopositivity was present in 54% of BPH specimens and in 99% of primary prostate carcinomas. The increased COX-2 expression correlated significantly with Gleason grade. In our study, high-grade neoplasms presented low to moderate VEGF staining intensity compared to COX-2 expression. These results suggest the activation of an angiogenic switch in poorly differentiated neoplasms, where COX-2 may play a crucial role compared to VEGF and the possible key role of COX-2 in poorly differentiated cancers. According to our findings, anti-VEGF therapy could prove to be more beneficial in patients with low-grade disease, while patients with high-grade prostate carcinoma are more likely to respond to selective COX-2 inhibitors. Immunohistochemical determination of VEGF-A and COX-2 content might prove a useful tool in the design of patient-tailored, anti-angiogenic treatments.

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Résumé L’angiogenèse est l’un des processus les plus importants pour le maintien de l’homéostasie de l’oxygène dans les tissus. Le facteur de croissance de l’endothélium vasculaire, VEGF, joue un rôle primordial dans la réponse angiogénique. Ce facteur de croissance mène à l’activation du récepteur de type 2 du facteur de croissance de l’endothélium vasculaire, VEGFR-2. Suite à une activation du VEGFR-2, plusieurs cascades de signalisation sont activées dans les cellules endothéliales. Afin d’atténuer cette signalisation, le VEGFR-2 est multi-ubiquitiné sur des résidus lysine et de cette manière, il est amené aux voies de dégradation, principalement dans les lysosomes. Cette ubiquitination est induite par l’association de l’ubiquitine ligase (E3) c-Cbl à un résidu tyrosine phosphorylé du domaine C-terminal du récepteur. Dans cette étude, nous avons identifié la tyrosine 1319 comme étant nécessaire pour l’association de c-Cbl au VEGFR-2 et son ubiquitination. Nos résultats démontrent aussi que dans des cellules endothéliales aortiques bovines, BAEC, la surexpression du récepteur mutant Y1319F ralentit la dégradation du VEGFR-2 et induit une activation plus forte et prolongée de la synthétase endothéliale du monoxyde d’azote (eNOS). Ces résultats nous permettent de mieux comprendre le déroulement de la régulation de la signalisation du VEGFR-2 au niveau intracellulaire. Mots-clés: [Angiogenèse, VEGFR-2, VEGF, c-Cbl, Ubiquitination, Tyrosine 1319, Dégradation] / Abstract Angiogenesis is one of the most important processes to maintain oxygen homeostasis throughout the different tissues. The different signaling pathways of the vascular endothelial growth factor receptor 2, VEGFR-2, play a primordial role in the angiogenic response induced by different angiogenic factors, one of which is the vascular endothelial growth factor, VEGF. Following VEGFR-2 activation, many signaling cascades are triggered in endothelial cells; in order to attenuate this response, VEGFR-2 undergoes multi ubiquitination on lysine residues and in this fashion it is brought into the degradation pathways, mainly through the lysosomes. This ubiquitination is induced by the association of the ubiquitin ligase (E3) c-Cbl to a phosphorylated tyrosine residue in the c-terminal domain of VEGFR-2. In this study, we identified tyrosine residue 1319 as being necessary for the association of c-Cbl to VEGFR-2 and for its ubiquitination. Our results show as well that overexpression of the mutant Y1319F version of VEGFR-2 in bovine aortic endothelial cells, BAEC, slows down the degradation process of VEGFR-2 and at the same time increases and prolongs the activation of endothelial nitric oxide synthase, eNOS. These results allow us to better understand the process of VEGFR-2 signaling regulation at the intracellular level. Keywords: [Angiogenesis, VEGFR-2, VEGF, c-Cbl, Ubiquitination, Tyrosine 1319, Degradation]

Neuropilin (Nrp) is an essential cell surface receptor with dual functionality in the cardiovascular and nervous systems. The first identified Nrp-ligand family was the Semaphorin-3 (Sema3) family of axon repulsion molecules. Subsequently, Nrp was found to serve as a receptor for the vascular endothelial growth factor (VEGF) family of pro-angiogenic cytokines. In addition to its physiological role, VEGF signaling via Nrp directly contributes to cancer stemness, growth, and metastasis. Thus, the Nrp/VEGF signaling axis is a promising anti-cancer therapeutic target. Interestingly, it has recently been shown that Sema3 and VEGF are functionally opposed to one another, with Sema3 possessing potent endogenous anti-angiogenic activity and VEGF serving as an attractive cue for neuronal axons. We hypothesized that direct competition for an overlapping binding site within the Nrp extracellular domain may explain the observed functional competition between VEGF and Sema3. To test this hypothesis we have separately investigated the mechanisms of VEGF and Sema3 binding to Nrp. Utilizing structural biology coupled with biophysics and biochemistry we have identified both distinct and common mechanisms that facilitate the interaction between Nrp and these two ligand families. Specifically, we have identified an Nrp binding pocket to which these ligands competitively bind. The Sema3 family uniquely requires proteolytic activation in order to engage this overlapping binding site. These findings provide critical mechanistic insight into VEGF and Sema3 mediated physiology. Additionally, these data have informed the development of small molecules, peptides, and soluble receptor fragments that function as potent and selective inhibitors of VEGF/Nrp binding with exciting therapeutic potential for treating cancer.

Les facteurs de croissance endothéliaux (VEGFs) sont produits par les tumeurs qui sont hypoxiques. Principaux responsables de la néo-vascularisation pathologique, ils régulent le stroma tumoral. Les nouvelles stratégies qui ciblent et inhibent le VEGF ouvrent vers la thérapie anti-cancéreuse moderne. Elles ont pour but de contrôler l'angiogenèse tumorale plutôt que la détruire. Le défi est donc de piéger sélectivement le VEGF produit en excès, dans le microenvironnement tumoral, sous l'effet de l'hypoxie. La thèse présentée dans ce manuscrit est consacrée à la réalisation d'une nouvelle stratégie ciblante par l'intermédiaire de cellules, aussi appelée " Cheval de Troie ". Elle combine dans la même entité, une unité de ciblage et un système de délivrance spécifique d'un gène/molécule thérapeutique. Dans le but d'adresser la thérapie aux cellules cancéreuses sans affecter les cellules saines, un modèle de cellules endothéliales de type précurseur (CEPs) a été utilisé comme cellules ciblantes capables d'atteindre spécifiquement le site tumoral. Les CEPs ont été " armées " pour exprimer un gène thérapeutique chargé d'inhiber le VEGF. La neutralisation a été obtenue par la production d'une forme soluble du récepteur-2 du VEGF (VEGFR2 soluble), agissant comme inhibiteur. Caractéristique des tumeurs solides se développant, l'hypoxie a été choisie pour déclencher/éteindre l'expression et la sécrétion du VEGFR2 soluble, en introduisant, en amont du gène thérapeutique, une séquence régulatrice : HRE. Adressé au site tumoral par les CEPs, le régulateur de l'angiogenèse qu'est la forme soluble du VEGFR2, est exprimé de manière conditionnée et réversible, à l'hypoxie. Ceci ouvre à de nouvelles stratégies de normalisation contrôlée et stable des vaisseaux tumoraux en vue de l'utilisation de thérapies combinées.