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

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Orientador: Miriane de Oliveira / Resumo: Introdução: O hormônio triiodotironina (T3) influencia o metabolismo e desenvolvimento do tecido adiposo (TA), modulando a proliferação e diferenciação de adipócitos, podendo agir sobre os reguladores do processo de adipogênese, como o receptor ativado por proliferador de peroxissomo (PPARy). O TA está envolvido na regulação da energia corporal, sintetizando e secretando substâncias denominadas adipocinas, dentre elas a adiponectina e leptina. A adiponectina está relacionada ao aumento da sensibilidade à insulina, enquanto a leptina está envolvida com o gasto energético. O T3 pode desencadear ações por ativação de vias extranucleares, dentre elas a via MAPK/ERK e integrina αVβ3. Objetivo: Verificar a ação do T3, com participação das vias extranucleares MAPK/ERK e integrina αVβ3, na modulação de adiponectina e leptina, além de avaliar os parâmetros relacionados ao perfil adipogênico e dano de DNA. Métodos: Adipócitos, 3T3-L1, foram tratados com T3 (10nM) por uma hora, na ausência ou presença dos inibidores de MAPK/ERK – PD98059 (PD, 50uM) e da integrina αvβ3 – ácido tetraiodotiroácetico (Tetrac, 10-4M). A ausência de qualquer tratamento foi considerada grupo controle (C). Após o período de tratamento foi realizado PCRq-RT para analisar a expressão de mRNA de adiponectina e leptina, e Western Blot para expressão proteica de adiponectina, leptina, PPARy, pAKT e pERK; a viabilidade celular foi realizada pelo ensaio de MTT; a quantificação do acúmulo lipídico pelos ens... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The hormone triiodothyronine (T3) influences the metabolism and development of adipose tissue (TA), modulating the proliferation and differentiation of adipocytes, and can act on regulators of the adipogenic differentiation process, such as the peroxisome proliferator activated receptor). TA is involved in the regulation of body energy, synthesizing and secreting substances called adipokines, among them adiponectin and leptin. Adiponectin is related to increased insulin synaptic, since leptin is involved in energy expenditure. T3 can trigger actions by activation of extranuclear pathways, including MAPK / ERK and integrin α Vβ3. Objective: Given the role of T3 in TA and the importance of adipokines, the objective of this study is to verify the action of T3 with the participation of extranuclear pathways in the modulation of adiponectin and leptin and the parameters related to the adipogenic profile. Methods: Adipocytes, 3T3-L1, were treated with a physiological dose of T3 (10nM) for one hour, in the absence or presence of MAPK / ERK-PD98059 (PD) and integrin αvβ3 - tetraiodothyrocetic (Tetrac) integrin inhibitors. The absence of any treatment was considered as a control group (C). After the treatment period PCRqRT was performed to analyze the expression of leptin and adiponectin mRNA, and Western Blot for protein expression of adiponectin, leptin, PPARγ, pAKT and pERK; cell viability was performed by the MTT assay; the quantification of lipid accumulation by the... (Complete abstract click electronic access below) / Mestre

In the current thesis, spatio-temporal evaluation of the endogenous angiogenic response to ischemia was perfomed. After vascular occlusion, ischemic angiogenesis is an important reparative mechanism and can ameliorate the outcome of ischemic disease. Diabetic foot ulcers affect almost 15% of diabetic patients and are the leading cause of amputations worldwide (Yoon et al., 2005). Diminished blood flow because of atherosclerotic occlusive disease of the peripheral arteries of diabetic patients, in conjunction with anatomic and functional microcirculatory impairments contribute to development of trophic ulcerations, infections and gangrene of the lower extremities, frequently requiring amputation of the leg (Sasso et al., 2005). Numerous studies have confirmed the impaired post-ischemic angiogenesis in diabetes (Yoon et al., 2005; Sasso et al., 2005). Consequently, wound healing patterns are disturbed in diabetes mainly due to decreased ischemia-driven angiogenesis (Yoon et al., 2005). Integrin ανβ3 is a promising imaging target of angiogenic activity which is up-regulated on activated endothelial cells (ECs) but not on quiescent ones. Molecular imaging (MI) of ανβ3 integrin expression with the aid of a dedicated high resolution gamma camera, is a very sensitive imaging approach for the evaluation of angiogenesis in the rabbit hindlimb ischemia model. Furthermore, diabetes mellitus (DM) was induced, to study the effects of this pathology on the spatio-temporal distribution of angiogenesis. In order to evaluate the whole spectrum of endogenous process of collateralization after occlusion of an artery, Digital Subtraction Angiography (DSA) was also used for the visualization of larger collaterals. During the first part of the study DM experimental protocol was investigated in order to find the appropriate protocol for the induction of long-term diabetic animal model, as it is a methodology that has not yet been standardized. At the same time a cohort of animals underwent endovascular embolization for the establishment of hindlimb ischemia and were imaged with the aid of a MI radiotracer technique in order to deal with unresolved issues and establish the imaging protocol. The study included seven New Zealand White (NZW) rabbits that underwent unilateral percutaneous endovascular embolization of the femoral artery, for the establishment of hindlimb ischemia that triggers the endogenous process of collateralization. The contralateral limb was not embolized and served as a control. The employed radiotracer for angiogenesis imaging, was a 99mTc labeled cyclic RGD peptide [c RGDfk-His]-99mTc that binds specifically to ανβ3 integrin via the three amino acid sequence Arginine-Glycine-Aspartic acid or RGD. Image acquisition was performed with a high resolution gamma camera and all animals underwent molecular imaging on the 3rd and the 9th day post-embolization. In all animals DSA was performed on the 9th day post-embolization. The acquired images demonstrated that retention of the radiotracer at the ischemic tissue is remarkably increased compared to the non-ischemic hindlimb (normal limb) (16020 ± 2309 vs. 13139 ± 2493 on day 3; p=0.0014 and 21616 ± 2528 vs. 13362 ± 2529 on day 9; p<0.0001, respectively). In addition, radiotracer retention in normal limbs seems to be increased at day 9 in normal limbs compared to day 3 (p=0.0112). DSA at day 9, demonstrated that the mean vessel length detected was significantly superior in the normal compared to the ischemic limb (mean value 3680 ± 369.8 vs.2772 ± 267.7; p< 0.0001, respectively). Angiogenesis was successfully detected using a 99mTc labeled cyclic RGD peptide MI technique and was significantly more pronounced in the ischemic compared to normal limbs, both at day 3 and day 9 after embolization. The peak of the phenomenon was detected at day 9. Increased mean vessel length in the normal compared to the ischemic limb demonstrates that although angiogenesis is pronounced in day 9, arteriogenesis is not sufficiently pronounced and that the phenomenon of arteriogenesis has just initiated. The study of the angiogenic response to ischemia, has not yet been completed as MI of diabetic animals with hindlimb ischemia is underway and not completed due to many difficulties and delay in different phases of the experiment. With the conclusion of the MI of diabetic animals with hindlimb ischemia, the study will be completed and we expect to demonstrate the effect of DM on the spatio-temporal pattern of angiogenesis, providing a valuable tool in clinical practice for the precise and early diagnosis and therapy assessment of the ‘diabetic foot’. / Σκοπός της παρούσας μελέτης ήταν η χωρο-χρονική εκτίμηση της ενδογενούς αγγειογενετικής διαδικασίας ως απόκριση στο ερέθισμα της προκλητής ισχαιμίας. Μετά από απόφραξη αρτηρίας, η επαγόμενη αγγειογένεση είναι ένα σημαντικός μηχανισμός αποκατάστασης που μπορεί να περιορίσει το αποτέλεσμα της ισχαιμίας. Το έλκος του ‘διαβητικού ποδιού’ εμφανίζεται στο 15% περίπου των ασθενών που πάσχουν από διαβήτη και αποτελεί την κύρια αιτία ακρωτηριασμού του κάτω άκρου, μετά τα ατυχήματα (Yoon et al., 2005). Η μειωμένη αιματική ροή λόγω της αποφρακτικής αρτηριοπάθειας που οφείλεται στην αθηροσκληρωτική προσβολή των περιφερικών αρτηριών των διαβητικών, σε συνδυασμό με ανατομική και λειτουργική φθορά του αγγειακού δικτύου της μικροκυκλοφορίας, οδηγούν στην ανάπτυξη ελκών, μολύνσεων και γάγγραινας των κάτω άκρων που πολύ συχνά οδηγεί στον ακρωτηριασμό του ποδιού (Sasso et al., 2005). Πολυάριθμες μελέτες όμως έχουν δείξει ότι η αγγειογένεση που επάγεται από κρίσιμη ισχαιμία, στην παθολογία του διαβήτη δεν είναι φυσιολογική (Yoon et al., 2005; Sasso et al., 2005). Συνεπώς στον διαβήτη και ο μηχανισμός επούλωσης πληγών δεν συμβαίνει φυσιολογικά κυρίως λόγω ελαττωματικής ενδογενούς αγγειογένεσης ως απόκριση στην ισχαιμία (Yoon et al., 2005). Το μόριο της ιντεγκρίνης ανβ3 υπερεκφράζεται στα ενεργοποιημένα ενδοθηλιακά κύτταρα που συμμετέχουν στην αγγειογενετική διαδικασία αλλά όχι στο ανενεργό ή αλλιώς «σιωπηλό» ενδοθήλιο. Συνεπώς αποτελεί έναν πολύ καλό μοριακό στόχο για απεικόνιση και δείκτη της αγγειογενετικής δραστηριότητας. Η Μοριακή Απεικόνιση (ΜΑ) του επιπέδου έκφρασης του μορίου ιντεγκρίνης ανβ3 με τη χρήση ραδιοϊσοτοπικών τεχνικών έχει το πλεονέκτημα υψηλής ευαισθησίας ανίχνευσης πολύ χαμηλών συγκεντρώσεων του ραδιοϊχνηθέτη σε σχέση με τις συμβατικές τεχνικές (x-ray computed tomography (CT) angiography, contrast-enhanced ultrasound και high-resolution magnetic resonance angiography). Ο σκοπός της μελέτης μας ήταν να ερευνήσουμε τις δυνατότητες της ΜΑ με την βοήθεια γ-κάμερας υψηλής διακριτικής ικανότητας και πειραματικού μοντέλου κονίκλου με ίσχαιμο οπίσθιο άκρο. Επιπλέον ένα σημαντικό μέρος της μελέτης αφορούσε την εφαρμογή του πρωτοκόλλου για τη πρόκληση διαβήτη ώστε να γίνει μελέτη της επίδρασης της συγκεκριμένης παθολογίας στην χωρο-χρονική κατανομή της αγγειογένεσης. Προκειμένου να αποδοθεί μία συνολική εκτίμηση του ενδογενούς μηχανισμού αποκατάστασης του αγγειακού δικτύου, εφαρμόστηκε Ψηφιακή Αφαιρετική Αγγειογραφία για την εκτίμηση της δημιουργίας παράπλευρου δικτύου. Στη πρώτη φάση της μελέτης, έγινε διερεύνηση του πρωτοκόλλου πρόκλησης διαβήτη δεδομένου ότι η μεθοδολογία παρουσιάζει έλλειψη προτυποποίησης. Παράλληλα εφαρμόστηκε το πρωτόκολλο ισχαιμίας σε μια ομάδα λευκών κονίκλων Νέας Ζηλανδίας για την διερεύνηση του πρωτοκόλλου ΜΑ. Στην μελέτη, χρησιμοποιήθηκαν συνολικά επτά κόνικλοι Νέας Ζηλανδίας οι οποίοι υπεβλήθησαν σε εμβολισμό της μηριαίας αρτηρίας ενός από τα δύο οπίσθια άκρα για την πρόκληση οξείας ισχαιμίας. Στους κόνικλους έγινε ενδοφλέβια έγχυση του κυκλικού επισημασμένου πεπτιδίου [c RGDfk-His]-99mTc που περιέχει την αλληλουχία τριών αμινοξέων Αργινίνης-Γλυκίνης-Ασπαρτικού οξέος (Arginine-Glycine-Aspartic acid or RGD), μέσω της οποίας δεσμεύεται το πεπτίδιο στο μόριο της ιντεγκρίνης ανβ3. Η απεικόνιση του επιπέδου έκφρασης του μορίου ιντεγκρίνης ανβ3 πραγματοποιήθηκε με γ-κάμερα υψηλής διακριτικής ικανότητας την 3η και την 9η ημέρα μετά την απόφραξη της μηριαίας αρτηρίας. Ψηφιακή Αφαιρετική Αγγειογραφία πραγματοποιήθηκε την 9η ημέρα μετά την απόφραξη της μηριαίας αρτηρίας. Τα δεδομένα από την ποσοτικοποίηση των απεικονιστικών δεδομένων, έδειξαν ότι υπάρχει αυξημένη πρόσληψη του ραδιοϊχνηθέτη στη περιοχή ισχαιμίας σε σχέση με τη περιοχή φυσιολογικής αιμάτωσης του ετερόπλευρου άκρου (16020 ± 2309 έναντι 13139 ± 2493 την 3η ημέρα, p=0.0014 και 21616 ± 2528 έναντι 13362 ± 2529 την 9η ημέρα, p<0.0001, αντίστοιχα). Επιπλέον η πρόσληψη του ραδιοϊχνηθέτη στα φυσιολογικά άκρα φαίνεται να είναι αυξημένη την 9η ημέρα σε σχέση με την 3η ημέρα (p=0.0112), γεγονός που μπορεί να αποδοθεί στην σταδιακή συγκέντρωση ενεργοποιημένου ενδοθηλίου και στους φυσιολογικούς ιστούς. Η Ψηφιακή Αφαιρετική Αγγειογραφία έδειξε ότι την 9η ημέρα που έγινε η λήψη δεδομένων, το μέσο μήκος αγγείων στα φυσιολογικά άκρα ήταν αρκετά μεγαλύτερο σε σχέση με τα ίσχαιμα άκρα (μέση τιμή 3680 ± 369.8 έναντι 2772 ± 267.7, p< 0.0001, αντίστοιχα). Η ραδιοϊσοτοπική τεχνική που εφαρμόστηκε για την απεικόνιση του επιπέδου έκφρασης του μορίου ιντεγκρίνης ανβ3 στη παρούσα μελέτη, έδειξε ότι υπάρχει αυξημένη πρόσληψη του ραδιοϊχνηθέτη στη περιοχή ισχαιμίας σε σχέση με τη περιοχή φυσιολογικής αιμάτωσης του ετερόπλευρου άκρου, την 3η ημέρα και την 9η ημέρα μετά την απόφραξη της μηριαίας αρτηρίας. Τα πειραματικά δεδομένα δείχνουν επίσης ότι το φαινόμενο της αγγειογένεσης κορυφώνεται την 9η ημέρα μετά την πρόκληση ισχαιμίας. Επιπλέον τα δεδομένα από τη Ψηφιακή Αφαιρετική Αγγειογραφία την 9η ημέρα, δείχνουν ότι ο ενδογενής μηχανισμός σχηματισμού παράπλευρου δικτύου αν και έχει πυροδοτηθεί δεν έχουν σχηματιστεί ακόμα μεγαλύτερα αγγεία και γι αυτό το λόγο η αρτηριογένεση υπολείπεται της αγγειογένεσης σε αυτή τη φάση. Για την ολοκλήρωση της μελέτης, εκκρεμεί η απεικόνιση των διαβητικών κονίκλων με ίσχαιμο οπίσθιο άκρο η οποία καθυστέρησε λόγω επιμέρους δυσκολιών στη διαδικασία των πειραμάτων. Η ΜΑ δεικτών της αγγειογένεσης σε άκρο που πάσχει από ισχαιμία παρουσία διαβήτη, δύναται να έχει τεράστια εφαρμογή στην κλινική πράξη για την ιατρική παρακολούθηση του ‘διαβητικού ποδιού’.

Large efforts have been made to target integrin αVβ3 of endothelial cells. We have successfully developed a new class of protein (Ref to as ProAgio) by rational protein design using a stable host protein, domain 1 of cell adhesion protein CD2. ProAgio is designed to target integrin αVβ3 at a novel site and induces angiogenic endothelial cell apoptosis by recruiting and activating caspase 8 to the cytoplasmic domain of the targeted integrins. Tests with tumor xenograft models show that ProAgio strongly inhibits tumor growth. Histology analyses indicate that tumor vessels are reduced, while the established vasculatures are not affected. Toxicity analyses demonstrate that ProAgio is not toxic to mouse. Our study develops an effective anti-angiogenesis agent and provides a new platform for development of therapeutics by targeting integrins. We have successfully developed an anti-angiogenesis protein targeting integrin αVβ3 at a novel site by rational protein design. The developed agent is not toxic to non-cancerous blood vessels and other tissue/organs, providing an excellent candidate for future potential clinical development. Our developed protein is one of the very few examples that do not act through targeting VEGF/VEGFR or any other RTK pathways. The βA groove is present in almost all other β integrins. This approach may be applicable to develop agents targeting the similar βA groove of other integrin pairs, which can address wide array of pathological conditions such as AMD, Rheumatoid Arthritis, Osteoporosis etc.

<p>This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.</p><p>The interstitial fluid pressure (P_IF) is of importance for control of tissue fluid balance. A lowering of P_IF <i>in vivo</i> will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling P_IF.</p><p>Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of P_IF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of P_IF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored P_IF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered P_IF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered P_IF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from <i>Streptococcus equi</i> modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction <i>in vitro</i> and control of P_IF <i>in vivo</i>, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence P_IF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.</p>

This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation. The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF. Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.

All blood vessels in the microvasculature are embedded in loose connective tissue, which regulates the transport of fluid to and from tissues. The intersti-tial fluid pressure (IFP) is one of the forces that control this transport. A lowering of IFP in vivo results in an increased transport of fluid from the circulation into the underhydrated connective tissues, resulting in edema formation. During homeostasis, contractile connective tissue cells exert a tension on the connective tissue fibrous network by binding with β1 in-tegrins, thereby actively controlling IFP. During inflammation, the IFP is lowered but platelet-derived growth factor (PDGF)-BB induces an IFP nor-malization dependent on integrin αVβ3. We demonstrate that extracellular proteins from Streptococcus equi subspecies equi modulated cell-mediated and integrin αVβ3-directed collagen gel contraction in vitro. One of these proteins, the collagen- and fibronectin binding FNE, stimulated contraction by a process dependent on fibronectin synthesis. This study identified a pos-sible novel virulence mechanism for bacteria based on the ability of bacteria to modulate the edema response. Another protein, the collagen-binding pro-tein CNE, inhibited contraction and this led to the identification of sites in collagen monomers that potentially are involved in connecting αVβ3 to the collagen network. PDGF-BB and prostaglandin E1 (PGE1) stimulate and inhibit collagen gel contraction in vitro and normalize and lower IFP, respec-tively. We showed that these agents affected both similar and different sets of actin-binding proteins. PDGF-BB stimulated actin cytoskeleton dynamics whereas PGE1 inhibited processes dependent on cytoskeletal motor and adhesive functions, suggesting that these different activities may partly ex-plain the contrasting effects of PGE1 and PDGF-BB on contraction and IFP. Mutation of the phosphatidylinositol 3’-kinase (PI3K), but not phospholipase C (PLC)γ activation site, rendered cells unable to respond to PDGF-BB in contraction and in activation of the actin binding and severing protein cofilin. Ability to activate cofilin after PDGF-BB stimulation correlated with ability to respond to PDGF-BB in contraction, suggesting a role for cofilin in this process downstream of PDGF receptor-activated PI3K. Many proteins can modulate contraction either by affecting the extracellular matrix and cell adhesions or by altering cytoskeletal dynamics. Knowledge on how these proteins might influence IFP is likely to be of clinical importance for treat-ment of inflammatory conditions including anaphylaxis, septic shock and also carcinoma growth.