Tesis sobre el tema "VEGF165b"

ABSTRACT The pivotal role of angiogenesis in the growth and spread of all solid tumors has driven the cancer research on applying antiangiogenic factors to suppress formation of new blood vessels in order to prevent or slow tumor growth. Glycoproteins, Interferon alpha (IFNα and VEGF165b are of particular interest in this study. IFNα is a multifunctional cytokine with many physiological effects including antiangiogenesis effects. VEGF165b is a competitive antagonist of the Vascular Endothelial Growth Factor (VEGF) receptor and has been reported to successfully inhibit angiogenesis. The thesis goal is to develop a system for simultaneous production of IFNα2b (IFNα) and VEGF165b and targeted delivery to enhance their antiangiogenic properties. For this purpose, HEK293 cells were developed to produce IFNα and VEGF165b simultaneously. The potential of a stable HEK293 cell line producing IFNα or VEGF165b to continuously deliver IFNα or VEGF165b after encapsulation in alginate-poly-l-lysine-alginate (APA) microcapsules was evaluated. For a better delivery system, co-encapsulation of HEK293 VEGF165b producing cells and HEK293 IFNα producing cells or a mixture of encapsulated HEK293 cells producing either IFNα or VEGF165b were studied. Attempts were also made to increase the bioactivity (pharmacodynamic) of IFNα by modifying its O-glycosylation to an N-glycosylation site and of VEGF165b by increasing its sialylation level. The bioactivity was investigated in experimental rats. The results suggest that one cell line, HEK293, can produce IFNα and VEGF165b simultaneously. This process has the advantage of ease of manipulation and low cost but is somewhat limited by the fact that production of IFNα and VEGF165b cannot be controlled. Microencapsulation of IFNα or VEGF165b producing cells demonstrates that encapsulated cells grow and remain viable within the microcapsules. The IFNα and VEGF165b released
RÉSUMÉLe IFNα est une cytokine multifonctionnelle avec plusieurs effets physiologiques incluant l'antiangiogenèse effets tandis que le VEGF165b est un antagoniste concurrentiel du récepteur de Vascular Endothelial Growth Factor (VEGF) et empêche avec succès l'angiogenèse. La thèse a pour but de développer un système pour la production simultanée du IFNα et du VEGF165b, de plus, que la livraison soit ciblée pour augmenter les propriétés antiangiogéniques. À cette fin, les cellules HEK293 ont été génétiquement modifiées pour produire simultanément l'IFNα et le VEGF165b. Le potentiel d'une lignée cellulaire HEK293 stable, produisant simultanément le IFNα ou VEGF165b pour livrer en perpétuité le IFNα et VEGF165b après l'encapsulation dans des microcapsules composé de l'alginate-poly-l-lysine-alginate (APA) a été évalué. Pour apporté des améliorations au système, deux mélanges ont été évalué. La co-encapsulation de cellules HEK293 produisant le VEGF165b et de cellules de HEK293 produisant IFNα et un mélange deux types de cellules encapsulées séparément ont été étudiés. Des tentatives ont été également accomplies pour augmenter leur bioactivité (pharmacodynamic) du IFNα en modifiant son O-glycosylation à un emplacement de N-glycosylation et de VEGF165b en augmentant son niveau de sialylation. La bioactivité a été étudiée chez les rats expérimentaux. Les résultats suggèrent que les cellules, HEK293, puissent produire l'IFNα et le VEGF165b simultanément. Ce processus a l'avantage de facilité la manipulation et de maintenir les coûts bas mais est légèrement limité par le fait que la production du d'IFNα et du VEG165Fb ne peut pas être contrôlée. Le microencapsulation de cellules produisant le IFNα ou VEGF165b démontrent que les cellules encapsulées se développent tout en retenant leur capacités de synthèses et demeurent viables d

Thèse (Ph. D.)--Université de Montréal, 2001.
"Thèse présentée à la faculté des études supérieures en vue de l'obtention du grade de philosophiae doctor (Ph. D.) en biologie moléculaire." Version électronique également disponible sur Internet.

Le VEGF-A joue un rôle clé au cours de l’angiogenèse physiologique mais aussi de la néo-vascularisation tumorale essentielle à la croissance des tumeurs malignes. Le VEGF-A et ses récepteurs (VEGFR1/2) représentent une cible de première importance pour le développement de thérapies anti-tumorales, et un certain nombre de médicaments anti-angiogéniques (AAG) inhibant le VEGF-A ou ses récepteurs sont actuellement utilisés en clinique dans le traitement des carcinomes pulmonaires. Parmi les thérapies anti-angiogéniques ciblant le VEGF-A, on peut lister soit l’anticorps monoclonal anti-VEGF Bevacizumab (BVZ) ou bien les inhibiteurs pharmacologiques du domaine tyrosine kinase des VEGFR: les VEGFR-TKI. Seuls les patients porteurs d’adénocarcinomes pulmonaires peuvent bénéficier de thérapies AAG, les patients porteurs de carcinomes squameux présentant de sévères complications (hémorragies pulmonaires). Le sVEGFR1, est un variant tronqué du VEGFR1 qui ne contient que les premiers six motifs N-terminaux extracellulaires de type Ig du domaine extracellulaire et il est dépourvu des domaines transmembranaire et tyrosine kinase. Le sVEGFR1 a éte initialement considéré comme un facteur anti-angiogénique qui neutralise les fonctions du VEGF-A dans les cellules endothéliales. Les hauts niveaux ont été corrélés avec un mauvais pronostic et une mauvaise réponse aux thérapies dans plusieurs types de cancer. Nous avons montré in vitro dans 4 lignées cellulaires de SCC que le bevacizumab, ainsi que les inhibiteurs VEGF-TKI (Semaxanib, KI8751) augmentent les niveaux intra- et extra-cellulaires du sVEGFR1. Nous avons confirmé ces résultats in vivo dans des modèles murins de xénogreffes squameux induits par NCTU. De façon intérssante, l’augmentation du sVEGFR1 en réponse aux thérapies anti-angiogénique est spécifique aux modèles squameux et n’a pas été observée dans les modèles d’adénocarcinomes in vitro et in vivo. Sur le plan moléculaire, nous avons montré que le VEGF165 par l’intermédiaire de SOX2 régule l’expression du sVEGFR1 en réponse aux thérapiesAAG. De plus, nous avons identifié une boucle autocrine 1 intégrine / VEGFR1 / VEGFR2 par laquelle sVEGFR1 contrôle différentiellement la prolifération cellulaire et la survie, permettant notamment de distinguer les cellules SCC sensibles ou résistantes aux thérapies AAG. Enfin, dans une série de 77 cancers bronchiques non à petites cellules, nous avons montré que 11% et 44% des patients SCC expriment de bas ou de hauts nivaux de sVEGFR1 respectivement. Les hauts niveaux ont été corrélés avec des stades pTNM avancés. Dans l'ensemble, nos résultats sont la première preuve que les thérapies AAG augmentent l'expression du sVEGFR1 dans les cellules SCC. En outre, nos données mettent en évidence une fonction pro-tumorale inattendue de sVEGFR1 grâce à l'activation d'une boucle autocrine VEGFR/ β1 intégrine. Ces résultats pourraient aider à comprendre pourquoi les SCC répondent différemment aux AAG que les ADC et d'identifier les patients SCC qui pourraient etre éligibles à ces thérapies
Vascular endothelial growth factors (VEGFs) and their receptors are regulators of physiological and pathological angiogenesis. In patients with squamous cell lung carcinoma (SCC), clinical trials evaluating anti-angiogenic therapies (AAG) have failed to identify strong benefits. Rather, these patients are at higher risk of bleeding complications when exposed to Bevacizumab (BVZ), a humanized monoclonal anti-VEGF-A antibody. The soluble VEGF receptor-1, namely sVEGFR1, is a truncated version of the cell membrane-spanning VEGFR1 that only retains the first six N-terminal Ig-like extracellular motifs of VEGFR1 owing to alternative splicing of its pre-mRNA. As a consequence, sVEGFR1 is mainly viewed as an anti-angiogenic factor that counteracts VEGF-A functions on endothelial cells. Moreover, high levels of sVEGFR1 were correlated with bad prognosis and bad response to therapies in many cancer types. Using various SCC cell lines, we showed that Bevacizumab as well as VEGFR-Tyrosine Kinase Inhibitors (Semaxanib, KI8751) increase the intra- and extra-cellular levels of sVEGFR1. We confirmed this up-regulation in NCTU-induced SCC murine tumorgrafts models treated with VEGFR-TKI (sunitinib) or anti-VEGFR2 (DC101). Of note, this effect was never observed in the lung adenocarcinoma histological sub-type (ADC), using either cell lines or a mouse model treated in the same conditions. At the molecular level, we identified the VEGF165 and SOX2 proteins as crucial upstream regulators of sVEGFR1 in response to AAG. Moreover, we unraveled an original and SOX2 proteins as crucial upstream regulators of sVEGFR1 in response to AAG. Moreover, we unraveled an original ines or a mouse model treato discriminate between AAG-sensitive or -resistant SCC cells. Finally, in a series of 77 Non Small Cell Lung Carcinoma, we provided the first description of a differential pattern of sVEGFR1 expression with 11% and 44% of SCC exhibiting no or high expression respectively, high levels of sVEGFR1 being correlated with advanced pTNM stages. As a whole, our results provide the first evidence that AAG therapies upregulate sVEGFR1 expression in SCC cells. In addition, our data highlight an unexpected pro-tumoral function of sVEGFR1 through the activation of a beta 1 integrin-dependent VEGFR autocrine loop. These results might help to understand why SCC are less responsive to anti-angiogenic drugs than ADC and to identify SCC patients eligible to these therapies

Several of the multitude of functions attributed to Vascular Endothelial Growth Factor-A (VEGF-A) are coordinated by its various isoforms, which are generated as a result of alternative splicing from a single gene. Despite the fact that the VEGF isoforms exhibit distinct biochemical properties, little has been done to clarify their functions and their contributions to physiological and pathological processes. In this thesis I describe the biochemical and biological characterization of the heparin-binding domain of mouse VEGF 164 through structure-function analysis. To investigate the functional significance of heparin binding, mutations were introduced into exon 7 of VEGF 164 to identify essential residues for heparin binding. Three key amino acids involved in this interaction were identified. Mutants with alterations in these amino acids were unable to bind heparin and were compromised in their ability to bind to cell-surface heparan sulfate. These mutants, however, retained wild-type like potency in inducing tissue factor expression in vitro and microvessel growth ex vivo, and maintained the capacity to bind to the receptors neuropilin-1, VEGFR-1, and VEGFR-2. A second goal of this work was to better define the role of VEGF 164 in mediating inflammatory processes during pathological vascularization of the retina. Analysis of VEGF 164-deficient (VEGF1ZU/ iao) mice subjected to neovascularization-inducing conditions and rats injected intravitreally with recombinant VEGF variants demonstrated that endogenous and exogenous VEGF 164 increases leukocyte adhesiveness to retinal vessels compared to the non-heparin-binding isoform, VEGF 120. Interestingly, the three basic residues that confer heparin binding of VEGF 164, appear to be critical for its pro inflammatory activity, but not for its angiogenic activity. In addition, both mutants (and VEGF 120) exhibited a reduced affinity for VEGFR-1, a leukocyte-expressed receptor that mediates VEGF-induced migration. Results of in vivo experiments using P1GF, VEGF-E, as well as a VEGFR-1 neutralizing antibody, further demonstrate a role for VEGFR-1 in retinal leukostasis.

L'hématopoïèse, processus de production des cellules sanguines à partir des cellules souches, est notamment régulée par les concentrations d'O2 médullaires comprises entre 0 à 5 % (hypoxie). Nous avons établi des liens entre des facteurs intrinsèques à la cellule impliqués dans le processus de différenciation (antigène CD34) et leur sensibilité à l'hypoxie, et étudié les effets sur l'hématopoïèse de facteurs inductibles par l'hypoxie (Vascular Endothelial Growth Factor, VEGF). La culture de cellules hématopoiétiques CD34+ à 1 % d'O2 augmente ou stabilise l'expression du gène cd34 qui diminue à 20 % d'O2. Le maintien prolongé de cette expression associé à une expression membranaire durable de la protéine sont corrélés avec le statut immature des cellules. D'autre part, le VEGF165 permet la survie de cellules souches murines en culture liquide à 1 % d'O2. Ainsi l'hypoxie freine la différenciation des cellules souches par l'expression du gène cd34 et favorise leur survie par le VEGF165
Hematopoiesis, the process of mature blood production from stem cells, is in part regulated by bone marrow oxygen concentrations, which vary from 0 to 5 % (hypoxia). We studied in this work the relationships between cell intrinsic factors involved in the maturation process (CD34 antigen) and their sensitivity to hypoxia, and the effects of molecules inducible by hypoxia (Vascular Endothelial Growth Factor, VEGF) on hematopoiesis. We showed that cultures of CD34+ cells at 1 % O2 induce or stabilize the cd34 gene expression that decreases at 20 % O2. The prolonged maintenance of this expression associated with the long-lasting membrane expression of the protein were correlated with the primitiveness of cells. We also showed that VEGF165 led to the survival of murine stem cells cultured at 1 % O2. This work suggests that hypoxia slows down the differentiation of stem cells by inducing cd34 gene expression, and favours their survival through VEGF165

HTLV-1(Human T cell Leukemia Virus type 1) est un rétrovirus oncogène qui infecte majoritairement les lymphocytes T CD4+. Il est transmis par contact entre deux cellules, via une synapse virale. L’entrée du virus dans une cellule cible met en jeu deux partenaires : les glycoprotéines d’enveloppe virales (Env) et plusieurs récepteurs cellulaires. Un premier récepteur a été décrit récemment : le transporteur de glucose de type 1 (Glut-1). Il a ensuite été montré que les Héparanes Sulfates ProtéoGlycanes (HSPGs) étaient un constituant essentiel du complexe récepteur, et interviennent dans les étapes précoces du mécanisme d’entrée. Pour notre part, nous avons démontré que la neuropiline-1 (NRP-1), récepteur de la sémaphorine 3A et du VEGF165 est également un constituant du HTLV-1 (article 1). Un des complexes récepteurs du VEGF165 est constitué de la NRP-1, des HSPGs et du récepteur de type 2 du VEGF. Le VEGF165 et la NRP-1 sont tous deux capables de lier les héparanes sulfates (HS), le VEGF165 se lie majoritairement à la NRP-1 de façon HS-dépendante. Néanmoins, une interaction directe du VEGF165 à la NRP-1, impliquant les derniers résidus du VEGF165, existe également. En nous basant sur le modèle du VEGF165, nous avons montré que les HS et la NRP-1 coopèrent dans le processus d’entrée. Par ailleurs, nous avons mis en évidence que l’Env partage un motif d’homologie avec le VEGF165 qui lui permet également de s’associer directement à la NRP-1. L’ensemble de ce travail nous permet de proposer un modèle d’entrée complètement nouveau impliquant les trois molécules préalablement décrites et un mimétisme moléculaire entre un domaine du VEGF165 et l’Env du HTLV-1 (article 2).

A cardiomioplastia (CDM) tem sido proposta com uma alternativa de tratamento cirúrgico para pacientes em estado avançado de cardiomiopatia dilatada e isquêmica. Os resultados clínicos e experimentais demonstram que este procedimento atenua o processo de remodelamento ventricular, através da compressão dinâmica ou passiva do miocárdio pelo grande dorsal (GD). Além disso, estudos observaram formação de vasos colaterais do GD para o coração após a CDM. O infarto do miocárdio (IM) induz disfunção e remodelamento ventricular e tem sido muito utilizado na literatura como modelo experimental de isquemia miocárdica. A aplicação de fatores angiogênicos diretamente no miocárdio isquêmico tem mostrado resultados positivos na estimulação da formação de colaterais. O objetivo do presente estudo foi avaliar os efeitos da CDM associada ao tratamento com VEGF165 na função ventricular e no desenvolvimento de fluxo colateral extramiocárdico em ratos infartados. Foram utilizados ratos machos Wistar (n=57, 220-250g) divididos em grupos infartados e controles. As alterações temporais induzidas pelo IM (ligadura da artéria coronária esquerda) foram avaliadas aos 14 (IM-14) e aos 56 (IM-56) dias pós IM sendo comparadas com seus respectivos controles (C-14 e C-56). Animais controles (C-CDM) e infartados (IM-CDM) foram submetidos à CDM passiva (sem estimulação do GD) após 14 dias de IM e avaliados aos 56 dias. Ratos controles foram submetidos à cirurgia fictícia de IM e de CDM (S-IMCDM) e ratos infartados à cirurgia fictícia de CDM (IM-SCDM) a fim de verificar eventuais alterações induzidas pelos procedimentos cirúrgicos. Um grupo de ratos infartados recebeu a administração de uma dose de 25µg de VEGF165 na artéria principal do GD imediatamente antes da CDM (14 dias de IM) e foi avaliados aos 56 dias (IMCDM-VEGF). Ao final do protocolo os animais foram anestesiados (pentobarbital sódico, 40mg/Kg) e a artéria carótida direita foi canulada para registro da PA. Logo após, esta cânula foi inserida no ventrículo esquerdo (VE) para registro da pressão ventricular. O registro e processamento dos sinais de pressão foram realizados utilizando-se um sistema de aquisição de sinais (CODAS, 1 Khz). O débito cardíaco (DC) e os fluxos regionais (coração e rins) foram avaliados através da infusão de 300 000 microesferas azuis no ventrículo esquerdo. Após a infusão de 50 000 microesferas amarelas na artéria principal do GD o fluxo colateral extramiocárdico do GD para o coração (FCO GD→coração) foi quantificado através da divisão do número de microesferas amarelas no coração pelo número de microesferas amarelas no GD. Após a oclusão da artéria do GD foram infundidas 300 000 microesferas azuis no VE e o fluxo colateral extramiocárdico do coração para o GD (FCO coração→GD) foi avaliado pela divisão do número de microesferas azuis no GD pelo número de microesferas azuis no coração. O IM induziu hipotensão e aumento da pressão diastólica final (PDF) nos grupos IM-14 (84±6 e 6,88±2,6 mmHg) e IM-56 (98±3 e 15,4±2 mmHg) em relação aos seus respectivos controles (C-14: 102±4 e –3,2±0,5; C-56: 114±3 e 0,5±1,7 mmHg). O débito cárdiaco (DC) foi menor no grupo IM-56 (49,5±9 ml/min) em relação ao grupo IM-14 (72±9 ml/min). A máxima velocidade de relaxamento do VE (-dP/dt) estava reduzida nos grupos IM-14 (-2416±415 vs -4141±309 mmHg/seg nos C-14) e IM-56 (-3062±254 vs -4191±354 mmHg/seg nos C-56) e a de contração do VE (+dP/dt) somente no grupo IM-56 (4191±354 vs 5420±355 mmHg/seg nos C-56). O IM não alterou o fluxo e a resistência vascular coronariana, no entanto, o fluxo renal estava reduzido e a resistência renal aumentada no grupo IM-56 quando comparados ao grupo C-56. Os animais com 56 dias de IM apresentaram aumento de massa ventricular (pv) e da razão peso ventricular/peso corporal (pv/pc) em relação aos controles (1,3±0,04 vs 0,98±0,04 23 g e 3,37±0,08 vs 2,54±0,09 mg/g nos C-56). O tamanho do infarto foi menor no grupo IM-14 (35±3 % do VE) em relação ao grupo IM-56 (44±2 % do VE). Os grupos sham não apresentaram alterações nos parâmetros avaliados em relação aos seus controles. Os ratos infartados submetidos à CDM não apresentaram hipotensão (105±2 mmHg), nem aumento da PDF (4,8±1,7 mmHg) conforme observado no grupo IM-56. O FC, o DC, a RVP e os fluxos e a resistência vascular coronariana foram semelhantes entre os grupos C-56, IM-56, C-CDM e IM-CDM. A +dP/dt e a –dP/dt mostraram-se reduzidas nos grupos C-CDM e IM-CDM em relação ao grupo C-56. O fluxo e a resistência vascular renal estavam normalizadas nos ratos IM-CDM. O pv (1,11±0,04g) e a razão pv/pc (2,94±0,09 mg/g) apresentaram-se similares aos valores do grupo C-56 e o tamanho do IM foi semelhante entre os grupo IM-56 e IM-CDM (44±2 vs 45±3 % do VE). O grupo IMCDM-VEGF apresentou normalização dos parâmetros hemodinâmicos e morfométricos de forma semelhante aos do grupo IM-CDM quando comparados ao grupo IM-56. A resistência coronariana mostrou-se reduzida nos animais IMCDM-VEGF (22,07±2,01 mmHg/ml/min/g) quando comparada ao grupo C-CDM (37,81±4 mmHg/ml/min/g), apesar do fluxo coronariano ter sido similar entre os grupos submetidos à CDM. O FCOcoração→GD ocorreu predominantemente nos animais dos grupo C-CDM e IMCDM-VEGF (70% e 83,3% vs 28,6% no IM-CDM) enquanto que o FCO GD-coração foi observado em todos os animais dos grupos IM-CDM e IMCDM-VEGF (20% no C-CDM). A administração de VEGF165 aumentou o FCO GD→coração em valores absolutos e normalizados por grama (24,85±10,3% e 62,29±23,27%/g) em relação aos grupos C-CDM (0,88±0,89% e 1,42±1,42%/g) e IM-CDM (4,43±1,45 % e 7,66±2,34 %/g). O FCO GD→coração normalizado foi maior nos animais IM-CDM em relação aos C-CDM. O grupo IMCDM-VEGF (4,47±1,46 %/g) apresentou maior FCO coração→GD normalizado em comparação ao grupo MI-CDM (2,43±1,44 %/g). O tamanho do infarto foi menor nos animais do grupo IMCDM-VEGF (36±3 % do VE) em relação aos grupos IM-56 e IM-CDM. Correlações positivas foram obtidas entre o FCO GD→coração e o volume sistólico e (r=0,7) e o fluxo coronariano (r=0,7), e entre a PDF e a razão pv/pc (r=0,8) e o tamanho do IM (r=0,6). Além disso, correlações inversas foram observadas entre o tamanho do infarto e o volume sistólico (r=0,8) e o FCO GD→coração (r=0,7). Estes resultados permitem concluir que a CDM passiva preveniu a disfunção e o remodelamento do VE em ratos infartados. A aplicação de VEGF165 induziu diminuição do tamanho do IM que pode estar associado ao aumento do fluxo colateral extramiocárdico do GD→coração observado neste grupo tratado com VEGF. Estes achados sugerem que o uso de fatores angiogênicos, como o VEGF165, pode induzir melhora da perfusão das regiões isquêmicas do coração infartado, limitando a perda tecidual. Este efeito associado ao da compressão passiva do VE infartado pelo GD pós CDM, pode prevenir as disfunções decorrentes de isquemias miocárdicas.

Oxaliplatin therapy of colorectal cancer induces a dose-dependent neuropathic syndrome in 50% of patients. Pharmacological treatments may offer limited relief; scientific efforts are being solicited to define a new therapeutic approach. Interestingly, adult mesenchymal stem cells offer a totipotent cellular source for replacing injured neural cells and at the same time represent a source of neuroprotective and anti-inflammatory mediators, opposing the effect of nerve damage. In our hands, adipose-derived stem cells (ASCs) injection (2x106 ASCs/rat i.v.) were able to counteract mechanical hyperalgesia induced by repeated oxaliplatin administration in rats (2.4 mg kg-1 i.p. for a total of 10 administrations). This effect reached a maximum 6h after ASCs administration and lasted up to 72h. Subsequent ASCs administrations induced a reduction of hypersensitivity, with a similar efficacy trend over time. Investigating a possible mechanism of action by which ASCs exert their effect, 2x106 ASCs labeled with 1 µM of the fluorescent probe 5-(and-6-9-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine were injected in order to evaluate the localization of ASCs in the rat body. Labelled ASCs were detectable in the bloodstream 1 and 3 h after injection, the percentage gradually decreased, 24 h after administration no cells were found. At this time, ASCs were detected in the liver digested homogenate. No ASCs were found in the central nervous system and in lungs. VEGF-A, EGF and TGF-β were assayed in plasma. EGF and TGF-β were not altered by oxaliplatin or ASCs treatments. On the contrary, VEGF-A concentration significantly increased in oxaliplatin-treated rats in comparison to the control group whereas ASCs were able to counteract this alteration, suggesting both a possible implication of VEGF modulation in the development of neuropathic pain and in ASCs pain relieving mechanism. This hypothesis was strengthened by the reduction of oxaliplatin-induced hyperalgesia after an acute i.p. administration of the VEGF-antibody bevacizumab (dose-dependently, 1-15 mg kg-1). Moreover, plantar injection of the pain-related isoform VEGF165b (10-100 ng) in naïve rats, significantly decreased the pain threshold up to 3 h after administration in a bevacizumab-reverted manner (15 mg kg-1). VEGF165b (30-100 ng) dose-dependently, significantly reduced the weight tolerated on the posterior paw also after an intrathecal administration. These data led us to further investigate the role of VEGF-A in modulating pain signaling and the correlation between ASCs anti-nociceptive efficacy and VEGF-A modulation in central nervous system. As evaluated by western blot analysis, oxaliplatin repeated treatment significantly increased VEGF165b expression in spinal cord and PAG while in DRG, cortex and thalamus the protein levels were not influenced with respect to control group. ASCs i.v. injected significantly counteracted the oxaliplatin-dependent VEGF165b increase only in spinal cord. Accordingly, an intrathecal administration of ASCs (75x103/rat) was able to revert hyperalgesia induced by repeated oxaliplatin treatments starting from 1 h after the injection and up to 6 h. To further investigate the link between central VEGF-A and the modulation of pain perception, the selective neutralization of VEGF-A and VEGF165b was performed both in oxaliplatin-treated and in control rats. The specific antibody against VEGF165b, i.t. injected (7.5 μg/rat), significantly decrease oxaliplatin-induced hyperalgesia with similar efficacy to bevacizumab (45 μg/rat i.t. injected) but with higher potency. The modulation of VEGF-A is suggested as a key mechanism in the complex response orchestrated by stem cells against neuropathy and the role of ASCs “secretome” as effector of their efficacy is highlighted. For this reason, an artificial and biocompatible niche in which ASCs can be maintained was set up and the efficacy of this engineered niche in relieving oxaliplatin-induced neuropathic pain was tested. As physiologically occurs in tissues, the artificial niches should promote ASCs proliferation, ensuring the maintenance of their stemness and meanwhile permit a bidirectional flux of solutes from the inner and outer environment without a direct delivery of ASCs. For this purpose, 4x106 rASCs were encapsulated into each artificial niche (AlgiMatrix® sponge), the day after AlgiMatrix® sponges were implanted s.c. in the back of neuropathic rats (two sponges per rat). Encapsulated rASCs significantly reduced mechanical hypersensitivity induced by oxaliplatin treatment beginning 6h after the surgery. The effect on hypersensitivity remained constant for 7 days after AlgiMatrix® sponges implantation and disappeared at 8th day. Enclose ASCs in an artificial niche allows to exploit their modulatory capabilities in response to tissue injuries reducing possible side effects associated to stem cell differentiation. So, the present data suggest an alternative approach in the use of ASCs for the treatment of oxaliplatin-induced neuropathic pain.

Neuropilina-1 (NRP-1) jest białkiem eksprymowanym przez szereg typów komórek somatycznych. Co więcej, wykazano jego nadekspresję w kilku rodzajach nowotworów złośliwych, np.: rak piersi. Uważa się również, że w patologicznej angiogenezie oddziaływanie NRP-1 z czynnikiem wzrostu śródbłonka naczyń (VEGF165) prowadzi do unaczynienia nowotworu i jego wzrost. Dodatkowo, ten kompleks sygnałowy jest także zaangażowany w wyciszanie odpowiedzi immunologicznej przeciwko nowotworowi. Dlatego też inhibitory kompleksu VEGF165/NRP-1 są bardzo interesującymi związkami posiadającymi właściwości blokowania kilku szlaków regulacji wzrostu nowotworu i tworzenia przerzutów. W ramach poprzednich projektów (Pracownia Peptydów) skupiono się na zaprojektowaniu silnych inhibitorów tworzenia się tego kompleksu o ogólnej sekwencji H-Lys(Har)-Xaa-Xaa-Arg-OH. Związki wiodące zostały zbadane we wstępnych testach mierzących odporność na proteolizę w ludzkim osoczu, w ramach których stwierdzono, że hydroliza zachodzi w obszarze łącznika (-Xaa-Xaa-). Głównym celem niniejszej pracy było zaprojektowanie i synteza takiego elementu strukturalnego, który będzie stabilny proteolitycznie. Do osiągnięcia tego wybrano 1,4-dipodstawione 1,2,3-triazole, a badania podzielono na kilka etapów. W pierwszym etapie zostały zaprojektowane dwie podgrupy triazolopeptydów podstawiane odpowiednio: 1) w obszarze łącznika cząsteczki: -Xaa-Xaa- 2) w obszarze ramienia cząsteczki: H-Lys(Har)-. W drugim etapie opracowano protokół syntetyczny otrzymywania triazolopeptydów na fazie stałej, który może być prowadzony na klasycznych żywicach polistyrenowych z linkerem Wanga. Pozwala to na uniknięcie konieczności prowadzenia syntezy w roztworze, która jest bardziej czasochłonna niż synteza na fazie stałej. Wszystkie reakcje na stałym podłożu zachodzą ilościowo i pozwalają otrzymać triazolopeptydy w 10-14 etapach z wydajnością sumaryczną ok. 20% po oczyszczaniu techniką HPLC. W trzecim etapie przeprowadzono badania aktywności inhibicyjnej na tworzenie się kompleksu VEGF165/NRP-1. Testy przeprowadzono przy użyciu techniki immunoenzymatycznej ELISA. Uzyskane wyniki wskazały, że aktywność inhibicyjna w stężeniu 10 μM waha się w przedziale 9,2-58,1%. W czwartym etapie wykorzystano technikę dynamiki molekularnej na superkomputerze ICM do wymodelowania oddziaływań między NRP-1 a triazolopeptydami. Wybrano reprezentacyjne ułożenie liganda, które może być wykorzystane do wyjaśnienia trendów zmierzonych w teście ELISA.W piątym etapie dokonano połączenia wyników z dwóch poprzednich. Wywnioskowano, ze triazolopeptydy wykazują słabszą aktywność ale najlepsze z nich cechują się podobną aktywnością co heptapeptyd A7R, którego działanie zostało sprawdzone w modelach zwierzęcych. W szóstym etapie przeprowadzono badania odporności na degradację proteolityczną najsilniejszych triazolopeptydów w ludzkim osoczu z zastosowaniem techniki HPLC-MS. Wyniki wskazują, że pierścienie triazolowe są kompletnie stabilne a czas półtrwania związków przekracza 48 godzin. Należy wziąć również pod uwagę fakt, że wydłużona ekspozycja komórek na działanie związków chemicznych może nieść pewien efekt toksyczny. Z tego powodu zdrowe komórki pochodzące ze szpiku kostnego (linia mysia 32D) zostały poddane inkubacji w 100 μM roztworze triazolopeptydu 4 przez 48 godzin i po tym eksperymencie nie zaobserwowano śmierci tych komórek. Podsumowując, triazolopeptydy można otrzymać w łatwy sposób dzięki opracowanemu protokołowi syntetycznemu na fazie stałej. Udokumentowano aktywność inhibicyjną zaprojektowanych triazolopeptydów na tworzenie się kompleksu VEGF165/NRR-1, a najlepsze analogi charakteryzują się podobną siłą działania co A7R. Co więcej, odporność na proteolizę otrzymanych triazolopeptydów jest znacznie większa niż związku wyjściowego. Wszystkie uzyskane wyniki sugerują, że ta klasa związków może łączyć w sobie aktywność tego typu peptydomimetyków oraz odporność proteolityczną charakterystyczną dla związków małocząsteczkowych.
Neuropilin-1 (NRP-1) is a protein expressed by a number of types of somatic cells. Moreover, it has been found to be overexpressed in several kinds of malignant tumors e.g. breast cancer and it is postulated that in pathological angiogenesis its interaction with the Vascular Endothelial Growth Factor 165 (VEGF165) leads to progression of tumor vascularization and growth. Additionally, this signaling complex is also involved in suppression of immune response against tumor cells. Inhibitors of the VEGF165/NRP-1 complex are very attractive compounds to block several processes regulating tumor growth and metastasis. In the frame of previous projects (Laboratory of Peptides) efforts have been made to design strong inhibitors of this interaction with general sequence H-Lys(Har)-Xaa-Xaa-Arg-OH. Lead compounds were tested in a preliminary assay toward proteolytic stability in human serum. It has been observed that proteolysis occurs in the linker site of the molecule (-Xaa-Xaa-). The main aim of this dissertation is to desing and synthesize mimetics of such structural element with the proteolytically resistant one. For this purpose a 1,4-disubstituted 1,2,3-triazole was chosen. To achieve this goal all presented research was devided in several stages. In the first stage two subseries of triazolopeptides were designed with major modifications in: 1) the linker site of the molecule: -Xaa-Xaa- 2) the arm site of the molecule: H-Lys(Har)- The second stage was to elaborate the synthetic protocol for triazolopeptide synthesis on solid support, which could be proceeded on traditional polystyrene resins with Wang linker. It allows to avoid synthesis in liquid phase, which is more time consuming related to solid phase approach. All reactions on solid support proceed quantitatively affording designed peptidotriazoles in 10-14 synthetic steps and final yield c.a 20% after HPLC purification. The third stage of this work was to study the inhibitory activity toward VEGF165 binding to NRP-1. This was achieved by immonoenzymatic assay ELISA. In the frame of x this step, it was observed, that inhibitory activity spans from 9.2% to 58.1% at concentration of 10 μM. The fourth stage was to model the interaction with supercomputer OKEANOS ICM using molecular dynamics approach. The representative binding pose was proposed, which could be used for explanation of observed trends in ELISA test. The fifth stage was to combine the results from two previous ones. It was concluded, that peptidotriazoles are in general weaker than the lead compound, however the best of them exhibit inhibitory activity on similar level in comparison with A7R heptapeptide, which activity was validated in in vivo model. The sixth stage of this dissertation was planned to study proteolytic resistance of the best triazolopeptides in human plasma with the use of HPLC-MS technique. The analysis showed that this triazole rings are completely stable under proteolytic environment and the half-life exceed 48h. It should be considered, that prolonged exposition for chemicals could be related with some toxic effects on normal cells. For this reason, normal, healthy bone marrow cells 32D (murine) were subjected to incubation in 100 μM solution of triazolopeptide 4 for 48h and no cells death was observed. In summary, triazolopeptides could be easily prepared with the use of elaborated synthetic protocol on solid support. It was proved, that those compounds exhibit inhibitory activity toward VEGF165 binding to NRP-1 and the best compounds exhibit similar level of activity in in vitro comparison to A7R. Moreover, proteolytic stability of triazolopeptides is considerably higher than the lead compound. All these findings suggest, that this class of compounds is able to link activity of such peptidomimetics and proteolytic resistance of small molecules, and might be a perspective in this field for the future.

Angiogeneza jest procesem biologicznym, który polega na tworzeniu się nowych naczyń krwionośnych z już istniejących w organizmie. Patologiczna forma angiogenezy jest odpowiedzialna za pojawienie się choroby nowotworowej. Jednym z głównych czynników proangiogennych jest czynnik wzrostu śródbłonka naczyń (VEGFA165, określany jako VEGF165), który w sposób selektywny oddziałuje z receptorem (VEGFR) oraz ko-receptorem, którym jest białko neuropilina 1 (NRP-1). Ekspresję NRP-1 obserwuje się na wielu typach nowotworów co sugeruje, że NRP-1 w komórkach nowotworowych może pełnić funkcje osobnego receptora dla VEGF165. Związki, które są w stanie zablokować w sposób selektywny interakcję VEGF165/NRP-1 mogą stać się w przyszłości lekami stosowanymi w chorobach nowotworowych. Celem mojej pracy doktorskiej było projektowanie, synteza oraz badania biologiczne cyklicznych peptydów, których struktura oparta jest na najkrótszym fragmencie heptapeptydu A7R (ATWLPPR) w celu znalezienia silnych inhibitorów układu VEGF165/NRP-1. Peptyd A7R został wyizolowany z biblioteki fagowej przez zespół prof. Perreta. Peptyd ten wykazuje w testach in vivo oraz in vitro właściwości antyangiogenne. Badania struktura – aktywność (SAR) peptydu A7R wykazały, że C- końcowy fragment LPPR jest kluczowy w wykazywaniu przez ten peptyd aktywności biologicznej. Moje badania dotyczyły syntezy cyklicznych peptydów, gdyż są one stabilniejsze w surowicy ludzkiej krwi w odróżnieni od ich liniowych odpowiedników. Ta właściwość czyni je bardziej odpowiednimi potencjalnymi kandydatami na leki. Struktura zaprojektowanych cyklicznych peptydów oparta była na sekwencji LPPR. Zaprojektowane cykliczne peptydy posiadały dwa rodzaje cyklizacji: łańcuch boczny –do- łańcucha bocznego oraz głowa – do – łańcucha bocznego. Każdy zaprojektowany peptyd posiadał na C-końcu resztę argininy, która okazała się być kluczowym aminokwasem w wykazywaniu aktywności biologicznej przez peptyd A7R oraz LPPR. Cyklizacja została przeprowadzona za pomocą utworzenia wiązania amidowego pomiędzy pierwszą a trzecią resztą aminokwasu obecnego w sekwencji LPPR. W zależności od przeprowadzonej cyklizacji peptydy różniły się ilością atomów w cyklu (10-15). Synteza liniowych peptydów oraz cyklizacja została przeprowadzona manualnie korzystając z metody syntezy peptydów na nośniku polimerowym (SPPS), wykorzystując w tym celu nośnik polimerowy Merrifielda oraz prowadząc całą syntezę w oparciu o strategię Fmoc/Boc. Cyklizacja została wykonana za pomocą TBTU. Peptydy były oczyszczane za pomocą RP HPLC i analizowane za pomocą spektrometrii mas. Wydajności zaprojektowanych cyklicznych peptydów były zazwyczaj niskie. W zależności od rodzaju cyklicznego peptydu w surowym produkcie znajdował się cykliczny monomer, mieszanina cyklicznego peptydu w postaci monomeru wraz z symetrycznym dimerem bądź tylko cykliczny dimer. Świadczy to o tym, że synteza małych cyklicznych peptydów jest wyzwaniem. Otrzymałam 15 peptydów (7 cyklicznych monomerów oraz 8 cyklicznych dimerów), których stopień inhibicji układu VEGF165/NRP-1 został zbadany za pomocą testu ELISA. Większość otrzymanych cyklicznych peptydów wykazywała wyższą aktywność niż peptyd A7R. Dla większości cyklicznych monomerów oraz jednego dimeru zostało wykonane modelowanie komputerowe. Struktury peptydów dokowane były do domeny b1 kokryształu NRP-1 z tuftsyną (tetrapeptyd TKPR ― jeden z inhibitorów NRP-1). Modelowanie komputerowe dowiodło, że reszta egzocyklicznej argininy odgrywa kluczową rolę w oddziaływaniu z receptorem a grupa aminowa znajdująca się na N-końcu odpowiedzialna jest za oddziaływanie z Glu348 znajdującym się w NRP-1. Wyniki testu ELISA oraz modelowanie komputerowe wykazało, że zarówno wielkość pierścienia jak i konfiguracja reszt aminokwasów obecnych w sekwencji peptydu jest kluczowa do wykazywania wysokiej aktywności biologicznej przez analizowane peptydy. Dla trzech najbardziej aktywnych peptydów zostały wykonane badania stabilności w surowicy ludzkiej krwi korzystając z metody LC MS. Badania wykazały, że peptydy te są stabilne w surowicy ludzkiej krwi. Czas półtrwania w zależności od badanego peptydu wahał się od 5 h (monomer) do 32 h (dimer). Ostatni etap mojej pracy dotyczył optymalizacji syntezy cyklicznych peptydów. W tym celu wykorzystałam różne rodzaje nośników polimerowych (Wang, Wang Tenta Gel), odczynników sprzęgających (TBTU, DIC/Oksyma, HATU), różne warunki prowadzenia reakcji (temperatura pokojowa, reaktor mikrofalowy). Wyniki moich badań wykazały, że wydajność otrzymania produktu monomerycznego zależy głównie od sekwencji oraz od rodzaju zastosowanej cyklizacji.
Angiogenesis is a biological process described as creation of new blood vessels from pre-existing ones. Pathological form of this process is crucial during cancer development process. One of the most important protein which takes part in angiogenesis is vascular endothelial growth factor (VEGFA165, also known as VEGF165), which selectively binds to its receptor (VEGFR) and co-receptor – protein called neuropilin-1 (NRP-1). In the recent years expression of NRP-1 has been demonstrated in various types of tumours what suggests that NRP-1 in tumour cells may serve as a separate receptor for VEGF165. Therefore compounds which are able to block selectively VEGF165/NRP-1 interaction seem to be promising candidates as a new anti-angiogenic and anti-tumour drugs. The aim of my thesis was design, synthesis and biological evaluation of cyclic peptides based on the shortest active part of heptapeptide A7R (ATWLPPR) to obtain potent inhibitors VEGF165/NRP-1 system. A7R peptide was isolated from the phage library by professor Perret’s group who showed in in vitro and in vivo tests anti-angiogenic and anti-tumour properties of this peptide. Structure activity relationship study (SAR) of A7R showed that the most important for biological activity is C-terminal tetrapeptide (LPPR). My research was focused on synthesis of cyclic peptides, because they are much more stable in human plasma than their linear counterparts what makes them more suitable as prospective candidate as drugs. However, the problem is always whether cyclic compounds will maintain (or improve) their biological activity. Based on the LPPR sequence two types of cyclic peptides were designed. One type with side chain-to-side chain type of cyclization, and second type with head-to-side chain type of cyclization. All designed cyclic peptides possessed exocyclic C-terminal arginine as this amino acid residue is essential for biological activity of A7R and LPPR. Cyclization was performed by an amide bond formation between 1st and 3rd amino acid residue in LPPR sequence. Depend on the type of cyclization peptides differed by the number of atoms in the cycle (10-15). Synthesis of linear peptide chains and cyclization were performed manually using SPPS methodology on Merrifield resin according to Fmoc/Boc strategy. Cyclization reaction was performed with the use of TBTU coupling reagent. Peptides were purified by RP HPLC method and analysed by MS methods. Yields of desired cyclic peptides were rather low. In the products of cyclization, depend on particular cyclic peptide, beside the desire cyclic monomer, mixture of monomer and symmetrical dimer, or only All obtained 15 peptides (7 cyclic monomers and 8 cyclic dimers) were examined in vitro for their inhibitory on VEGF165/NRP-1 binding using competitive enzyme-linked immunosorbent assay (ELISA). Most of synthesized cyclic peptides were found as potent inhibitors and showed higher inhibitory effect than A7R. To have more deep insight into the SAR results of the synthetized peptides molecular modeling was performed for most cyclic monomers and one cyclic dimer. The structures of peptides were docked into b1 domain of NRP-1 co-crystalized with tuftsin (one of the NRP-1 inhibitors, with the sequence TLPRR). Docking analysis suggested that for cyclic peptides, besides Arg which position was similar as for Arg of tuftsin, the key structural feature for the inhibitory effect is the N-terminal amino group of the cyclic peptides that can interact with Glu348 residue in NRP-1. The results of ELISA test and molecular modelling showed that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect. Three of the most active peptides were tested for their stability in human plasma by LC MS methods. These cyclic peptides turned to be quite stable, their half-life were approximately from 5 h for monomers, up to 32 h in the case of cyclic dimer. The last part of my research concerned the optimization of the synthesis of cyclic peptides. The effect of type of resin (Wang, Wang Tenta Gel), coupling reagents (TBTU, DIC/Oxyma, HATU) and different reaction conditions (room temperature and microwave radiation) on the yield of desired cyclic peptides were examined. The results of my study showed that the yield of monomeric product depends mostly on sequence of peptide and type of cyclization.

碩士
國立臺灣大學
毒理學研究所
96
Angiogenesis is a process of new blood vessel formation which has essential roles in development, reproduction and repair. In our previous studies, several that the leukocyte cell-derived chemotaxin 2 (LECT2) gene expression was down-regulated with vascular invasion. The levels of LECT2 are clearly correlated with regulating tumor size and overall survival of HCC patients. Using CAM assay, we found that LECT2-transfected cells conditioned media exhibited extremely low angiogenic activity as compared to control cells. This result suggesting that LECT2 may down-regulate certain angiogenic factors. Hence we proposed to investigate the molecular mechanism underlying LECT2 mediated anti-angiogenic factors-induced angiogenesis in vitro and in vivo. Further, we also observed report the expression of LECT2 in others cancer cells. In this study we evaluated the function of recombinant human LECT2 (rLECT2) proteins on angiogenesis by using human umbilical vein endothelial cells (HUVEC). We demonstrated a selective and significant inhibition of VEGF165 mediated angiogenic activity in HUVEC by rLECT2 protein through inhibiting the VEGF165-induced proliferation, migration and tube formation. The rLECT2 protein also suppressed VEGF165-induced angiogenesis in CAM assay and matrigel plug assay. Both in vitro and in vivo, we found that LECT2 suppressed the VEGF165-induced vascular permeability. Our results demonstrated that rLECT2 protein could reduce the VEGF165-induced VEGFR-2 phosphorylation and inhibited the expression of downstream ERK and AKT phosphorylation in HUVEC. In addition, rLECT2 protein reduced cancer cell conditioned media-induced tube formation in HUVEC and LECT2 also decreased tumor growth of melanoma cells. In conclusion, we for the first time found that LECT2 played an important role in anti-angiogenesis. Moreover the LECT2 might have broad therapeutic applications in diseases characterized by excessive angiogenesis.

碩士
國立陽明大學
口腔生物研究所
96
Autogenic and allogenic bone grafts used traditionally for repairing bony defects have many limitations. Moreover, their regenerative effects in treating large bony defects are still less than ideal. Recently, bone tissue engineering has been regarded as a potential alternative for bone grafting. Cells, scaffolds and signaling molecules are employed in tissue engineering approach to enhance regeneration. In order to maximize the regenerative potential in treating large defects, the combined use of three elements in tissue engineering may be needed. Bone marrow stem cells (BMSCs) are multipotential cells widely used in bone tissue engineering. Gene-activated matrix (GAM) technology is a gene therapy strategy used for sustained delivery of signals. GAM is composed of matrix and plasmid DNA encoding gene of interest. Type I collagen, with excellent cell attachment property, is the most used matrix materials for GAMs. However, collagen GAMs suffer from poor mechanical strength. PLGA scaffolds fabricated by frozen compressed deposit manufacturing (FCDM), with superior mechanical properties and controllable pore size, can be used to reinforce the collagen GAM. Previous studies have indicated that both bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor 165 (VEGF165) enhance bone formation synergistically. We hypothesized that the combined use of human BMSCs and FCDM-PLGA reinforced collagen GAM encoding BMP4 and VEGF165 may act cooperatively on bone regeneration. The combined effect was determined in vivo using SCID mice subcutaneous ectopic bone formation model. The bone mineral content of samples after 3, 8, and 15 weeks of implantation, measured by dual-energy X-ray absorptiometry (DEXA) and radiographic analysis, demonstrated that BMSCs and FCDM-PLGA reinforced BMP4/VEGF165 collagen GAM exerted a synergistic effect in bone formation. The results suggested that the developed combinational approach may be useful in repairing large bony defects in the future.