The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of vital angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor (VEGF) and acidic fibroblast growth factor (aFGF). Transforming growth factor beta-1 (TGF-β1) secretion was relatively low and there was negligible production of basic fibroblast growth factor (bFGF). Histology revealed direct correlation between cell infiltration into scaffolds and the PDO concentration. There was greater macrophage infiltration through fibrous networks of the PDO and 90:10 scaffolds. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Macrophages (MФ) and mast cells are important cell types in the context of tissue remodeling and regeneration. Mast cells participate in the early stages of wound healing and modulate the acute inflammatory responses to biomaterials. Mast cells can secrete a myriad of different cytokines by the process of degranulation; the process of regulated secretion in which preformed contents stored in their granules are rapidly released by exocytosis. Some of these cytokines such as IL-4, IL-13 and TNF-α can modulate the MФ phenotype. Macrophages (MΦ) are innate immune cells, crucial for tissue homeostasis, presentation of foreign and self-antigens following infection/injury, pathogen clearance, inflammation resolution, angiogenesis, and wound healing. MΦ display plasticity and can acquire pro-inflammatory (M1) or angiogenic/wound healing (M2) phenotypes depending upon the environmental stimuli. The phenotypic profile of MФ as M1 or M2 following exposure to the biomaterial can dictate the downstream processes of tissue remodeling and angiogenesis. An analysis of how these two cell types interact with electrospun biomaterials and how different properties of an electrospun biomaterial impacts the MΦ phenotype is the focus of this thesis. Mast cells synthesize several potent angiogenic factors and can also stimulate fibroblasts, endothelial cells and macrophages. An understanding of how they participate in wound healing and angiogenesis is important to further our knowledge about in situ vascular prosthetic regeneration. The adhesion, proliferation and cytokine secretion of bone marrow derived murine mast cells (BMMC) on electrospun polydioxanone (PDO), polycaprolactone (PCL) and silk scaffolds, as well as tissue culture plastic (TCP) has been investigated in the presence or absence of IL-3, SCF, IgE and IgE with a crosslinking antigen, dinitrophenol-conjugated albumin (DNP). It was previously believed that only activated BMMCs exhibit adhesion and cytokine secretion. However, this study shows non-activated BMMC adhesion to electrospun scaffolds. Silk scaffold was not found to be conducive for mast cell adhesion and cytokine secretion. Activation by IgE and DNP significantly enhanced mast cell adhesion, proliferation, migration and secretion of TNF-α, MIP-1α and IL-13. This indicates that mast cells might play a role in MФ polarization (M1/M2), biomaterial integration into the host tissue, regeneration, and possibly angiogenesis. In the next study, bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) electrospun from varying polymer concentrations (60, 100, and 140 mg/ml). Scaffold evaluation showed that large polymer concentrations led to larger fiber diameters, which in turn led to larger pore-sizes and porosity but a smaller surface area to volume ratio. After 24 hrs of culture, the cell lysates were analyzed for Arginase (Arg1) and inducible nitric oxide synthase (iNOS) expression by western blot and cell culture supernatants were analyzed for Nitric oxide (NO2-), Tumor Necrosis Factor – alpha (TNF-α), Interleukin-6 (IL-6), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor – beta1 (TGF-β1) and basic fibroblast growth factor (bFGF) levels. The results indicated a correlation between Arg1 expression and increasing fiber/pore-size, indicating that the larger fiber/pore-sizes polarize towards a M2 phenotype. Also, the expression of iNOS was downregulated on the larger fiber/pore-size. The levels of NO2- were significantly higher on the lower fiber/pore-sizes indicating an M1 phenotype. The levels of VEGF, TGF-β1 and bFGF increased with increasing fiber/pore-sizes. The results showed higher Arg1 expression in M2s on the 60 mg/ml scaffold created by the air-flow impedance method compared to the 60 mg/ml scaffold created on the solid mandrel created by traditional electrospinning. The Arg1 expression was reduced on the compressed 140 mg/ml PDO scaffold compared to the normal 140 mg/ml scaffold. This result indicates that pore-size might be playing a greater role compared to fiber diameter in BMMФ phenotype modulation. In order to assess the angiogenic potential of BMMΦs cultured on PDO scaffolds, a 3D angiogenesis bead assay was performed using conditioned media from the BMMΦ:PDO interaction. The results of the 3D angiogenesis bead assay showed that the conditioned media from BMMΦs of M0 and M2 phenotypes cultured on the 140 mg/ml PDO scaffold induced larger sprouting and higher percentage density of sprouts when compared to the 60 mg/ml PDO scaffold and TCP. To investigate the signaling mechanism involved in this phenotypic switch, BMMΦs were isolated from the bone marrow of MyD88 knockout (KO) mice (Jackson Laboratories) and cultured on PDO (60 and 140 mg/ml) scaffolds (106 /disc) and TCP for 24 hrs and their Arg1 and iNOS expression was analyzed by western blot. The expression of Arg1 and iNOS was severely impaired on the BMMΦs from MyD88-/- mice cultured on the 140 mg/ml scaffold when compared to the 60 mg/ml PDO scaffold and TCP. This result indicates that scaffolds with different fiber/pore-sizes signal differently. A subcutaneous mouse model (described in Chapter 6) was used to evaluate the angiogenic and regenerative potential of PDO scaffolds in vivo. The DIVAA assay showed statistically higher FITC-dextran signal intensity for the 140 mg/ml scaffold compared to the 60 mg/ml scaffold indicating greater angiogenic response in the 140 mg/ml tube. However, problems of high background were observed in this assay with the use of electrospun PDO. The observed high background was probably due to the formation of complexes between dextran and adsorbed plasma proteins on the surface of the PDO. More studies are needed to optimize this assay for use with biomaterials such as PDO. H&E staining of the harvested PDO tubes (60 mg/ml and 140 mg/ml) was also performed. The cross-sections of these tubes showed greater cell recruitment and infiltration into the fibrous structures of the 140 mg/ml tube compared to the 60 mg/ml tube. This result corroborates the in vitro result of BMMФ infiltrating deeper into the structures of the 140 mg/ml scaffold compared to the 60 mg/ml scaffold. The scaffolds were also analyzed by immunostaining for iNOS (indicative of M1 phenotype of MФs). The results showed statistically higher ratios of iNOS positive:negative areas on the 60 mg/ml scaffold compared to the 140 mg/ml scaffold. Overall, these studies indicate that 140 mg/ml scaffold supports greater cell recruitment and cell infiltration in vivo but a smaller ratio of iNOS positive:negative areas compared to the 60 mg/ml scaffold, which supports a predominately M1 MФ phenotype. The studies indicate that varying properties of PDO can alter both the phenotype and function of BMMΦs in vitro and in vivo. We have also shown that the 140 mg/ml scaffold signal BMMΦs through MyD88-dependent mechanisms. A complete understanding of the way materials signal would allow us to control or modulate undesirable immune reactions to biomaterials in vivo. These studies would also help engineer biomaterials that promote angiogenesis and regeneration.

The focus of this thesis is the development of two molecularly targeted imaging methods, in both cases based on contrast agents encompassing micron-sized microparticles of iron oxide (MPIO). MPIO are obligate intravascular agents and as presented in this thesis the half-life in the blood circulation is < 1min. In the first approach described in the thesis, the overall goal was to detect metastasis in mouse lungs, very early in metastatic development, by targeting vascular cell adhesion molecule 1 (VCAM-1) using conjugates of an anti-VCAM-1 antibody and 1 μm MPIO (VCAM-MPIO). In Chapter 3, I demonstrate specific retention of VCAM-MPIO in the vasculature of a lung metastasis model, and also the very short blood half-life of the contrast agent; both of which suggest the potential for in vivo detection. In Chapter 4, I show that whilst the bound VCAM-MPIO do not sufficiently dephase the signal obtained with the bright lung MRI approaches used (hyperpolarized ³He/¹²⁹Xe or ¹⁹F MRI), it is possible to sensitively detect the presence of lung metastases in vivo using radiolabelled VCAM-MPIO (⁸⁹Zr-DFO-VCAM-MPIO) in combination with PET imaging. The overall goal of the second approach described, was to detect and characterize tumour angiogenesis by targeting &alpha;_v&beta;₃-expressing endothelium in vivo, using a conjugate of cyclic penta-peptides c(RGDyK) and 2.8 &mu;m MPIO [c(RGDyK)-MPIO]. To this end, I demonstrate in Chapter 5 that c(RGDyK)-MPIO specifically binds to &alpha;_v&beta;₃-expressing endothelium in subcutaneous tumours and yields quantifiable contrast effects on T₂&ast;-weighted MRI. Furthermore, I have implemented in this approach gadolinium DCE imaging, providing dynamic vascular information. To date there is no reported detection method for pulmonary metastasis at the micrometastastic stage, as presented in this thesis. Translation of this method into clinic could allow for earlier therapeutic intervention and, thus, more effective treatment. The angiogenesis characterization imaging method presented here may provide a sensitive approach for the characterization of heterogeneity in tumour angiogenesis/vascularity and monitoring of anti-angiogenic therapies.

Les glioblastomes sont des tumeurs fatales du fait de leur agressivité et du manque de traitements efficaces. La prolifération accrue, le caractère invasif et la résistance à la mort cellulaire leur confèrent une croissance rapide et une invasion du parenchyme cérébral environnant, à l’origine de leur systématique récidive. Exprimée par la composante tumorale en hypoxie mais également par la composante vasculaire, l’AM participe de façon autocrine et paracrine au développement des glioblastomes en favorisant la croissance des cellules tumorales et l’angiogenèse tumorale.Il a été montré que des anticorps polyclonaux dirigés contre les récepteurs de l’AM inhibent in vitro la croissance, la migration et la formation de pseudo-capillaires des cellules endothéliales, suggérant une neutralisation par ces anticorps de certaines étapes de l’angiogenèse. De même, il a été montré in vivo que ces anticorps inhibent la croissance tumorale en supprimant l’angiogenèse et la croissance des cellules tumorales suggérant ainsi que les récepteurs de l’AM constitueraient une bonne cible thérapeutique. Des anticorps capables de reconnaître et neutraliser à la fois l’AM, les CLR, RAMP2 et RAMP3 agissant de la même manière sur la croissance tumorale et l’angiogenèse représenteraient un bénéfice thérapeutique majeur. Des anticorps dirigés contre un peptide chimérique constitué de l’enchainement de séquences peptidiques des protéines CLR, RAMP2, RAMP3 et du peptide AM sont en cours. Le traitement par ces anticorps diminue la croissance des cellules tumorales ainsi que leurs migration et invasion. Ces résultats très encourageants nous permettent pour le moment de valider la faisabilité du concept d’anticorps développés à partir d’un peptide chimérique pour neutraliser le système AM/AMR dans le but d’envisager dans le futur une application thérapeutique. / Glioblastoma are fatal tumors because of their aggressiveness and lack of effective treatments. The increased proliferation, the invasiveness and resistance to cell death gives them a rapid growth and invasion of brain parenchyma surrounding the origin of their systematic recurrence. Expressed by the tumoral component in hypoxia but also by the vascular component, the AM participate by an autocrine and paracrine way, the development of glioblastoma by promoting tumor ell growth and tumor angiogenesis.It was shown that polyclonal antibodies directed against the AM receptor inhibit in vitro growth, migration and the formation of pseudo-capillary of endothelial cells, suggesting neutralization by theses antibodies in certain stages of angiogenesis. Similarly, it has been shown in vivo that these antibodies inhibit tumor growth by suppressing angiogenesis and tumor cell growth, suggesting that the AM receptor would be a good therapeutic target. Antibodies that recognized and neutralized both the AM, the CLR, RAMP2 and RAMP3 acting the same way on tumor growth and angiogenesis represent a major therapeutic benefit. Antibodies against a chimeric peptide consisting of peptide sequence of CLR, RAMP2, RAMP3 and AM peptide are in progress. Treatment with these antibodies decreases the growth of tumor cells, their migration and invasion. These encouraging results allow us the time to validate the feasibility of the concept of antibodies developed from a chimeric peptide to neutralize the AM/AMR system in order to consider in the future therapeutic application.

No description available.

Os vasos sanguíneos são formados, entre outros componentes, por células endoteliais as quais fazem parte da microvasculatura óssea e são capazes de regular o desenvolvimento ósseo tendo em vista de que os processos de osteogênese e angiogênese estão interligados. Os leucotrienos (LTs) são mediadores lipídicos envolvidos no recrutamento de leucócitos e na regulação da síntese de citocinas. O tratamento com o leucotrieno B4 (LTB4) induz a angiogênese pela superexpressão do fator de crescimento endotelial vascular (VEGF). Assim, o objetivo do trabalho foi investigar o efeito das células endoteliais reguladas pelo LTB4 na diferenciação osteogênica. Para isso, células endoteliais primárias de aorta foram isoladas e cultivadas por até 4 dias e, quando apropriado, foi realizado o tratamento das mesmas com o LTB4. O meio condicionado das células endoteliais foi armazenado para os experimentos com osteoblastos. Células osteoblásticas foram isoladas da calvária e cultivadas por até 21 dias, avaliando-se, portanto, os efeitos das células endoteliais reguladas ou não pelo LTB4 e a resposta dos estímulos exógenos LTB4, o inibidor da síntese de LTs MK 886 e o antagonista do receptor do LTB4, o U75302. Tais respostas foram observadas na fase de crescimento celular, por meio da viabilidade, proliferação e produção de marcadores osteogênicos e angiogênicos como o RANKL, OPG e o VEGF por meio da redução do MTT, citometria de fluxo e western blotting, respectivamente. A diferenciação foi avaliada por meio dos ensaios de fosfatase alcalina (ALP) e expressão gênica por meio de ensaio enzimático e qRT-PCR e mineralização por Vermelho de alizarina. Resultados mostraram que tanto as células endoteliais, mediadas ou não pelo LTB4, quanto os estímulos exógenos não foram capazes de modular a proliferação dos osteoblastos. Porém, durante a diferenciação, o LTB4 inibiu a atividade da ALP, a expressão gênica do BLT1, ALP, BGLAP (osteocalcina) e OPG (osteoprotegerina) foi aumentada, e os genes RANKL e VEGF tiveram a sua expressão diminuída pelo tratamento com o meio condicionado das células endoteliais, mediadas ou não pelo LTB4 (P<0,05). Além disso, a mineralização dos osteoblastos foi aumentada pelas células endoteliais e diminuída pelas células endoteliais mediadas pelo LTB4 (P<0,05). Assim, podemos concluir que os fatores angiogênicos das células endoteliais, mediadas ou não pelo LTB4, exercem um papel importante na regulação da diferenciação osteogênica e formação óssea contribuindo, portanto, para a compreensão de mecanismos que regulam a patofisiologia de doenças ósseas. / Endothelial cells make blood vessels and are involved in the regulation of tissue metabolism. Endothelial cells from bone microvasculature are capable of regulating bone development in view of the fact that the processes of osteogenesis and angiogenesis are interconnected. Leukotrienes (LTs) are lipid mediators involved in leukocyte recruitment and regulation of cytokine synthesis. It is known that the treatment with LTB4 induces angiogenesis by overexpression of vascular endothelial growth factor (VEGF). Thus, the aim of this study was to investigate the effect of LTB4-regulated endothelial cells on osteogenic differentiation. For this, primary endothelial cells from aorta were isolated and cultured for up to 4 days and, where appropriate, the treatment with LTB4 was done. The conditioned medium of these cells was stored for osteoblast experiments. Osteoblastic cells were isolated from calvaria and cultured for up to 21 days, assessing the effects of endothelial cells regulated or not by LTB4 and the response of exogenous LTB4 stimuli, the inhibitor of LTs synthesis MK 886 and the antagonist of LTB4 receptor, U75302. Such responses were observed in the cell growth phase through the viability, proliferation and production of osteogenic and angiogenic markers such as RANKL, OPG and VEGF by MTT assay, flow cytometry and western blotting, respectively. The cell differentiation was evaluated by alkaline phosphatase (ALP), gene expression and mineralization assay through ALP enzymatic assay, qRT-PCR and Alizarin Red staining. Results showed that both endothelial cells, mediated or not by LTB4 and exogenous stimuli were not able to modulate the osteoblasts proliferation. However, during the differentiation, LTB4 inhibited ALP activity, the gene expression of BLT1, ALP, BGLAP (osteocalcin) and OPG (osteoprotegerin) was increased, and the RANKL and VEGF genes had their expression decreased by the treatment with the endothelial cells conditioned medium, mediated or not by LTB4 (P <0.05). In addition, the osteoblasts mineralization was increased by endothelial cells conditioned medium (CM-EC) and decreased by LTB4-mediated endothelial cells conditioned medium (CM-EC-LTB4) (P <0.05). Thus, we can conclude that the angiogenic factors of the endothelial cells, mediated or not by LTB4, play an important role in the regulation of osteogenic differentiation and bone formation, thus contributing to the understanding of mechanisms that regulate the pathophysiology of bone diseases.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.

A terapia fotodinâmica é uma técnica que utiliza uma substância fotossensibilizadora, luz de comprimento de onda adequado e oxigênio para produzir um efeito citotóxico, sendo uma alternativa aos tratamentos convencionais para o câncer. Este tratamento, quando realizado nos vasos sanguíneos, leva à destruição deles. No entanto, a recuperação dos vasos é observada algum tempo depois, o que pode ser um processo angiogênico (formação de novos vasos sanguíneos) induzido pela própria terapia. Os vasos sanguíneos fornecem oxigênio e nutrientes às células, levando ao crescimento de tecidos, inclusive tumorais. Para a investigação da angiogênese após a terapia fotodinâmica, foi utilizado o modelo de membrana corioalantoica de ovos de galinha, pois possui alta vascularização e fácil acesso aos vasos sanguíneos. A terapia fotodinâmica foi feita nos vasos da membrana com o fotossensibilizador Photogem&reg;, em uma concentração de 10 &mu;g/mL, e subdoses de luz para não levar o embrião à morte. As doses de luz de 6 e 15 J/cm2 foram estabelecidas para os experimentos e foi observada diminuição na densidade de vasos 3 horas após a terapia fotodinâmica, com um aumento 24 horas depois. Para a quantificação desses efeitos, uma rotina no MATLAB&reg; foi elaborada para determinar a porcentagem de área ocupada pelos vasos sanguíneos nas imagens da membrana, que foram realizadas antes, a cada 30 minutos durante as primeiras 3 horas após o tratamento e 24 horas depois. Além disso, para uma análise da distribuição de grandes e pequenos vasos, o comprimento e o diâmetro de cada vaso nas imagens foram medidos com o software ImageJ&reg;, que permitiu verificar que os menores vasos são os mais afetados 3 horas depois da terapia, com aumento no número desses vasos após 24 horas. Como isso poderia ser um indício de um processo angiogênico após a terapia fotodinâmica, marcadores de angiogênese foram utilizados em Western Blot. Apesar de esse método molecular não ter mostrado diferença entre o grupo com terapia fotodinâmica e os grupos controle, as análises por imagem indicam a formação de novos vasos 24 horas após a terapia, com uma rede vascular diferente da que havia antes. / Photodynamic therapy is a technique that uses a photosensitizing substance, light of adequate wavelength and oxygen to produce a cytotoxic effect, being an alternative to conventional treatments for cancer. This treatment, when carried out in the blood vessels, leads to their destruction. However, vessel recovery is observed some time later, which may be an angiogenic process (formation of new blood vessels) induced by the therapy itself. The blood vessels supply oxygen and nutrients to the cells, leading to tissue growth, including tumoral. For the investigation of angiogenesis after photodynamic therapy, the chorioallantoic membrane model of chicken eggs was used, because it has high vascularization and easy access to the blood vessels. Photodynamic therapy was performed on membrane vessels with the Photogem&reg; photosensitizer, at a concentration of 10 &mu;g/mL, and light subdoses to avoid leading the embryo to death. Light doses of 6 and 15 J/cm2 were established for the experiments and a decrease in vessel density 3 hours after photodynamic therapy was observed, with an increase 24 hours later. For quantification of these effects, a routine in MATLAB&reg; was designed to determine the percentage of area occupied by blood vessels in the membrane images, which were performed before, every 30 minutes for the first 3 hours after treatment and 24 hours later. Furthermore, for an analysis of the distribution of large and small vessels, the length and diameter of each vessel in the images were measured with the ImageJ&reg; software, which allowed to verify that the smaller vessels are most affected 3 hours after the therapy, with an increase in the number of these vessels after 24 hours. Since this could be an indication of an angiogenic process after photodynamic therapy, angiogenesis markers were used in Western Blot. Although this molecular method showed no difference between the group with photodynamic therapy and the control groups, the image analysis indicates the formation of new vessels 24 hours after the therapy, with a vascular network different from before.

As proteínas S100A8/A9 são expressas em diferentes tipos celulares e quando sozinhas ou complexadas e em baixas concentrações, promoveram proliferação, migração celular e formação de estruturas capilares. Por outro lado, quando em altas concentrações, esse complexo inibe o crescimento de diversos tipos de células tumorais murinas e humanas. Ainda, tanto a proteína S100A9 humana, quanto um peptídeo sintético idêntico a porção C-terminal da proteína S100A9 murina (pS100A9m) possuem efeitos antinociceptivo e imunorregulatório. Apesar dessas evidencias, até o momento não foi investigado o efeito do pS100A9m sobre a angiogênese e a tumorigênese. Portanto, o objetivo do presente estudo foi investigar, in vitro, o efeito do pS100A9m sobre os eventos fundamentais envolvidos com a angiogênese e o desenvolvimento tumoral. Para tanto, a fim de avaliar o efeito do pS100A9m sobre a angiogênese foi utilizada a linhagem de células endoteliais tímicas murinas (tEnd.1) nos ensaios de proliferação, migração da célula endotelial em meio de cultura, avaliada nos modelos de wound healing e transwell ou migração em meio condicionado, obtido de células tumorais LLC WRC256, avaliada no modelo de transwell, ensaio de adesão (aos componentes de matriz, tais como o colágeno tipo I, fibronectina e laminina) e formação de tubos em matrigel tridimensional (3D). Para os estudos sobre o efeito do pS100A9m sobre as células tumorais, foi utilizada a linhagem de células LLC WRC256 para realização dos ensaios funcionais de proliferação, migração (wound healing) e adesão (sobre os componentes da matriz extracelular). Os resultados obtidos demonstraram que o pS100A9m inibe a proliferação, migração, adesão sobre os componentes de matriz e, consequentemente, a formação de estruturas capilares em matriz 3D. Em relação às células tumorais LLC WRC256, foi observada, novamente, a ação inibitória do pS100A9m sobre os eventos de proliferação e migração. Em relação à adesão, o peptídeo aumentou a capacidade de adesão das células tumorais sobre o colágeno tipo I e fibronectina, porém inibiu a adesão dessas células sobre laminina. Em conclusão, os dados aqui obtidos demonstram que o pS100A9m inibe in vitro os eventos fundamentais envolvidos com a angiogênese e com a progressão tumoral. Desta forma, o peptídeo da porção C-terminal da proteína S100A9 pode ser considerado uma nova ferramenta para o estudo da angiogênese e tumorigênese, além apresentar potencial para uma possível aplicação terapêutica nesses processos / The S100A8/A9 proteins are expressed in different cell types and alone or when complexed, and at low concentrations promoted proliferation, cell migration and formation of capillary structures. On the other hand, at higher concentrations, this compound inhibits the growth of many types of murine and human tumor cells. Moreover, both human S100A9 protein and a synthetic peptide identical to the C-terminal portion of murine S100A9 (mS100A9p) present antinociceptive and immunomodulatory effects. Despite these evidences, the effect of mS100A9p on angiogenesis and tumorigenesis has not been investigated. Therefore, the aim of this study was to investigate the in vitro effect of mS100A9p on crucial events involved in angiogenesis and tumor development. For this, in order to evaluate the effect of mS100A9p on angiogenesis was used the murine endothelial cell line derived from thymus hemangioma (tEnd.1) for proliferation assays, endothelial cell migration in the presence of culture medium (scratch wound healing and chemotaxis assays) or in conditioned medium prevenient from LLC WRC256 tumor cells (chemotaxis assays), adhesion assay (on extracellular matrix components, such as type I collagen, fibronectin and laminin) and tube like-structure formation in 3D matrix. For the analyzes of the effect of mS100A9p on tumor cells, the cell line LLC WRC256 was used to perform functional assays such as proliferation, migration (scratch wound healing model) and adhesion (on components of the extracellular matrix). The results showed that the mS100A9p inhibits the proliferation, migration and adhesion of endothelial cells to the matrix components and consequently the formation of capillary structures in 3D matrix. Regarding LLC WRC256 tumor cells, it was observed again the inhibitory action of the mS100A9p on proliferation and migration events. In relation to cellular adhesion, this peptide increased this parameter of tumor cells on type I collagen and fibronectin. However mS100A9p inhibited the adhesion of these cells on laminin. In conclusion, the data obtained show that the mS100A9p inhibits in vitro crucial events involved in angiogenesis and tumor progression. Thus, the C-terminal portion of murine S100A9 protein may be considered as a new tool for the study of tumorigenesis and angiogenesis besides presenting potential to a possible therapeutic application in these processes

A angiogênese é um processo fundamental e fisiológico de organismos vertebrados, sendo responsável pela formação de novos vasos sanguíneos a partir dos já existentes. Entretanto, a angiogênese pode ocorrer também em condições patológicas, como causa ou consequência de doenças. Um exemplo disso está nos tumores, que para crescer além de alguns milímetros cúbicos, necessitam de um suprimento adequado de oxigênio e nutrientes, e, portanto, dependem da angiogênese. Por isso, compostos que inibem a angiogênese já estão em uso na clínica, não só para o tratamento de tumores, mas também de outras doenças dependentes da angiogênese, as retinopatias. Neste projeto, daremos continuidade à linha de pesquisa do nosso grupo, que procura identificar e validar peptídeos com potencial translacional (pré-fármacos), por apresentarem atividade anti-angiogênica. Utilizando a metodologia do Phage Display, nosso grupo identificou e caracterizou um hexapeptídeo, que foi selecionado por interagir com os receptores do principal fator iniciador da angiogênese, o VEGF (fator de crescimento endotelial vascular). Os receptores de VEGF (ou VEGFR) são proteínas do tipo receptor tirosina quinase, expressos em células endoteliais e essenciais para a iniciação e progressão da neovascularização. O hexapeptídeo identificado em nosso laboratório liga-se ao VEGFRs e inibe a formação de vasos sanguíneos in vivo em modelos animais de angiogênese. Neste trabalho, procuramos estender os estudos com este hexapeptídeo para identificar o sítio de ligação do mesmo no VEGFR e avançar em modelos que permitam a determinação dos requisitos estruturais de interação peptídeoreceptor. Com estes conhecimentos, poderemos num futuro próximo, caminhar para o desenvolvimento racional de moléculas peptideomiméticas com propriedades anti-angiogênicas. / Angiogenesis is a fundamental and physiological process for vertebrate organisms, being responsible for the formation of new blood vessels, sprouting from the existent ones. However, angiogenesis may occur in pathological conditions, being cause or consequence of diseases. One example is tumor development. To grow beyond a few cubic millimeters, tumors need a suitable supply of oxygen and nutrients, and, therefore, they are dependent of angiogenesis. In fact, anti-angiogenic compounds are already in therapeutic use, targeting not only tumors but other angiogenesis dependent diseases, like retinopathies. In this project, we expand research from our own group to identify and develop anti-angiogenic peptides with translational potential (pre-drugs). Using Phage Display methodology, our group identified and characterized a hexapeptide, that was selected based on its capacity to interact with the receptors for the main initiator factor of angiogenesis, the VEGF (Vascular Endothelial Growth Factor). VEGF receptors (VEGFR) are tyrosine kinase proteins, expressed by endothelial cells and essential for neovascularization initiation and progress. The hexapeptide identified in our lab binds to VEGFRs and inhibit blood vessel formation in vivo when tested in an angiogenesis animal model. In this study, we seek to further understand the interaction of this hexapeptide with its receptor by identifying its binding domain on VEGFR and develop models that will allow the determination of the structural requirements for interaction of this receptor ligand pair. With this knowledge, we can in a near future progress to a rational development of novel peptidemimetic molecules with angiogenic properties similar to this hexapeptide.