Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model.

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Advances and Challenges on Cancer Cells Reprogramming Using Induced Pluripotent Stem Cells Technologies.

Cancer cells transformation into a normal state or into a cancer cell population which is less tumorigenic than the initial one is a challenge that has been discussed during last decades and it is still far to be solved. Due to the highly heterogeneous nature of cancer cells, such transformation involves many genetic and epigenetic factors which are specific for each type of tumor. Different methods of cancer cells reprogramming have been established and can represent a possibility to obtain less tumorigenic or even normal cells. These methods are quite complex, thus a simple and efficient method of reprogramming is still required. As soon as induced pluripotent stem cells (iPSC) technology, which allowed to reprogram terminally differentiated cells into embryonic stem cells (ESC)-like, was developed, the method strongly attracted the attention of researches, opening new perspectives for stem cell (SC) personalized therapies and offering a powerful in vitro model for drug screening. This technology is also used to reprogram cancer cells, thus providing a modern platform to study cancer-related genes and the interaction between these genes and the cell environment before and after reprogramming, in order to elucidate the mechanisms of cancer initiation and progression. The present review summarizes recent advances on cancer cells reprogramming using iPSC technology and shows the progress achieved in such field.

Estabelecimento e caracterização de células tumorais de pacientes portadores da síndrome de Li-Fraumeni / Establishment and characterization of cancer cells from patients with Li-Fraumeni syndrome

A Síndrome de Li-Fraumeni (LFS) é uma síndrome rara de predisposição hereditária ao câncer relacionada a mutações germinativas no gene TP53. Portadores de mutações neste gene apresentam alto risco para o desenvolvimento de um amplo espectro tumoral em idade precoce. No sul do Brasil, 0,3% da população local é portadora de uma mutação germinativa no códon 337 do gene TP53 (p.R337H) que foi descrita como tendo um efeito fundador no país. Sabe-se que a proteína p53 desempenha um papel crítico na regulação da diferenciação e do padrão de divisão das células tronco (CT). As CT são células capazes de se autorrenovarem e de se diferenciarem em diversos tipos celulares. Estas células têm sido apontadas como responsáveis pela iniciação e progressão tumoral, sendo resistentes aos tratamentos quimioterápicos. Levantou-se a hipótese de que pacientes LFS possuem alto risco para o desenvolvimento de tumores malignos devido a presença de CT em tecidos específicos. O objetivo do presente trabalho foi isolar e caracterizar células tronco tumorais (CTT) de pacientes LFS portadores de mutações germinativas no gene TP53. As amostras tumorais foram obtidas mediante consentimento informado e no total, 12 culturas de células primárias de 11 pacientes LFS com e sem a mutação p.R337H foram estabelecidas. Uma cultura de células oriunda de um carcinoma de mama foi o enfoque do presente estudo. O novo protocolo nomeado "ALTERNADO" (cultura em monocamada ­ cultura em suspensão (mamoesferas) ­ cultura em monocamada) foi elaborado e permitiu o cultivo prolongado das células obtidas sem sinais de senescência. Os métodos de imunofluorescência e citometria de fluxo demonstraram que estas células apresentam o mesmo perfil imunofenotípico CD44high/+, CD24low/- e ALDH+ previamente descrito para CTT de mama. Ainda, observamos a expressão dos seguintes marcadores: nanog, sox2, oct3/4, p53 e Ki67. A videomicroscopia por time-lapse permitiu a observação de divisões simétricas frequentes e ausência de fenótipos senescentes na cultura obtida. A análise do padrão de divisão celular utilizando o anti-oct3/4 demonstrou que ambos os tipos de divisões (simétrica e assimétrica) ocorrem, porém a divisão simétrica se mostrou mais frequente. Além disso, as células apresentaram capacidade de diferenciação in vitro para as linhagens adipogênica e osteogênica, bem como alta capacidade clonogênica. O perfil da expressão dos receptores de estrógeno e progesterona, além da capacidade proliferativa demonstrada pelo anti-Ki67 se mostrou idêntica ao observado no anatomopatológico do tumor, porém, diferentemente do tumor, algumas células tiveram marcação citoplasmática para o receptor HER2. A análise de expressão gênica em larga escala revelou perfil diferencial de expressão entre as mamoesferas e as células aderentes, sendo que os principais genes diferencialmente expressos na mamoesfera estavam relacionados com o aumento da capacidade de metástase e invasão. Por meio do ensaio de invasão demonstramos que as mamoesferas possuem células com maior capacidade de invasão quando comparadas as mesmas células cultivadas em monocamada. Estabelecemos, pela primeira vez, um banco de células a partir de diferentes tipos tumorais de pacientes LFS. As culturas celulares primárias estabelecidas e os dados apresentados no presente trabalho são de extrema importância para o melhor entendimento do papel desta mutação específica e também do gene TP53 na biologia dos tumores desta coorte de pacientes

Li-Fraumeni Syndrome (LFS) is a rare syndrome of hereditary predisposition to cancer which is related to germline mutations in TP53 gene. Carriers of mutations in this gene are at high risk for the development of a broad spectrum of tumors in early ages. In southern Brazil, 0,3% of the local population is carrier of a germline mutation in codon 337 of TP53 gene (p.R337H) which was described as having a founder effect in the country. It is known that p53 plays a critical role in the regulation of differentiation and the pattern of stem cells (SC) division. SC are cells capable of self renewing and differentiating to different cell types. These cells have been identified as responsible for tumor initiation and progression, being resistant to chemotherapy treatments. It was hypothesized that LFS patients are at high risk for developing malignant tumors due to the presence of SC in specific tissues. The objective of the present study was to isolate and characterize tumor stem cells (CSC) of LFS patients carriers of germline mutations in TP53 gene. Tumor samples were obtained with informed consent and in total, 12 primary cultures from 11 patients with and without LFS p.R337H mutation were established. A culture of cells derived from a breast carcinoma was the focus of the present study. The new protocol named "ALTERNATE" (monolayer culture - suspension culture (mammospheres) - monolayer culture) was prepared and allowed extended cultivation of obtained cells without signs of senescence. Immunofluorescence and flow cytometry assays demonstrated that these cells shared the same immunophenotypic profile CD44high/+, CD24low/- and ALDH+ previously described for breast CSC. We also observed the expression of the following markers: nanog, sox2, oct3/4, p53 and ki67. The videomicroscopy by time-lapse allowed the observation of frequent symmetric divisions and the lack of senescent phenotypes in established culture. The cell division pattern analysis by using anti-oct3/4 showed that both types of divisions (symmetric and asymmetric) occur, but symmetric division was more frequently observed. Moreover, the cells had the ability to differentiate in vitro to osteogenic and adipogenic lineages, as well as possess high clonogenic capacity. The expression profile of estrogen and progesterone receptors, as well as proliferative capacity demonstrated by anti-ki67 showed identical pattern to that observed in the tumor, however, some cells began to express HER2 receptor in cytoplasm. The microarray analysis revealed distinct gene expression profile between mammospheres and adherent cells, while the major differentially expressed genes in mammospheres were associated with increased metastasis and invasion ability. Through the invasion assay we demonstrated that our cells cultured as mammospheres possess high invasive capacity when compared to the same population of cells but which were cultured in monolayer. Herein, we established for the first time a bank of cells from different tumor types of LFS patients. These obtained cell cultures and the results presented in this study are extremely important to better understand the role of this specific mutation and also the TP53 gene in tumor biology of this cohort of patients

Células tronco do tecido adiposo de equinos: Estudo do seu potencial para o tratamento da endometrose / Stem cells from equine adipose tissue Study of their potential for the treatment of endometrosis

A aplicação terapêutica de Células Tronco ( CT) em equinos é um campo emergente. Nestes animais, as CT são promissoras para o tratamento de lesões nos tendões e rupturas de ligamentos...

In horses, Stem Cell (SC) therapies are a promising tool to the treatment of many injuries, as tendon lesions and ligaments rupture...