Hypoxia is a common feature of solid tumors and one of the hallmarks of tumor microenvironment. Tumor hypoxia plays an important role in angiogenesis, malignant progression, metastatic development, chemo-radio resistance and favours immune evasion by the emergence of tumor variants with increased survival and anti-apoptotic potential. There is very little work done on the impact of tumor hypoxia on the regulation of tumor susceptibility to the lysis induced by cytotoxic antitumor response. Therefore, we asked whether hypoxia confers tumor resistance to cytotoxic T lymphocyte (CTL)-mediated killing. We demonstrated that exposure of target cells to hypoxia has an inhibitory effect on the CTL-mediated autologous target cell lysis. Such inhibition was not associated with an alteration of CTL reactivity and tumor target recognition. We also showed that the concomitant hypoxic induction of Signal transducer and activator of transcription 3 (STAT3) phosphorylation on tyrosine 705 residue (pSTAT3) and hypoxia inducible factor 1 alpha (HIF-1α) is functionally linked to the alteration of Non small cell lung carcinoma (NSCLC) target susceptibility to CTL-mediated killing. We also showed that hypoxia-induced resistance of lung tumor to CTL-mediated lysis was associated with autophagy induction in target cells. Inhibition of autophagy resulted in impairment of pSTAT3 (via inhibition Src kinase) and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine (HCQ) in B16F10 tumor bearing mice and mice vaccinated with TRP2 peptide dramatically increased tumor growth inhibition. Collectively, the current study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen specific T cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.Finally, as resistance of tumor targets to killer cells is likely to be regulated by multiple factors, we further aimed to identify the microRNA's regulated by hypoxia in NSCLC and melanoma and their putative involvement in the regulation of tumor susceptibility to antigen-specific CTL-mediated killing. MicroRNA-210 (miR-210) was significantly induced in a HIF-1α dependent manner in NSCLC and melanoma cells and miR-210 was expressed in hypoxic zones of human NSCLC tissues. Moreover, we demonstrated that hypoxia-induced miR-210 regulates tumor cell susceptibility to CTL-mediated lysis in part by suppressing PTPN, HOXA1 and TP53I11 expression indicating that miR-210 plays a potential role in the regulation of anti-tumor immune response.

A galectina-3 (gal-3) pertence a uma família de proteínas com domínios de ligação a beta-galactosídeos e está relacionada com diversos aspectos tumorais, como proliferação e adesão celular, angiogênese e proteção contra morte celular. Estudos mostram sua relação com o fenômeno da hipóxia, característica de diversos tumores sólidos que apresentam altas taxas de proliferação celular. A adaptação à hipóxia é mediada principalmente pelo Fator Induzido por Hipóxia (HIF-1), a qual atua na indução de diversos genes de sobrevivência em ambientes com baixas concentrações de oxigênio. Além de HIF, outros fatores são importantes nesse processo, como NF-kB, por exemplo, sendo um fator de transcrição responsivo a diversos estresses celulares, entre eles, a hipóxia. Alguns modelos tumorais apresentam-se ideais para o estudo dos efeitos da hipóxia no microambiente tumoral, como os glioblastomas. Estes são tumores do sistema nervoso central com altas taxas de letalidade, são refratários aos principais métodos de tratamento por sua plasticidade, crescimento infiltrativo e heterogeneidade. Histologicamente, estes tumores apresentam atipia nuclear, altas taxas de mitose e áreas de pseudopaliçada. Postula-se que estas áreas sejam compostas por células migrantes de ambientes necróticos, os quais são também hipóxicos devido a sua distância de vasos sanguíneos e é demonstrado que estas células expressam tanto HIF-1alfa quanto gal-3. Ensaios in vitro realizados por nosso grupo demonstraram que a gal-3 é positivamente regulada pela hipóxia em uma linhagem de glioma híbrido, NG97ht, além de demonstrar que esta proteína é um fator chave na proteção destas células contra a morte celular induzida pela privação de oxigênio e nutrientes, mimetizando condições necróticas de pseudopaliçada in vivo, destacando-se as habilidades antiapoptóticas desta proteína. Embora uma de suas possíveis funções tenha sido elucidada, os mecanismos de atuação e de indução da gal-3 ainda são obscuros. Deste modo, este projeto visa explorar os papéis pró-tumorais da gal-3, podendo torná-la um possível alvo em terapias anti-neoplásicas, entendendo melhor seus mecanismos de proteção contra a morte celular e controle de expressão em ambientes hipóxicos, além de estudar suas possíveis funções in vivo no desenvolvimento de tumores, e também estendendo seus estudos para outras linhagens de glioblastoma. Nossos resultados demonstraram que a gal-3 está co-localizada com mitocôndrias nestas linhagens de glioma, podendo sofrer alterações pós-traducionais em hipóxia, como a fosforilação e que houve acúmulo de HIF-1alfa nuclear nestas células em hipóxia. Vimos também que a gal-3 na linhagem NG97ht apresentou-se proveniente de dois alelos diferentes e que fatores intermediários deveriam ser expressos previamente pela célula antes da indução de gal-3 em hipóxia. Também demonstramos que houve dependência de NF-kB na indução transcricional de gal-3 nestas condições. Estes experimentos também demonstraram que a exposição de células à hipóxia e privação de nutrientes é capaz de induzir tanto espécies reativas de oxigênio como o aumento da autofagia nestas células, fatores importantes na indução da morte celular, além de demonstrar que na linhagem NG97ht a indução da morte nestas condições ocorreu por necrose, sem apresentar apoptose celular. Expandimos esta teoria da participação da gal-3 como molécula protetora contra a morte em hipóxia e privação de nutrientes para outra linhagem de glioma humano, a T98G. E finalmente, demonstramos que a diminuição da expressão de gal-3 em células tumorais da linhagem U87MG levou a diminuição das taxas de estabelecimento e crescimento tumoral in vivo / Galectin-3 (gal-3) belongs to a family of proteins with beta-galactoside binding domains and is related to various tumoral aspects, such as cell proliferation and adhesion, angiogenesis and protection against cell death. Studies show its relationship with the hypoxia phenomenon, a characteristic of many solid tumors that have high cell proliferation rates. The adaptation to hypoxia is mainly mediated by Hypoxia Induced Factor (HIF-1), which acts in the induction of several survival genes in environments with low oxygen concentrations. In addition to HIF, other factors are important in this process, such as NF-kB, for example, which is a transcription factor responsive to various cellular stresses, including hypoxia. Some tumor models are ideal for studying the effects of hypoxia in the tumor microenvironment, e.g. glioblastomas. These central nervous system tumors with high mortality rates are refractory to the main treatment methods due to their plasticity, heterogeneity and infiltrative growth. Histologically, these tumors exhibit nuclear atypia, high mitotic rates and pseudopalisading areas. It is postulated that these areas are composed of migrating cells out of necrotic microenvironments, which are also hypoxic due to their distance from the blood vessels and it is shown that these cells express both HIF-1alfa and gal-3. In vitro assays performed by our group demonstrated that gal-3 is positively regulated by hypoxia in a hybrid glioma cell line, NG97ht, and demonstrated that this protein is a key factor in protecting these cells against cell death induced by oxygen and nutrient deprivation conditions mimicking necrotic pseudopalisading areas in vivo, highlighting the pro-survival abilities of this protein. Although one of its possible functions has been elucidated, gal-3 mechanisms of action and induction are still unclear. Thus, this project aims to explore the gal-3 pro-tumoral effects, which may make it a possible target for anti-neoplastic therapies, better understanding the mechanisms of protection against cell death and expression in hypoxic environments, and also study its possible functions in vivo, extending these studies to other glioma cell lines. Our results demonstrated that gal-3 is located within the mitochondria in these glioma cell lines and may undergo posttranslational modifications in hypoxia, such as phosphorylation and that there is accumulation of nuclear HIF-1alfa in these cells under hypoxia. We have also seen that gal-3 in the NG97ht cell line presents two different alleles and that intermediate factors must be expressed previously by the cell before gal-3 induction in hypoxia. We also demonstrated that there is dependence on the NF-kB transcriptional factor for the gal-3 induction under these conditions. These experiments also demonstrated that exposure of cells to hypoxia and nutrient deprivation is capable of inducing reactive oxygen species and increased autophagy in these cells, which are important factors in the induction of cell death. In addition, we demonstrated that the induction of the NG97ht cell death in these conditions is due to necrosis. We expanded this theory of the participation of gal-3 as a protective molecule against cell death in hypoxia and nutrient deprivation to another human glioma cell line, T98G. And finally, we demonstrated that decreased expression of gal-3 in the U87MG glioma cell line leads to lower tumor establishment rates and decreased growth in vivo

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

O presente estudo avaliou a capacidade de invasão de hepatócitos de rato por Shigella flexneri (S. flexneri) nas condições de normóxia e hipóxia. O estudo do microambiente de hipóxia tem grande importância, por estar presente em muitas doenças hepáticas, além de aumentar a translocação quando presente no lúmen intestinal. Bactérias invasivas como S. flexneri podem romper a barreira intestinal e chegar ao fígado através da circulação portal. O efeito da invasão bacteriana das células hepáticas é pouco conhecido. Neste trabalho buscamos pesquisar as alterações morfológicas e funcionais de hepatócitos de rato após infecção por S. flexneri na presença e na ausência de hipóxia. Para esta finalidade foram utilizados hepatócitos de rato cultivados pela técnica de cultura primária. Vários parâmetros foram analisados, tais como: taxa de invasão celular pela bactéria, quantificação da produção e liberação de DHL, produção de TNF-, taxa de morte celular por apoptose e a expressão do fator de transcrição HIF-1a. Os resultados mostraram que a metodologia empregada para a obtenção do microambiente hipóxico foi satisfatória, com redução de 70% da pO2 inicial (atingindo 43.2 mmHg in vitro ou 6.5% O2). A invasão de hepatócitos de rato por S. flexneri foi menor nas células previamente expostas à hipóxia quando comparada com a invasão das células cultivadas em normóxia. A viabilidade dos hepatócitos não apresentou diferenças significativas entre os grupos experimentais, variando entre 74 e 86%. A liberação de TNF- nas situações de normóxia e hipóxia foi similar, embora as células infectadas em normóxia tenham aumentado a liberação desta citocina. Na condição de hipóxia + infecção a liberação de TNF- foi menor do que na condição de normóxia + infecção, porém ambos os grupos produziram aumento significativo da citocina em relação aos controles normóxicos e hipóxicos. Este resultado sugere que a presença da bactéria no interior das células aumenta significativamente a liberação de TNF-pelos hepatócitos. A produção de DHL também foi maior de forma significativa no grupo hipóxico em relação ao grupo normóxico, porém não apresentou alteração nos grupos infectados por S. flexneri após uma hora. As taxas de apoptose aumentaram nos grupos hipóxia e nos grupos infectados com S. flexneri de maneira similar, variando entre 24 e 31%, quando comparados aos grupos controle em normóxia. A expressão do fator de transcrição HIF ocorreu nos grupos: hipóxia, normóxia + infecção e hipóxia + infecção, evidenciando que a infecção por S. flexneri induz a expressão deste fator. Em seu conjunto, nossos resultados buscam contribuir para o maior conhecimento da interação entre S. flexneri e hepatócitos em condição de hipóxia e normóxia. Tal conhecimento poderá ser útil na construção de futuras estratégias para auxiliar no combate a esta importante bactéria invasiva, principalmente nos casos de septicemia / This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-a production, cell death rate by apoptosis and expression of the transcription factor HIF-1a. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-a in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-a was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-a by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF- 1a transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria

No description available.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

O presente estudo avaliou a capacidade de invasão de hepatócitos de rato por Shigella flexneri (S. flexneri) nas condições de normóxia e hipóxia. O estudo do microambiente de hipóxia tem grande importância, por estar presente em muitas doenças hepáticas, além de aumentar a translocação quando presente no lúmen intestinal. Bactérias invasivas como S. flexneri podem romper a barreira intestinal e chegar ao fígado através da circulação portal. O efeito da invasão bacteriana das células hepáticas é pouco conhecido. Neste trabalho buscamos pesquisar as alterações morfológicas e funcionais de hepatócitos de rato após infecção por S. flexneri na presença e na ausência de hipóxia. Para esta finalidade foram utilizados hepatócitos de rato cultivados pela técnica de cultura primária. Vários parâmetros foram analisados, tais como: taxa de invasão celular pela bactéria, quantificação da produção e liberação de DHL, produção de TNF-, taxa de morte celular por apoptose e a expressão do fator de transcrição HIF-1a. Os resultados mostraram que a metodologia empregada para a obtenção do microambiente hipóxico foi satisfatória, com redução de 70% da pO2 inicial (atingindo 43.2 mmHg in vitro ou 6.5% O2). A invasão de hepatócitos de rato por S. flexneri foi menor nas células previamente expostas à hipóxia quando comparada com a invasão das células cultivadas em normóxia. A viabilidade dos hepatócitos não apresentou diferenças significativas entre os grupos experimentais, variando entre 74 e 86%. A liberação de TNF- nas situações de normóxia e hipóxia foi similar, embora as células infectadas em normóxia tenham aumentado a liberação desta citocina. Na condição de hipóxia + infecção a liberação de TNF- foi menor do que na condição de normóxia + infecção, porém ambos os grupos produziram aumento significativo da citocina em relação aos controles normóxicos e hipóxicos. Este resultado sugere que a presença da bactéria no interior das células aumenta significativamente a liberação de TNF-pelos hepatócitos. A produção de DHL também foi maior de forma significativa no grupo hipóxico em relação ao grupo normóxico, porém não apresentou alteração nos grupos infectados por S. flexneri após uma hora. As taxas de apoptose aumentaram nos grupos hipóxia e nos grupos infectados com S. flexneri de maneira similar, variando entre 24 e 31%, quando comparados aos grupos controle em normóxia. A expressão do fator de transcrição HIF ocorreu nos grupos: hipóxia, normóxia + infecção e hipóxia + infecção, evidenciando que a infecção por S. flexneri induz a expressão deste fator. Em seu conjunto, nossos resultados buscam contribuir para o maior conhecimento da interação entre S. flexneri e hepatócitos em condição de hipóxia e normóxia. Tal conhecimento poderá ser útil na construção de futuras estratégias para auxiliar no combate a esta importante bactéria invasiva, principalmente nos casos de septicemia / This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-a production, cell death rate by apoptosis and expression of the transcription factor HIF-1a. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-a in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-a was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-a by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF- 1a transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria

Breast cancer is the most common malignancy among women in the U.S. While many successful treatments exist for primary breast cancer, very few are available for patients with metastatic disease. The purpose of this study was to understand the role of Insulin Receptor Subtrate-2 (IRS-2) in breast cancer metastasis. IRS-2 belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, many of which have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with cancer cell invasion, glycolysis and tumor metastasis. In the current work, we identified IRS-2 as a novel hypoxia-responsive gene in breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have shown that IRS-2 upregulation in response to hypoxia promotes Akt signaling and tumor cell viability and invasion. We identified a cell context-dependent role for Hypoxia Inducible Factor (HIF) in the regulation of IRS-2 expression in hypoxia, with HIF-2 playing a more dominant role than HIF-1. We also demonstrate that binding of Snail, a regulator of the EMT, to the IRS-2 promoter keeps the chromatin in an open conformation that is permissive for HIF-dependent transcription of IRS-2 in hypoxia. IRS-2 is not upregulated by hypoxia in well-differentiated epithelial-like carcinoma cells that do not express Snail, implicating IRS-2 gene expression as part of the EMT programming. In summary, we have identified an endogenous mechanism by which cancer cells can shift the balance of IRS-1 and IRS-2 to favor IRS-2 expression and function, which promotes survival, invasion, and ultimately metastasis. Understanding the mechanism of IRS-2 regulation by hypoxia may reveal new therapeutic targets for metastatic breast cancer.

No description available.

Lípides nitrados (NO2-FA) são apontados como uma nova classe de mediadores lipídicos, podendo atuar como reservatórios endógenos de óxido nítrico (&#8226NO) bem como moduladores pluripotentes de sinalização celular. Recentemente, tem sido sugerido que os doadores de &#8226NO estariam envolvidos na regulação da angiogênese. Evidências contundentes indicam ainda que o processo de neovascularização poderia contribuir para a patogênese de uma serie de condições clínicas, entre elas a aterosclerose. Contudo, apesar de diversos estudos terem explorado os efeitos biológicos dos NO2-FA, os efeitos destes compostos sobre o processo de angiogênese não haviam sido descritos. Dessa maneira, o presente trabalho investigou os efeitos dos NO2-FA (derivados da nitração do ácido linoléico e oléico) noprocesso de angiogênese. Demonstrou-se que os NO2-FA podem atuar como mediadores pró-angiogênicos. Este efeito foi caracterizado em células endoteliais humanas, assim como, em modelos ex vivo e in vivo. Nas células endoteliais, observou-se que os No2-FA não influenciaram a proliferação ou a viabilidade celular, ao passo que estimularam a migração. Demonstrou-se também que os NO2-FA podem modular o brotamento ex vivo de novos vasos, em cultura de anéis de aorta de rato, bem como o processo angiogênico in vivo observado na membrana corioalantóica de embrião de galinha. Adicionalmente, os NO2-FA induziram a expressão do fator de crescimento endotelial vascular (VEGF), que é o principal mediador do processo de angiogênese. Em relação ao mecanismo de ação, os achados sugerem que os efeitos demonstrados seriam via mecanismos dependentes de &#8226NO, uma vez que foram abolidos na presença de um seqüestrador de &#8226NO, enquanto concentrações equivalentes dos lípides precursores não demonstraram qualquer influência nas condições experimentais utilizadas neste estudo. Por fim, os efeitos pró-angiogênicos dos NO2-FA foram mediados pela estabilização da proteína do fator induzível por hipóxia -1α (HIF-1α), uma vez que estes compostos promoveram acúmulo desta proteína e falharam em demonstrar efeitos indutores em células knockdown para o gene HIF-1α. Em conjunto, estes resultados indicam que os NO2-FA podem modular a migração de células endoteliais e estimular o processo de angiogênese resultante da ativação de HIF-1a via mecanismo dependente de &#8226NO. / Nitrated lipids (NO2-FA) are described as a new class of Iipid mediators that are able to act as endogenously nitric oxide (&#8226NO) reservoirs as well as pluripotent cell signaling modulators. Furthermore, recent findings suggest that &#8226NO donors could be involved in the regulation of angiogenesis. Compelling evidence also indicate that the neovascularization process might contribute to the pathogenesis of many clinical conditions, such as atherosclerosis. However, although several studies have explored the NO2-FA biological properties, the effects of these compounds on the angiogenic process remain unknown. Hence, the present study investigated the effects of the NO2-FA (derivates from the nitration of Iinoleic and oleic acids at physiological concentrations) on angiogenesis processo It is demonstrated that the No2-FA could act as pro-angiogenic mediators. This effect was observed not only in human endothelial cells but also in ex vivo and in vivo models. Using endothelial cells, it is showed that NO2-FA failed to affect cell proliferation ar influence cellular viability, but significantly stimulated cell migration. It was also found that the NO2-FA might modulate the ex vivo sprouting of new vessels as well as the in vivo angiogenic process, while inducing the expression of the vascular endothelial growth factor, the main mediator of angiogenesis. The data are consistent with the hypothesis that the observed effects mediated by NO-dependent mechanisms, since the presence of a &#8226NO scavenger abrogated the induced effects, whereas equimolar concentrations of its precursors, showed no effect on angiogenesis under our experimental conditions. Finally, the pro-angiogenic effects of NOrFA were mediated by the stabilization of the hypoxia inducible factor-1α (HIF-1α) protein, because these compounds increased the protein amount and failed to show inductive effects in HIF-1α knockdown cells. Taken together, these findings indicated that NO2-FA might modulate the endothelial cell migration and stimulate the process of angiogenesis by the HIF-1α induction through a &#8226NO-dependent mechanism.