Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

Tie-2 expressing monocytes (TEMs) were identified to be a subset of monocytes that was potent inducer of tumor angiogenesis in various types of cancer. On the other hand, monocytes and macrophages are crucially involved in the innate immune response, but the function of TEMs in this process is not well understood. Therefore, we studied the role of Tie-2 in regulating the inflammatory response initiated by lipopolysaccharide in murine macrophages. First, we observed that a small percentage of bone marrow-derived macrophages express Tie-2. Furthermore, we observed that Tie-2 ligands were able to induce phosphorylation of signalling proteins in macrophages. However, we found that Tie-2 ligands were not able to regulate pro-inflammatory cytokine secretion, nor were the ligands able to prevent apoptosis induced by lipopolysaccharide in macrophages. These results suggest that contrary to current evidence in the literature, Tie-2 ligands may not play a role in modulating the pro-inflammatory response in TEMs.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Background. Recent studies in cancer research have focused on the reciprocal interaction between cancer cells and their microenvironment. Tumour growth is angiogenesis dependent and the rate of angiogenesis correlates with a poor prognosis in many different cancers. We have shown that the rate of angiogenesis correlates with prognosis in Prostate Cancer (PC). We have also observed that the vasculature is involved during the involution of the prostate in rodents subsequent to hormonal ablation. Patients with metastatic PC are subjected to hormonal ablation therapy – a therapy unfortunately not curative. Our ambition is therefore to find means to enhance the effects of castration therapy of prostate tumours, possibly by a simultaneous inhibition of angiogenesis and of growth factors populating the tumour stroma. The angiopoietins are a family of growth factors that regulate angiogenesis by direct effects on endothelial cells in a context dependent manner. The purpose of this thesis was therefore to examine the role of the angiopoietins and the stroma in general in PC and to explore their potential as novel targets. Materials and Methods. We have had at our disposal access to clinical materials in the form of paraffin embedded samples from untreated PC patients with a long follow up. We have also used animal tumour models and in vitro cell culture systems followed by immunohistochemistry, in situ hybridization, western blotting, laser micro dissection, and quantitative real-time PCR for evaluation of the experiments. Results. In paper I, we found a significant correlation between high levels of angiopoietin 2 (Ang 2) and high vascular density, histological grade, metastases and poor prognosis in PC patients. In the second paper we found that the receptor for the angiopoietins, Tie 2, and the ligand Ang 1 mediated the decrease in vascular stability observed after castration treatment. This was not observed in prostate tumours subsequent to hormonal ablation (paper III), nor was there a decrease of other growth factor receptors. In summary (paper III), we found that a combined inhibition of the tumour stroma in terms of an inhibition of the PDGF-Rs by the use of Imatinib, and the vasculature in terms of a perturbed Tie 2 signalling, inhibited tumour growth. Finally, in paper IV, we found that Imatinib inhibited the castration induced influx of mast cells after castration therapy. The mast cells expressed high levels of FGF 2 and epiregulin, and inhibition of mast cell function inhibited tumour growth, by inhibiting angiogenesis. Conclusions. We have observed that the tumour stroma is of particular importance for tumour growth in PC. Targeting the tumour microenvironment, and in particular by a simultaneous inhibition of the vasculature and stroma, could prove beneficial for patients with advanced PC.

Orientador: Erich Vinicius de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T16:41:35Z (GMT). No. of bitstreams: 1 Fiusa_MaiaraMarxLuz_M.pdf: 4773403 bytes, checksum: 75096ce572a7e0682b295f48ac895796 (MD5) Previous issue date: 2013 / Resumo: A neutropenia febril (NF) em pacientes com neoplasias hematológicas é caracterizada pelo alto risco de sepse e choque séptico. Embora a utilização de escores clínicos como o MASCC permita a identificação de pacientes de baixo risco, este escore é menos informativo em pacientes de alto risco, onde se encaixam a maioria dos pacientes com neoplasias hematológicas, além daqueles submetidos a esquemas intensivos de quimioterapia. Ao mesmo tempo, a aplicação de biomarcadores de gravidade como a procalcitonina, validados em pacientes não-neutropênicos, é controversa em pacientes com NF. A quebra da barreira endotelial é um elemento chave no choque séptico, de modo que proteínas envolvidas neste processo são candidatos atrativos como biomarcadores de gravidade na sepse. Neste estudo, avaliamos prospectivamente o valor da dosagem de VEGF-A, sFlt-1, Ang-1 e Ang-2 como biomarcadores da evolução para choque séptico em 120 pacientes com NF. Pacientes internados nas enfermarias de Hematologia e Transplante de Medula Óssea do HC da UNICAMP para tratamento de NF entre março de 2011 e 2012 foram convidados a participar. As amostras foram coletadas na manhã seguinte à entrada no estudo, junto com a coleta de exames de rotina. O estudo foi desenhado com o objetivo de mimetizar as condições de coleta e processamento das amostras, que seriam encontradas na prática clínica real. Foi avaliada a evolução para choque séptico e mortalidade em 28 dias. Os resultados foram comparados com marcadores de prognóstico clássicos como proteína C reativa, e escores MASCC e SOFA. No total, 99 pacientes preencheram os critérios de inclusão, dos quais 19,8% evoluíram com choque séptico. Não foram observadas diferenças clínicas e demográficas entre os pacientes com NF não-complicada e choque séptico, exceto pelo escore SOFA, significativamente mais elevado no segundo grupo. Os níveis de VEGF-A e sFlt-1 foram semelhantes entre os dois grupos. Em contraste, os níveis séricos de Ang-2 estavam aumentados em pacientes com choque séptico, ao passo que os níveis séricos de Ang-1 estavam diminuídos. Considerando o papel antagônico destas angiopoietinas na regulação da permeabilidade endotelial, a relação Ang-2/Ang-1 foi calculada, como indicador de desequilíbrios na concentração destes moduladores, que pudessem indicar um estado mais ou menos propenso à queda da barreira endotelial. De fato, um aumento na relação Ang-2/Ang-1 foi encontrada em pacientes com choque séptico (5,29 - variação de 0,58 a 57,14) em relação aos pacientes com NF não-complicada (1,99 - variação de 0,06 a 64,62; P=0,01). Na análise univariada e multivariada, a relação Ang-2/Ang-1 provou ser um fator de risco independente para o desenvolvimento de choque séptico e mortalidade em 28 dias. Desta forma, concluímos que a elevação dos níveis séricos de Ang-2 e da relação Ang-2/Ang-1 está associada ao risco aumentado de choque séptico e mortalidade em pacientes com NF, mesmo quando coletada sob condições próximas àquelas encontradas a prática clínica / Abstract: Febrile neutropenia (FN) in patients with hematologic malignancies is characterized by a high risk of sepsis complications and septic shock. Although the use of clinical scores such as the MASCC allows the identification of low-risk patients, this score is much less informative in high-risk patients, a category in which most patients with hematologic malignancies, and those undergoing intensive chemotherapy regimens, fit in. At the same time, the use of classical biomarkers such as procalcitonin in non-neutropenic patients is controversial in patients with FN. Endothelial barrier breakdown is a key element in septic shock, so that proteins involved in this process are attractive candidates as biomarkers of sepsis severity. In this study, we prospectively evaluated the value of VEGF-A, sFlt-1, Ang-1 and Ang-2 serum levels as biomarkers of progression to septic shock in 120 patients with FN. Patients hospitalized in the Hematology and Bone Marrow Transplantation in-patient units of a university hospital (HC-UNICAMP) for the treatment of FN between March 2011 and March 2012 were invited to participate. Samples were collected in the following morning after study entry, along with the collection of routine labs. The study was designed to mimic the conditions of blood sample collection and processing that would be encountered in "real-world" clinical practice. Clinical outcomes were (1) progression to septic shock and (2) death within 28 days from fever onset. Results were compared with classical prognostic markers such as C-reactive protein, and MASCC and SOFA scores. In total, 99 patients met the inclusion criteria, of which 19.8% progressed to septic shock. No differences clinical and demographic differences were observed between patients with uncomplicated-FN or septic shock, except for a higher SOFA scores in the latter group. Levels of VEGF-A and sFlt-1 were similar between the two groups. In contrast, serum levels of Ang-2 were increased in patients with septic shock, whereas serum levels of Ang-1 were decreased in these patients. Considering the antagonistic role of angiopoietins 1 and 2 in regulating endothelial permeability, the Ang-2/Ang-1 ratio was calculated as an indicator of imbalances in the concentration of these modulators. In fact, a high Ang-2/Ang-1 ratio was found in patients with septic shock (5.29 - range 0.58 to 57.14) compared to patients with uncomplicated FN (1.99 - range 0.06 to 64.62, P = 0.01). In univariate and multivariate analysis, the Ang-2/Ang-1 ratio proved to be an independent risk factor for the development of septic shock and for 28-day sepsis-related mortality. Thus, we concluded that a high Ang-2/Ang-1 ratio is associated with increased risk of septic shock and mortality in patients with FN, even when collected under conditions close to those encountered in real-world clinical practice / Mestrado / Clinica Medica / Mestra em Ciências

La dérégulation de la formation et l'intégrité des vaisseaux sanguins peut conduire à un état pathologique tel qu’observé dans de nombreuses maladies ischémiques telles que: la croissance de tumeur solide, l’arthrite rhumatoïde, le psoriasis, les rétinopathies et l'athérosclérose. Par conséquent, la possibilité de moduler l'angiogenèse régionale chez les patients souffrant d'ischémie est cliniquement pertinente. Un élément clé dans l'induction de l'angiogenèse pathologique est une inflammation qui précède et accompagne la formation des nouveaux vaisseaux. Ce phénomène est démontré par l'augmentation de la perméabilité vasculaire et le recrutement de monocytes/ macrophages et cellules polynucléaires (neutrophiles). En collaboration avec d'autres groupes, nous avons montré que différents facteurs de croissance tels que le facteur de croissance endothélial vasculaire et les angiopoïétines peuvent non seulement promouvoir l'angiogenèse mais aussi induire diverses étapes connexes au processus de la réaction inflammatoire, y compris la synthèse et la libération des médiateurs inflammatoires et la migration des neutrophiles. Les objectifs de notre étude étaient d'adresser si le vascular endothelial growth factor (VEGF) et les angiopoïétines (Ang1 et Ang2) sont capables de promouvoir la formation des nouveaux vaisseaux sanguins au fil du temps et d'identifier la présence de différentes cellules inflammatoires dans ce processus. Des éponges d'alcool polyvinylique stérilisées et imbibées de Matrigel appauvri en facteur de croissance (contenant PBS, VEGF, Ang1 ou Ang2 (200 ng/200 μl)) ont été insérées sous la peau de souris C57/Bl6 anesthésiées. Les éponges ont ensuite été retirées aux jours 4, 7, 14 ou 21 après la procédure pour des analyses histologiques, immunohistologiques et cytométriques. La formation des nouveaux vaisseaux a été validée par la coloration au Trichrome de Masson et des analyses histologiques et immunohistologiques contre les cellules endothéliales (anti-CD31). De plus, la maturation des vaisseaux a été démontrée par la coloration séquentielle contre les cellules endothéliales (anti-CD31) et musculaires lisses (anti-alpha-actine). Nous avons effectué la même procédure pour caractériser le recrutement de neutrophiles (anti-MPO), et de macrophages (anti-F4/80). Afin de mieux délimiter la présence de différents sous-ensembles de leucocytes recrutés dans les éponges, nous avons utilisé une technique de cytométrie en flux sur des préparations de cellules isolées à partir de ces éponges. Nous avons observé que le VEGF et les angiopoïétines favorisent le recrutement de cellules endothéliales et la formation de nouveaux vaisseaux plus rapidement qu’en présence de PBS. Une fois formé au jour 7, ces nouveaux vaisseaux restent stables en nombre, et ne subissent pas une réorganisation importante de leur surface. Ces vaisseaux maturent grâce au recrutement et au recouvrement par les cellules musculaires lisses des néovaisseaux. En outre, le microenvironnement angiogénique est composé de cellules inflammatoires, principalement de neutrophiles, macrophages et quelques cellules de type B et T. Donc, le VEGF, l’Ang1 et l’Ang2 induisent séparément la formation et la stabilisation de nouveaux vaisseaux sanguins, ainsi que le recrutement de cellules inflammatoires avec des puissances différentes et une action temps-dépendante dans un modèle d’éponge/Matrigel. / A deregulation in blood vessel formation and integrity can lead to a pathological state as seen in many ischemic diseases such as tumor growth, rheumatoid arthritis, psoriasis, retinopathies and atherosclerosis. Therefore, the possibility to modulate regional angiogenesis in patients suffering from ischemia is clinically relevant. One key feature in the induction of pathological angiogenesis is that inflammation precedes and accompanies the formation of neovessels as evidenced by increased vascular permeability and the recruitment of monocytes/macrophages and neutrophils. Along with other groups, we have previously shown that selected growth factors, namely vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) can not only promote angiogenesis but can also induce inflammatory responses, including the synthesis/release of inflammatory mediators and neutrophil migration. The objectives of our study were to address how VEGF and angiopoietins are capable of promoting the formation of neovessels over time and to identify the presence of different inflammatory cells in this event. Sterilized polyvinyl alcohol (PVA) sponges soaked in growth factor-depleted Matrigel containing PBS, VEGF, Ang1 or Ang2 (200 ng/200 μl) were subcutaneously inserted into anesthetized C57/Bl6 mice. The sponges were then removed at day 4, 7, 14 or 21 post-procedure for histological, immunohistological (IHC) and flow cytometric analyses. The formation of neovessels was validated by Masson’s Trichrome staining and by IHC against endothelial cells (anti-CD31) and its maturation was elucidated by sequential IHC staining against endothelial cells and smooth muscle cells (anti-alpha-actin). Likewise, we performed IHC to characterize the recruitment of neutrophils (anti-MPO), and macrophages (anti-F4/80). To better delineate the presence of different leukocyte subsets recruited in the sponges, we utilized multicolor flow cytometry procedure on single cell preparation from the sponges. We observed that both VEGF and angiopoietins favors the recruitment of endothelial cells and the formation of new vessels more rapidly as compared to PBS. Once formed by day 7, these neovessels remain stable in number, do not undergo reorganization in their cross sectional area and mature through the recruitment and ensheathing of smooth muscle cells. In addition, the angiogenic microenvironment is comprised of inflammatory cells, mainly neutrophils, macrophages, and sparsly T and B cells. Hence, VEGF, Ang1 and Ang2 individually promote the formation and stabilisation of neovessels and the venue of inflammatory cells with different potency in a temporal dependant manner in a sponge/Matrigel model.

A pré-eclâmpsia afeta 3 a 5% das gestantes em todo o mundo, contribuindo para complicações materno-fetais graves. Sendo a isquemia placentária considerada um dos fatores primordiais para o desenvolvimento da doença. Essa isquemia está associada à alterações de fatores pró e anti angiogênicos, o presente estudo avaliou os fatores pró angiogênicos angiopoetina 1 e 2 (Ang-1 e Ang-2), que atuam na formação e no crescimento de novos vasos durante a placentação. O objetivo do estudo foi avaliar as concentrações plasmática de Ang-1 e Ang-2 na predição de pré-eclâmpsia e verificar a sensibilidade e especificidade dos mesmos por meio da curva ROC. Foram avaliadas 120 gestantes com idade gestacional entre 20 e 25 semanas, que participaram do projeto Coortes BRISA, que contava com um banco de 1400 gestantes, sendo que 30 gestantes com diagnóstico de pré-eclâmpsia (PE) e que realizaram o parto no Hospital das Clínicas de Ribeirão Preto e 90 gestantes saudáveis (GS) que realizaram parto na MATER ( Maternidade do Centro de Referência da saúde da Mulher). As concentrações plasmáticas de Ang-1 e de Ang- 2 foram determinadas utilizando o método ELISA. Para a análise dos dados, foi realizado ANOVA quando comparamos os grupos quanto às variáveis quantitativas. Para as comparações dos níveis pressóricos das gestantes com pré-eclâmpsia grave x pré-eclâmpsia não grave, no momento do recrutamento e com a doença estabelecida, foi utilizado o modelo de regressão linear com efeitos mistos (efeitos aleatórios e fixos). Para as comparações dos dados foi utilizado o pós-teste por contrastes ortogonais. Na comparação das concentrações de Ang-1 entre GS e PE não houve diferença estatística entre os grupos (P= 0,185) o mesmo foi observado para Ang-2 (P= 0,583). Em relação à razão Ang-1/Ang-2, também não observamos diferença estatística (P= 0,107). A capacidade preditiva dos biomarcadores foi avaliada através da curva ROC e a área sobre a curva para Ang-1, Ang-2 e a razão Ang-1/Ang-2 foram 0,47, 0,52 e 0,57 respectivamente. Nosso estudo não encontrou diferença significativa nas concentrações de Ang-1 E Ang-2 e nem na razão entre Ang-1/Ang-2. Ao realizar a curva ROC observamos, que esses biomarcadores não são bons preditores para pré-eclâmpsia. / Preeclampsia affects 3 to 5% of pregnant women worldwide, contributing to severe maternal-fetal complications. As placental ischemia, a set of primordial factors for the development of the disease was proposed. This ischemia is associated with changes in pro and anti-angiogenic functions, the present study is angiography and angiopoietin 1 and 2 (Ang-1 and Ang-2), which act in the formation and growth of new vessels during placentation. The evaluation of the evaluation of Angio-1 and Ang-2 in the prediction of pre-eclampsia and to verify their sensitivity and specificity by means of the ROC curve. There were 20 pregnant women with gestational age between 20 and 25 weeks, who participated in the BRISA Cohorts project, which had a bank of 1400 pregnant women, 30 pregnant women diagnosed with preeclampsia (PE) and who gave birth at Hospital das Clínicas of Ribeirão Preto and 90 healthy pregnant women (GS) who performed part of MATER (Maternity of the Reference Center for Women\'s Health). Plasma Ang-1 and Ang-2 tests were already using the ELISA method. For an analysis of the data, we performed ANOVA when comparing the groups as the quantitative variables. For the comparisons of pressure levels of pregnant women with severe preeclampsia vs. non-severe preeclampsia at the moment of recruitment and with the disease installed, the linear regression model with mixed effects (random and fixed lexus) was used. For the data comparisons, the orthogonal contrasts post-test was used. When comparing the Ang-1 combinations between GS and PE, it was not possible to compare the groups (P = 0.185). The same was observed for Ang-2 (P = 0.583). In relation to the Ang-1 / Ang-2 ratio, we also did not observe the statistical difference (P = 0.107). The ability to distribute the biomarkers was evaluated through the ROC curve and the area over the curve for Ang-1, Ang-2 and the Ang-1 / Ang-2 ratio were 0.47, 0.52 and 0.57 respectively. Our needs were not as significant at Ang-1 and Ang-2 concentrations nor at the Ang-1 / Ang-2 ratio. When performing a ROC curve we observed that these biomarkers are not good predictors of preeclampsia.

A pré-eclâmpsia afeta 3 a 5% das gestantes em todo o mundo, contribuindo para complicações materno-fetais graves. Sendo a isquemia placentária considerada um dos fatores primordiais para o desenvolvimento da doença. Essa isquemia está associada à alterações de fatores pró e anti angiogênicos, o presente estudo avaliou os fatores pró angiogênicos angiopoetina 1 e 2 (Ang-1 e Ang-2), que atuam na formação e no crescimento de novos vasos durante a placentação. O objetivo do estudo foi avaliar as concentrações plasmática de Ang-1 e Ang-2 na predição de pré-eclâmpsia e verificar a sensibilidade e especificidade dos mesmos por meio da curva ROC. Foram avaliadas 120 gestantes com idade gestacional entre 20 e 25 semanas, que participaram do projeto Coortes BRISA, que contava com um banco de 1400 gestantes, sendo que 30 gestantes com diagnóstico de pré-eclâmpsia (PE) e que realizaram o parto no Hospital das Clínicas de Ribeirão Preto e 90 gestantes saudáveis (GS) que realizaram parto na MATER ( Maternidade do Centro de Referência da saúde da Mulher). As concentrações plasmáticas de Ang-1 e de Ang- 2 foram determinadas utilizando o método ELISA. Para a análise dos dados, foi realizado ANOVA quando comparamos os grupos quanto às variáveis quantitativas. Para as comparações dos níveis pressóricos das gestantes com pré-eclâmpsia grave x pré-eclâmpsia não grave, no momento do recrutamento e com a doença estabelecida, foi utilizado o modelo de regressão linear com efeitos mistos (efeitos aleatórios e fixos). Para as comparações dos dados foi utilizado o pós-teste por contrastes ortogonais. Na comparação das concentrações de Ang-1 entre GS e PE não houve diferença estatística entre os grupos (P= 0,185) o mesmo foi observado para Ang-2 (P= 0,583). Em relação à razão Ang-1/Ang-2, também não observamos diferença estatística (P= 0,107). A capacidade preditiva dos biomarcadores foi avaliada através da curva ROC e a área sobre a curva para Ang-1, Ang-2 e a razão Ang-1/Ang-2 foram 0,47, 0,52 e 0,57 respectivamente. Nosso estudo não encontrou diferença significativa nas concentrações de Ang-1 E Ang-2 e nem na razão entre Ang-1/Ang-2. Ao realizar a curva ROC observamos, que esses biomarcadores não são bons preditores para pré-eclâmpsia. / Preeclampsia affects 3 to 5% of pregnant women worldwide, contributing to severe maternal-fetal complications. As placental ischemia, a set of primordial factors for the development of the disease was proposed. This ischemia is associated with changes in pro and anti-angiogenic functions, the present study is angiography and angiopoietin 1 and 2 (Ang-1 and Ang-2), which act in the formation and growth of new vessels during placentation. The evaluation of the evaluation of Angio-1 and Ang-2 in the prediction of pre-eclampsia and to verify their sensitivity and specificity by means of the ROC curve. There were 20 pregnant women with gestational age between 20 and 25 weeks, who participated in the BRISA Cohorts project, which had a bank of 1400 pregnant women, 30 pregnant women diagnosed with preeclampsia (PE) and who gave birth at Hospital das Clínicas of Ribeirão Preto and 90 healthy pregnant women (GS) who performed part of MATER (Maternity of the Reference Center for Women\'s Health). Plasma Ang-1 and Ang-2 tests were already using the ELISA method. For an analysis of the data, we performed ANOVA when comparing the groups as the quantitative variables. For the comparisons of pressure levels of pregnant women with severe preeclampsia vs. non-severe preeclampsia at the moment of recruitment and with the disease installed, the linear regression model with mixed effects (random and fixed lexus) was used. For the data comparisons, the orthogonal contrasts post-test was used. When comparing the Ang-1 combinations between GS and PE, it was not possible to compare the groups (P = 0.185). The same was observed for Ang-2 (P = 0.583). In relation to the Ang-1 / Ang-2 ratio, we also did not observe the statistical difference (P = 0.107). The ability to distribute the biomarkers was evaluated through the ROC curve and the area over the curve for Ang-1, Ang-2 and the Ang-1 / Ang-2 ratio were 0.47, 0.52 and 0.57 respectively. Our needs were not as significant at Ang-1 and Ang-2 concentrations nor at the Ang-1 / Ang-2 ratio. When performing a ROC curve we observed that these biomarkers are not good predictors of preeclampsia.

Nous avons identifié l’expression du récepteur des angiopoïétines, le récepteur Tie2, à la surface des neutrophiles humains. De plus, nous avons démontré qu’Ang1 et Ang2 induisent des activités pro-inflammatoires sur les neutrophiles, comme l’adhésion aux cellules endothéliales (CEs) et la synthèse du facteur d’activation plaquettaire (PAF). Puisque le PAF augmente la viabilité des neutrophiles et que les angiopoïétines modulent la survie des CEs, nous avons voulu évaluer l’effet des angiopoïétines sur la survie des neutrophiles. Des neutrophiles humains ont été isolés à partir du sang de donneurs sains en accord avec le comité d’éthique de l’Institut de cardiologie de Montréal. La viabilité des neutrophiles a été mesurée par cytométrie en flux à l’aide de marqueurs d’apoptose et de nécrose. Un traitement avec des témoins positifs, soit l’interleukine 8 (IL-8; 25 nM) ou le PAF (100 nM), a augmenté la survie basale des neutrophiles de 34 et 27%, respectivement. De plus, un traitement avec Ang1 (1 pM – 10 nM) a augmenté la survie des neutrophiles jusqu’à 35%, alors qu’Ang2 n’a eu aucun effet. La combinaison de l’IL-8 ou du PAF avec Ang1 (10 nM) a eu un effet additif sur la viabilité des neutrophiles et a augmenté la survie de 56 et 60%, respectivement. Un prétraitement avec des anticorps bloquants contre l’IL-8 a permis d’inhiber l’activité anti-apoptotique de l’IL-8 et d’Ang1 de 92 et 80%, respectivement. Ainsi, notre étude est la première à démontrer la capacité d’Ang1 à prolonger la viabilité des neutrophiles, qui est principalement causée par la relâche d’IL-8. / We reported the expression of angiopoietin receptor Tie2 on the surface of human neutrophils. In addition, we reported that Ang1 and Ang2 are both capable to promote pro-inflammatory activities in neutrophils, namely their adhesion onto endothelial cells (ECs) and platelet-activating factor (PAF) synthesis. PAF is known to promote pro-survival activity on neutrophils and since both angiopoietins can modulate ECs viability, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Human neutrophils were isolated from blood of healthy volunteers in accordance with the guidelines of the Montreal Heart Institute’s ethical committee. Neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers. Treatment with anti-apoptotic mediators such as interleukin 8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 27%, respectively. In addition, treatment with Ang1 (1 pM – 10 nM) increased neutrophil viability by up to 35%, while Ang2 had no effect. Combination of IL-8 or PAF with Ang1 (10 nM) provided an additive effect on neutrophil viability and further increased viability by 56 and 60%, respectively. Pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 80%, respectively. In summary, our data are the first one to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

L’angiogenèse, caractérisée par la formation de nouveaux vaisseaux sanguins à partir de vaisseaux préexistants, est un processus accompagné par l’inflammation, impliquant la synthèse et la relâche de différents facteurs de croissance par les cellules inflammatoires. Parmi ces facteurs, seuls le vascular endothelial growth factor (VEGF) et les angiopoïétines (Ang1 et Ang2) peuvent participer à la régulation de l’inflammation et de l’angiogenèse. La famille des angiopoïétines comporte quatre membres, desquels l’Ang1 et l’Ang2 ont été les plus étudiés. Ces deux médiateurs inflammatoires sont capables d’activer le récepteur Tie2, dont l’expression a initialement été rapportée sur les cellules endothéliales (CE). Notre laboratoire a été le premier à démontrer l’expression de Tie2 à la surface des neutrophiles, ainsi que sa capacité, suite à son activation par l’Ang1 ou l’Ang2, à induire la synthèse du facteur d’activation plaquettaire (PAF), l’activation de la β2-intégrine, la migration des neutrophiles ainsi que leur adhésion aux CE. D’autres études ont montré que les CE emmagasinent et relâchent le VEGF et l’Ang2, tandis que les péricytes et les cellules musculaires lisses contiennent l’Ang1. Puisque les neutrophiles relâchent le VEGF et que les deux angiopoïétines ont la capacité d’activer Tie2 sur ces derniers, nous avons voulu déterminer si les neutrophiles contiennent l’Ang1 et/ou l’Ang2 et si elles peuvent être relâchées suite à une stimulation avec des agonistes proinflammatoires. Nous avons découvert que l’Ang1, mais pas l’Ang2 est présente dans les neutrophiles, et qu’elle est relâchée suite à une stimulation au phorbol myristate acetate (PMA). De plus, nous avons démontré que l’Ang1 est localisée au niveau du cytosol et que sa relâche est calcium-indépendante, contrairement au VEGF, qui est localisé dans les granules β et sa relâche est calcium-dépendante. Cette étude démontre pour la première fois l’expression et la localisation de l’Ang1 dans les neutrophiles. Une récente étude effectuée dans notre laboratoire a démontré que les angiopoïétines induisent la migration des neutrophiles en activant le récepteur Tie2 et la voie de la PI3K. De plus, les angiopoïétines ont potentialisé la migration induite par l’IL-8. Ainsi, nous avons émis l’hypothèse que l’Ang1 et/ou l’Ang2 seraient capables d’induire la relâche et/ou la synthèse de l’IL-8 par les neutrophiles. Nous avons démontré pour la première fois, la capacité de l’Ang1 à induire l’expression de l’ARNm, ainsi que la synthèse et la relâche d’IL-8 par les neutrophiles. Cependant, un traitement avec l’Ang2 seule ou en combinaison avec l’Ang1 n’a eu aucun effet sur les activités mentionnées ci-dessus. Nous avons aussi observé que la synthèse et la relâche d’IL-8 induite par l’Ang1 requièrent la transcription de l’ADN en ARNm, suivie par la stabilisation de ce dernier, qui ultimement induit la traduction de l’ARNm de l’IL-8 en sa protéine. Finalement, nous avons démontré que la stimulation des neutrophiles avec l’Ang1 induit ces activités en activant la voie de la p42/44 MAPK, tout en étant indépendantes de la p38 MAPK et la PI3K/Akt. Ces résultats sont en lien direct avec une récente étude dans laquelle nous avons observé que l’Ang1, mais pas l’Ang2 est capable d’augmenter la survie des neutrophiles via la relâche d’IL-8. / Angiogenesis is known as the formation of new blood vessels from pre-existent ones. This process is accompanied by inflammation, which involves the synthesis and release of numerous growth factors by inflammatory cells. Among the growth factors involved in these activities, only the vascular endothelial growth factor (VEGF) and the angiopoietins (Ang1 and Ang2) can modulate both the inflammatory and the angiogenic processes. The angiopoietins family has four fully characterized members, from which Ang1 and Ang2 have been the most extensively studied. Ang1 and Ang2 are both capable to activate the receptor Tie2, initially discovered on the endothelial cell (EC) surface. We were the first group to report the expression of Tie2 on neutrophils along with its activation by Ang1 and Ang2 which can enhance platelet-activating factor (PAF) synthesis, β2-integrin activation, neutrophil migration and adhesion onto EC. Other studies have shown that EC have endogenous stores of VEGF and Ang2, whereas pericytes and smooth muscle cells contain intracellular pools of Ang1. Since neutrophils can release VEGF and that both angiopoietins can activate Tie2 receptor, we wanted to assess if neutrophils contain Ang1 and/or Ang2, and if so, investigate their capacity to be released under inflammatory stimuli. We observed that Ang1, but not Ang2, is found in the neutrophils and that it can be only released upon phorbol myristate acetate (PMA) stimulation. Moreover, using the nitrogen sub-cellular fractionation technique, we demonstrated that Ang1 is found in the cytosolic fraction and its release is calcium-independent, while VEGF is found in β-granules and its release is calcium-dependent. This study demonstrates for the first time the expression and release of Ang1 from the neutrophils and its localization in the cytosol. In one of our recent studies, we have shown that angiopoietins are capable to induce neutrophil migration through Tie2 activation and via the PI3K/Akt signalling pathway. Moreover, both angiopoietins were shown to potentiate IL-8-induced neutrophil migration. Thus, we sought to investigate the capacity of Ang1 and/or Ang2 to induce IL-8 synthesis and/or release from human neutrophils. We demonstrated for the first time, the capacity of Ang1 to induce IL-8 mRNA expression, along with its protein synthesis and release from the neutrophils. However, a treatment with Ang2, alone or in combination with Ang1, had no effect on these aforementioned activities. We also observed that Ang1-induced IL-8 protein synthesis and release requires the transcriptional mechanism from IL-8 DNA to mRNA, followed by the mRNA stabilization, which ultimately enhances its translation into IL-8 protein. Finally, we also observed that neutrophil stimulation with Ang1 enhances IL-8 mRNA expression, protein synthesis and release by activating the p42/44 MAPK signalling pathway, while being independent from p38 MAPK and PI3K/Akt. These results are in line with one of our recent studies, in which we observed that Ang1, but not Ang2, is capable to enhance neutrophil survival, by diminishing their apoptosis through the release of IL-8 by the neutrophils.