Lectins from the Red Marine Algal Species Bryothamnion seaforthii and Bryothamnion triquetrum as Tools to Differentiate Human Colon Carcinoma Cells

The carbohydrate-binding activity of the algal lectins from the closely related red marine algal species Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL) was used to differentiate human colon carcinoma cell variants with respect to their cell membrane glyco-receptors. These lectins interacted with the cells tested in a dose-dependent manner. Moreover, the fluorescence spectra of both lectins clearly differentiated the cells used as shown by FACS profiles. Furthermore, as observed by confocal microscopy, BTL and BSL bound to cell surface glycoproteins underwent intense internalization, which makes them possible tools in targeting strategies

Biology of vascular endothelium isolated from transgenic mice YAC67 and YAC84 -mouse models for Down syndrome

GIRK2 est situé sur le chromosome 21, dont la trisomie cause le syndrome de Down (DS). Les proportionss des sous-populations de lymphocytes T sont altérées, le nombre de lymphocytes B circulants est diminué. Notre hypothèse est un défaut de contrôle de la domiciliation/recirculation des leucocytes par les cellules endothéliales (CE). Les CE formant la paroi des vaisseaux, assurent la néovascularisation, interagissent avec les cellules circulantes, initient l adhésion donc, la réponse immune. Pour élucider l influence de GIRK2 sur la fonction des CE, un modèle cellulaire in vitro a été mis au point. Des lignées de CE furent établies à partir de: moelle osseuse, thymus, ganglions lymphatiques périphériques, plaques de Peyer et cerveau de souris transgéniques dotées de copies additionnelles du gène et de souris contrôles. La biologie de l endothélium fut abordée quant aux molécules d adhésion, et processus d adhésion et d angiogenèse. Les CE issues des souris transgéniques expriment différents niveaux de CD29, CD34, leurs propriétés d adhésion des lymphocytes ainsi que d angiogenèse sont dramatiquement affectées. Le profil d expression des gènes des CE de souris transgéniques montrent que parmi les molécules d adhésion, chimiokines et récepteurs, VEGFs et récepteurs, plus d un quart des ARNm est considérablement modifié par rapport aux contrôles. Nos résultats montrent clairement que le gène GIRK2 influence la function endothéliale des patients atteints de DS.GIRK2 is located on chromosome 21, which trisomy is the cause of Down syndrome (DS). In DS, among other features, proportions of T lymphocytes subpopulations are altered and number of circulating B cells are decreased. We hypothesized that it is due to the disturbed control of homing/recirculation of lymphocytes by endothelial cells (ECs). ECs constitute the vessel wall, achieve the neovascularisation, interact with circulating cells, initiate the adhesion process thus, immunological response. To assess the GIRK2 gene influence on the function of ECs, an in vitro cellular model was established. ECs lines were established from bone marrow, thymus, peripheral lymph nodes, Peyer s patches and brain from transgenic mice with additional copies of the gene and from normal control mice. Endothelium biology was investigated in the aspect of adhesion molecules as well as processes of adhesion and angiogenesis. ECs from transgenic mice have altered levels of CD29, CD34, their adhesive properties towards lymphoid cells are affected and their angiogenic properties are drastically different. cDNA microarray display for the gene expression pattern of ECs from transgenic mice showed that among adhesion molecules, chemokines, chemokine receptors, VEGFs and VEGFs receptors, more than one fourth of the mRNA was significantly modified compared to controls. Presented results give clear evidence that GIRK2 gene can influence the function of endothelial cells in DS patients.ORLEANS-SCD-Bib. electronique (452349901) / SudocSudocFranceF

Study on NK cells cytotoxicity toward endothelial cells

Plusieurs mécanismes peuvent réduire l angiogenèse tumorale d où les stratégies visant à bloquer les cellules endothéliales (CE). Les cellules tueuses naturelles (NK) (natural killer cells) stimulées, s arment pour l élimination des cellules dangereuses . Notre hypothèse est qu en conditions pathologiques (tumeur), les CE, acteurs de l angiogenèse tumorale seraient reconnues comme telles et candidates à l attaque par les NK. Les interactions entre les NK et les CE sont abordées à l aide de CE humaines in vitro, quant aux mécanismes moléculaires de l adhésion des NK en conditions statiques et conditions de flux. Ceci montre que les NK activées par l IL-2 reconnaissent et adhérent aux CE selon leur origine tissulaire. Ce mécanisme est indépendant des sélectines mais dépend soit des intégrines, soit des co-récepteurs similaires aux lectines de type C. La cytotoxicité des NK vis-à-vis des CE s exerce par la voie perforine-granzyme. En outre, stimulées par l IL-2, les NK induisent la translocation de Bid et libération du cytochrome C dans les CE cibles lesquelles expriment les récepteurs de mort , voie alternative d apoptose. Ce modèle in vitro est validé avec des NK du sang humain. A visée in vivo, les expériences réalisées avec des CE murines et des NK de la rate de souris indiquent que l efficacité des NK activées par l IL-2 est directement reliée à leur adhésion, laquelle dépend de l origine tissulaire des CE. Nous démontrons que l IL-12 (interleukine connue pour inhiber l angiogenèse tumorale) active les NK en synergie avec l IL-2. Les NK reconnaissant et tuant les CE in vitro suggère l hypothèse qu in vivo elles inhibent l angiogenèse tumorale.There are several mechanisms by which tumor angiogenesis may be reduced. They are addressed to tumor endothelial cell (EC) to block their activation and proliferation. Among the natural protection mechanisms, natural killer (NK) cells play an important role. Stimulated NK cells may eliminate potentially dangerous cells. Hence, we hypothesized that ECs involved in tumor angiogenesis may be recognized as dangerous cells and killed by NK cells. First, the adhesive interactions between NK cells and ECs were investigated. Using human in vitro cell lines, molecular mechanisms of NK cells adhesion to ECs under static and flow in vitro conditions were examined, showing that IL-2 activated NK cells were able to adhere to ECs. The molecular mechanism was shown not to be selectin - dependent but integrin and/or C type lectin like - dependent. Subsequently, cytotoxicity of NK cells toward ECs was analyzed. Its molecular mechanism was mainly perforin-granzyme dependent. NK cells were shown to induce in target ECs Bid translocation and cytochrome c releasing. To validate in vitro cellular model most of these experiments were repeated with freshly isolated human blood NK cells. The in vivo approach necessitated that adhesion and cytotoxicity were performed using murine ECs lines and freshly isolated NK cells from murine spleen. It was found that IL-12 synergizes with IL-2 to stimulate NK cells killing activity toward ECs. The demonstration that, under specific conditions stimulated NK cells are able to kill ECs in vitro allows hypothesizing that in vivo NK cells may participate in tumor angiogenesis inhibition.ORLEANS-SCD-Bib. electronique (452349901) / SudocSudocFranceF

CD133 positive progenitor endothelial cell lines from human cord blood.

International audienceEndothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB.1 and HEPC-CB.2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells

Increased Endothelial Progenitor Cell Number in Early Stage of Endometrial Cancer

International audienceObjectives: It is generally believed that circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) reflect the state of the endothelium, its injury and/or repair possibilities. In different types of cancers, increased numbers of CECs and EPCs were found, suggesting their participation in cancer angiogenesis. The objective of this study was to determine whether, in the blood circulation of women with early endometrial cancer, CEC and EPC levels differ from those of healthy women of similar age.Methods: For CEC number evaluation, samples of peripheral blood cells of women with endometrial carcinoma and control subjects were labeled with anti-CD31 and anti-CD45 antibodies; for EPCs, with anti-VEGFR2(vascular-endotheliumgrowth factor receptor 2)/KDR and anti-CD34 antibodies. The CEC and EPC cells were then quantified by flow cytometry.Results: Endothelial progenitor cell numbers (CD34(+), VEGFR2/KDR+) in the peripheral blood of women with endometrial carcinoma were significantly augmented as compared with those of control healthy women and CEC numbers (CD31(+), CD45(-)) were similar in both groups. Cancer patients were divided according to the grading into G1 and G2 groups and according to the stage into International Federation of Gynecology and Obstetrics (FIGO) stage IA and FIGO IB groups. Statistically significant augmented EPC numbers were demonstrated only in G1 and FIGO IA patients.$Conclusions: These results strongly suggest new vessel formation from recruited endothelial precursors as being involved mainly at the early stages of tumor progression

From Primary MSC Culture of Adipose Tissue to Immortalized Cell Line Producing Cytokines for Potential Use in Regenerative Medicine Therapy or Immunotherapy

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors

The antiangiogenic activtivity of IL-12 is increased in iNOS-/- mice and involves NK cells

International audienceWe have previously reported that the in vivo transfer of murine interleukin-12 (IL-12) gene using a Semliki Forest virus vector induced tumor regression through inhibition of tumor blood vessel formation. To examine whether IL-12 anti-angiogenic activity interferes with the NO pathway, we used inducible nitric oxide synthase-deficient mice (iNOS−/−) and demonstrated that the anti-tumor effect of IL-12 is more pronounced in these mice. In addition, despite the increased level of intratumoral VEGF in iNOS−/− mice, IL-12 induced a stronger inhibition of blood vessel formation. Histological analysis of SFV-IL-12-treated tumors showed an increase in natural killer (NK) perivascular infiltration in iNOS−/− as compared to control mice. In vitro IL-12-stimulated murine splenic NK cells displayed significant killing activity towards established murine endothelial cells used as targets. These studies indicate that the anti-angiogenic activity of IL-12 interferes with iNOS pathway and involves NK cell recruitment