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.

Antiangiogenic tomor therapies have attracted intense interest for their broad-spectrum action, low toxicity, and in the case of direct endothelial targeting, an absence of drug resistance. Among the growing list of antiangiogenic agents, endostatin has attracted most attention and been under the spotlight of numerous debates. Like other angiogenesis inhibitors, endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on cancer cells. Above all, therapy of experimental cancer with endostatin in rodents leads to tumor dormancy and does not induce resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E.coli expression vector to express and generate large quantities of recombinant GST-fused endostatin (GST-endostatin). Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting various endothelial cell lines including HUVEC, EA.hy926 and BAEC with IC50 ~ 20 nM. Flow cytometry analysis indicated GST-endostatin induced apoptosis in EA.hy926 cells. GST-endostatin also inhibited the cell migration of EA.hy926 cells toward chemoattractant bFGF with IC50 ~ 0.5 nM. Further more, GST-endostatin inhibited in vivo angiogenesis in chicken chorioallantoic membrane and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. After functional characterization of GST-endostatin, we decided to use GST-endostatin and EA.hy926 cells as a model system to study the inhibitory mechanism of endostatin in endothelial cells. By using fura-2 fluorescence probe, GST-endostatin was shown to elevate the cytosolic calcium in dose-dependent manner from extracellular source. Chelation of extracellular Ca2+ by EGTA or inhibition of calcium channel by nifedipine abolished the cytotoxic effect endostatin, suggesting the calcium rise by endostatin play an important role in its inhibitory mechanism. Besides, endostatin also stimulated activity of a large- conductance calcium-activated potassium (Bkca) channel, further supporting endostatin initiated serial changes in ion channels activities in endothelial cells. Respiratory enzyme activities and endogenous ATP synthesis in endothelial cells were significantly inhibited by GST-endostatin treatment, indicating GST-endostatin depleted the energy source for endothelial cells. In summary, present study demonstrated GST-endostatin caused dramatic changes in electrophysiologic properties and decreased endogenous ATP synthesis in endothelial cells, which may participate in its inhibitory mechanism.

L’athérosclérose est caractérisée par la formation d’une plaque d’athérome au niveau de la paroi d’un vaisseau sanguin. L’infiltration monocytaire participe à la phase précoce du développement de la plaque d’athérome. Le risque de rupture de la plaque est accru suite à sa néo-vascularisation. La chimiokine Regulated upon Activation, Normal T-cell, Expressed and Secreted (RANTES/CCL5) est impliquée dans le recrutement monocytaire. De plus, elle exerce un effet pro-angiogénique de manière dépendante de sa fixation aux glycosaminoglycanes (GAG) endothéliaux. Les syndécanes (SDC) sont des protéoglycanes transmembranaires à chaînes GAG exprimés par les cellules endothéliales. Le but de cette étude est de déterminer l’implication du domaine intracellulaire du SDC-4 dans l’angiogenèse et le recrutement monocytaire induits par la chimiokine RANTES/CCL5. Des mutants intracellulaires du SDC-4 ont été développés pour étudier le rôle de la PKC-α, de la liaison du PIP2 ou de protéines à domaine PDZ dans l’angiogenèse et le recrutement monocytaire induits par RANTES/CCL5. Nos résultats montrent que la chimiokine RANTES/CCL5 stimule l’angiogenèse et le recrutement monocytaire par le biais d’une signalisation intracellulaire induite par le domaine intracellulaire du SDC-4, activant notamment la PKC-α. Le développement de thérapies ciblant l’activation du SDC-4 pourrait permettre de réguler les effets physiopathologiques exercés par RANTES/CCL5. / Atherosclerosis is a pathology characterized by the formation of an atheroma plaque in the blood vessel wall. Monocyte infiltration participates to the early phase of atheroma plaque development. Plaque rupture is induced by its neovascularization. The chemokine Regulate upon Activation, Normal T-cell, Expressed and Secreted (RANTES/CCL5) is involved in monocyte recruitment. Moreover, RANTES/CCL5 induces angiogenesis through its binding to endothelial glycosaminoglycans (GAG) chains. Syndecans (SDC) are transmembrane proteoglycans with GAG chains expressed at the membrane of endothelial cells. The aim of this study is to analyze the involvement of SDC-4 intracellular domain in RANTES/CCL5-induced angiogenesis and monocyte recruitment. SDC-4 intracellular mutants were developed to analyze the role of PKC-α, PIP2 binding or PDZ domain proteins in RANTES/CCL5-induced angiogenesis and monocyte recruitment. Our data show that RANTES/CCL5 induces angiogenesis and monocyte recruitment through intracellular signaling, including the activation of PKC-α, mediated by SDC-4 intracellular domain. The development of new therapeutic strategy targeting SDC-4 activation may counteract RANTES/CCL5 physiopathological effects.

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