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Etude du remodelage tumoral associé aux effets du peptide vasoactif urotensine II sur la néoangiogenèse et la croissance des glioblastomes

Abstract : The mechanisms leading to new blood vessel formation is a prerequisite for adequate oxygenation and nutrient distribution in whole organism tissues. Starting during embryonic development, blood vessel formation results of the association of tubulogenic structures composed of endothelial cells, surrounded by smooth muscle cells and fibroblasts that stabilize the final network. The whole process is orchestrated by molecular factors, coordinating cell migration and orientation until adhesion at their final localization. These identified pro- and anti-angiogenic factors belong to growth factor and peptide families, and altogether tightly regulate the equilibrium of the angiogenic balance. In adults, such phenomenon occurs in physiological functions, such as menstrual cycles or vascular remodeling, and also in diseases like diabetes, age-related macular degeneration or cancer. Among cancers, particular subtypes are characterized by neo-angiogenic capacities, correlated with chemo resistance and aggressiveness. Deriving the normal preexisting vascular network for its own purpose, glial-derived brain tumors are highly proliferative and malignant neoplasms, incurable at this date. In particular, glioblastoma multiform (GBM) is a World Health Organization classified grade IV brain tumor, composed of pernicious infiltrative gliomal cells generating massive but diffuse tumor mass that literally crush normal brain vasculature, leading to necrotic foci and edema formation. Tumor cells are known to release pro-angiogenic molecules such as stromal-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF), in concentrations so high that the newly formed blood vessels appear aberrant, leaky and hemorrhagic. Consistent with the cerebral tissue sensibility for aneurisms and edema, this last point is a source of concern in the clinical management of this disease. The urotensinergic system is composed of two neuropeptides, urotensin II UII) and its paralog urotensin II-related peptide (URP) activating a class 1 GPCR called UT. Classically, this vasoactive system is involved in the cardiovascular parameters and tonic activity of blood vessels modulations. It has been demonstrated an up-regulation of UII and UT in multiple peripheral and central neoplasms. Recent work conducted in the research group “Astrocyte and Vascular Niche”, coordinated with Dr Hélène Castel, demonstrated that UII neuropeptide is a potent chemokine involved in glioma development, presenting a dual profile i) induction of directional migration of gliomal cells in a gradient concentration and ii) promotion of cell adhesion in homogenous concentration. These characteristics, supplemented with numerous reports indicating a central role of UII in vascular remodeling, strongly suggest that urotensinergic system is implied in GBM tumorogenesis. The present PhD thesis work aims to characterize the ability of UT agonist and antagonist to modulate glioma development. Experiments conducted on human endothelial cells from central and peripheral origins as well as on glioma cells showed that UII stimulated directional migration in vitro. Tridimensional cultures of endothelial cells reproducing extracellular matrix interaction allowed the recreation of capillaries-like tubes. We identified that UII is the only urotensinergic ligand that can orchestrate tubular structures formation. Subcutaneous implantation of plugs enriched with UT ligands in C57/Bl6 mice, showed that human and murine UII are recruiting peptides for peripheral macrophages as well as smooth muscle and endothelial cells. Human glioblastoma cell line U87-MG, naturally expressing UT, were grafted in immunodepressed Swiss Nude mice. The subcutaneous formed neoplasms overexpressed UT and UII in the vascular and gliomal components. We then dissected the impact of a series of agonists (hUII, URP, UII4-11) and antagonists (palosuran, urantide) to modulate xenografts growth and animal survival. Our results first demonstrated that daily intra-tumoral injections of hUII can greatly accelerate tumorigenesis, correlated with weak survival. Blockage of UT with non peptidic antagonist palosuran, and in a more extent with peptidic antagonist/biased ligand urantide were both able to slow down GBM tumorigenesis. Histological features of such treated tumors were then analyzed. We showed that the natural ligand hUII enhanced mitotic index, supported by an intense densification of the tumoral vascular network, whereas UT antagonists, and urantide in particular, diminished tumor proliferation and intra-tumoral blood vessel formation. Paradoxically, the angiogenic balance described as equivalent concentrations of pro- and anti-angiogenic molecules in the neighborhood of growing blood vessels, was greatly perturbed by high doses of proangiogenic UII, and generated poorly functional and hemorrhagic capillaries, forming hypoxic necrotic areas within xenografts. Administered during long time periods, urantide also induced such necrosis, probably provoked by the insufficient tumor perfusion due to an inadequate angiogenesis. The development of theranostic tools to follow GBM growth in patients has been initiated in our research group, in collaboration with the LITIS laboratory (EA 4108, QuanTiF team, Pr Pierre Véra and Dr Pierre Bohn, Rouen, France). MicroSPECT imaging of αVβ3 integrins with a specific 99mTc-RGD probe have been validated in U87-MG xenografted Nude mice, treated either with hUII or urantide. We showed that in early tumoral growth, integrins were confined in newly forming blood vessels and can be a confident marker of GBM angiogenesis. After a prolonged development period, αVβ3 is also overexpressed in non necrotic gliomal cells, despite a lower global 99mTc-RGD capitation due to massive necrosis and weak probe diffusion by poorly functional blood vessels. Indeed, immunohistochemical staining of integrins in these tumors brought to the conclusion that UII and urantide can induce a massive switch of integrin expression in the entire tumor. While UII and urantide present divergent activities, integrin quantification and measurement of necrosis both suggest that urantide may act as a biased ligand. Altogether, our data participate to better understanding of GBM development biology, and identification of new candidates for therapeutic options. Use of biased synthetic ligands for multiple vasoactive systems could lead to better clinical management of this lethal disease.
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Vadim Le Joncour. Etude du remodelage tumoral associé aux effets du peptide vasoactif urotensine II sur la néoangiogenèse et la croissance des glioblastomes. Sciences du Vivant [q-bio]. Université de Rouen, France, 2015. Français. ⟨tel-02388480⟩

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