Les interactions cellule-cellule et cellule-matrice extracellulaire (MEC) sont cruciales pour entretenir la cohésion tissulaire. Ces deux types d'adhésions sont fonctionnellement interconnectés par un dialogue permanent qui met en jeu des voies de signalisation convergentes régulant notamment l'architecture et la contractilité du cytosquelette d'acto-myosine sous-jacent. Ce dialogue permet d'établir un équilibre de forces intracellulaires en réponse à la tension appliquée par le milieu extérieur. L'endothélium des vaisseaux sanguins est un tissu soumis à des conditions mécaniques particulières. En plus des compressions intercellulaires subies par tout épithélium, les cellules endothéliales (CEs) doivent également subir et résister aux forces hémodynamiques du flux sanguin et à la rigidité de la lame basale – deux signaux mécaniques agissant de part et d'autre de l'endothélium. Les Cerebral Cavernous Maformations (CCM) ou encore angiomes caverneux sont des lésions vasculaires hémorragiques d'origine génétique qui se développent au niveau des capillaires du système nerveux central et qui se caractérisent par des défauts dans l'environnement proche des CEs. La perte des jonctions intercellulaires et du recouvrement par les cellules murales, l'organisation aberrante de la membrane basale aussi que la stagnation du flux sanguin sont les caractéristiques des CCM. C'est pourquoi nous avons choisi cette pathologie comme modèle intéressant de mécanotransduction mettant en jeu le dialogue entre les intégrines et les cadhérines. En effet, les trois gènes indifféremment mutés dans cette pathologie codent pour des protéines, CCM1-3, qui s'associent en un complexe ternaire et qui sont reconnues comme des acteurs importants de la régulation des jonctions adhérentes. Des études moléculaires et protéomiques montrant que le complexe CCM interagit avec la protéine ICAP-1, un régulateur négatif de l'intégrine β1, nous ont conduit à formuler l'hypothèse selon laquelle ce complexe jouerait un rôle pivot dans la signalisation croisée entre ces intégrines et cadhérines. Les études effectuées pendant ma thèse ont démontré que les protéines CCM régulent l'homéostasie tensionnelle médiée par les structures d'adhérence intercellulaires et à la MEC par leur action inhibitrice sur l'intégrine β1 et en controlant une balance d'activité entre les deux isoformes de ROCK, ROCK1 et ROCK2. Nous avons montré que, suite à la perte des protéines CCMs, la suractivation de l'intégrine β1 augmente la sensibilité des CEs aux signaux mécaniques comme la rigidité de la MEC ou les forces hémodynamiques du flux sanguin. Il en résulte une suractivation de la contractilité cellulaire dépendante de ROCK1 déclenchant une boucle de rétrocontrôle mécanique conduisant à l'amplification des tensions intra- et extracellulaire et brisant ainsi l'homéostasie tensionnelle pour favoriser le phénotype malin. / Cell-cell or cell-matrix interactions have crucial roles in the maintenance of the physical cohesion of any tissue. In addition, growing body of evidence indicates that these two adhesion systems do not act independently, but rather are functionally interconnected by a permanent crosstalk. This dialog usually operates via common molecules that trigger convergent signaling as well as by actomyosin network which, by providing physical link, contributes to establishment of intracellular force counterbalancing tension applied by extracellular surrounding. Blood vessels endothelium is a particular tissue in term of mechanical conditions. Apart from intracellular compression, endothelial lining needs to resist hemodynamic forces as well as rigidity of the basal membrane - two mechanical inputs acting from opposite sides of the endothelial layer. Cerebral Cavernous Malformation (CCM) is a sporadically acquired or inherited disease of venous capillaries within neuro-vascular unit characterized by defects in all aspects of local microenvironment. Loss of intra-endothelial junctions and mural cell coverage, aberrant organization of basal lamina as well as stagnant blood flow are features of CCM lesions. Thereby, CCM became for us an interesting model to study mechanotrasduction process and in this context, the cross-talk between integrin and cadherin mediated adhesion structures. Indeed, CCM proteins are well recognized players involved in a control of VE-cadherin mediated intracellular junctions. In addition, CCM1 was found to interact with ICAP-1, a negative regulator of β1 integrin, raising the possibility that this complex most likely acts as molecular node regulating β1 integrin/ VE-cadherin convergent signaling pathways.Studies performed during this thesis have demonstrated that CCM complex coordinates cadherin- and integrin-mediated tensional homeostasis by repressing β1 integrin activation and maintaining a balance of activity between the two isoforms of RhoA-associated kinases ROCK1 and ROCK2. We have found that β1 integrin sustained over-activation upon CCM proteins loss contributes to increased ECs sensitivity to mechanical cues, such as ECM physical reorganization or hemodynamic force that in turn activates ROCK1-dependent contractility. This establishes a positive feedback mechanical loop that breaks tensional homeostasis and switches on the malignant phenotype.

La survie des patients atteints de cancer et traités avec des thérapies conventionnelles reste faible dans plusieurs types de tumeurs. Récemment, une nouvelle approche immunothérapeutique a été développée pour cibler le système immunitaire au lieu de la tumeur elle-même, afin de restaurer la fonctionnalité des cellules immunitaires et la destruction des cellules cancéreuses. L’immunothérapie ciblant le récepteur inhibiteur PD-1 occupe une place privilégiée dans les thérapies anticancéreuses en raison de sa haute spécificité et de sa faible toxicité par rapport aux thérapies conventionnelles. Cependant, le taux de réponse reste faible avec seulement 20 à 25% de patients répondant à une immunothérapie anti-PD-1. Il est donc important de comprendre les mécanismes associés à la résistance à ces thérapies et d’identifier des biomarqueurs prédictifs de réponse. L'expression du ligand de PD-1, PD-L1, sur les cellules tumorales, la charge mutationnelle tumorale et l'infiltration tumorale par les lymphocytes ont déjà été décrits, mais de nouveaux biomarqueurs sont nécessaires pour mieux déterminer la sous-population de patients susceptible de bénéficier de ces traitements. Au cours de ce travail, nous avons établi une cohorte de 118 patients atteints d'un cancer du poumon non à petites cellules (CBNPC) traités avec une immunothérapie anti-PD-1/PD-L1, et nous avons étudié l'expression de plusieurs biomarqueurs potentiels, en particulier les cellules T mémoires résidentes dans le tissu (TRM) CD8+CD103+. Ces cellules constituent un candidat potentiel car elles représentent une population privilégiée de lymphocytes T CD8 grâce à l’expression de PD-1 et une forte capacité cytotoxique vis-à-vis des cellules tumorales autologues suite à la neutralisation de l’interaction de PD-1 avec PD-L1. Nous montrons qu’une forte infiltration de tumeurs de CBNPC avec des cellules TRM corrèle à une survie sans progression plus élevée (PFS) et une réponse plus efficace à anti-PD-1 que les tumeurs avec une faible infiltration par des TRM. De plus, les tumeurs qui expriment fortement ICAM-1, un ligand de l’intégrine LFA-1 exprimée sur les lymphocytes T CD8, sont hautement infiltrées par des TRM. Par ailleurs, il est bien connu que le signal TGF-β est crucial pour l’induction de CD103 et la formation de TRM CD8+CD103+. Je me suis donc intéressée à l'activation du TGF-β par les intégrines αV exprimée par les cellules tumorales humaines et murines. À l'aide des modèles in vitro et in vivo, nous montrons que les cellules tumorales exprimant les intégrines αV activent le TGF-β et induisent l'expression de CD103 à la fois par les cellules T CD8+ provenant de cellules mononucléées du sang périphérique (PBMC) et de lymphocytes infiltrant la tumeur (TIL). L’expression plus faible de CD103 par les TIL CD8+ de souris greffées avec des tumeurs déficientes pour l’expression d'αV n'a pas d'effet sur le contrôle de la croissance tumorale. De manière intéressante, nous montrons dans des modèles de tumeurs déficientes pour l’expression d’αV, que le traitement avec des anticorps anti-PD-1 bloquants corrèle avec un meilleur contrôle de la croissance tumorale et une meilleure réponse à l'immunothérapie anti-PD-1 qui sont associés à une infiltration plus forte de TIL et un état d'activation plus élevé des TIL CD8+ exerçant une activité cytotoxique spécifique. De plus, une expression élevée de l'intégrine αV dans les tumeurs corrèle avec une réponse plus faible des patients atteints de CBNPC à une immunothérapie anti-PD-1/PD-L1. Ces données montrent comment trois marqueurs distincts, cellules TRM, ICAM-1 et les intégrines αV, régulent le microenvironnement tumoral et l’immunité T CD8 avec des implications potentielles pour potentialiser les réponses aux immunothérapies. / The survival of cancer patients treated with conventional therapies remains low in multiple cancers. Recently, a new immunotherapeutic approach has been developed to target the immune system instead of the tumor itself, in order to restore immune cell functions in cancer destruction. Immunotherapy targeting the T cell inhibitory receptor PD-1 occupies a privileged place in cancer therapy thanks to its high specificity and low toxicity compared to chemotherapies. However, the response rate remains low with only 20-25% of patients responding to anti-PD-1 immunotherapy. An important issue is therefore to understand the mechanisms associated with resistance to these therapies and to identify the predictive biomarkers of response. The expression of the PD-1 ligand, PD-L1, on tumor cells, tumor mutational burden (TMB) and tumor infiltration by lymphocytes have been described to predict the response to immune checkpoint blockade (ICB). However, new biomarkers are needed to better determine patient subpopulation which could benefit from this treatment. To address this question, we established a cohort of 118 non-small cell lung cancer (NSCLC) patients treated with anti-PD-1/PD-L1 immunotherapy and studied the expression of several potential biomarkers. Tissue-resident memory T (TRM) cells are a potential candidate because they represent a distinct population of CD8+ T cells highly expressing integrin αEβ7 (CD103) and PD-1; and showing strong cytotoxic capacity towards autologous tumor cells upon neutralisation of PD-1/PD-L1 interaction. Results from the present study show that high infiltration of TRM cells in NSCLC tumors correlates with higher progression-free survival (PFS) and a better response to anti-PD-1/PD-L1 immunotherapy. Moreover, tumors with high expression levels of ICAM-1, the ligand of integrin LFA-1 expressed on T cells, show higher TRM infiltration. TGF-β is a cytokine directly involved in CD103 induction on activated tumor-specific T cells. Therefore, I also investigated the role of αV integrins in activating TGF-β and thereby in controlling TRM differentiation and anti-tumor T cell immunity. Using human and mouse models, we show that tumor cells expressing αV integrins activate TGF-β, which can in turn induce expression of CD103 on CD8+ T cells in vitro on peripheral blood mononuclear cells (PBMCs) and in vivo on tumor infiltrating lymphocytes (TIL). However, lower CD103 expression on CD8+ TIL and thus CD103+ TRM cell formation in C57BL/6 mice engrafted with αV-lacking cancer cells had no effect on tumor growth control. Remarkably, αV-deficient tumors responded more effectively to anti-PD-1 immunotherapy than αV-efficient tumors and this response correlates with higher tumor infiltration by activated CD8+ T cells and stronger cytotoxic activity toward autologous cancer cells. Moreover, high expression of αV integrins in NSCLC tumors correlates with worse response to anti-PD-1/PD-L1 immunotherapy. These data show how three distinct markers, TRM cells, ICAM-1, and αV integrins regulate the tumor microenvironment and CD8+ T cell immunity, with potential implications in improving response to ICB immunotherapies.

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease worldwide and occurs in up to 40% of patients with diabetes. The standard of care for DKD treatment has not kept up with the current health epidemic, which has led to a heavy economic toll and substantial health burden. Targeting either cell surface (cs)GRP78, activated α2-macroglobulin (α2M*) or preventing their interaction may provide a novel anti-fibrotic therapeutic target for the treatment of DKD and potentially non-diabetic chronic kidney disease (CKD) as well. Previously our lab has shown that HG-induced csGRP78 is a mediator of PI3k/Akt signaling and downstream extracellular matrix (ECM) protein production in glomerular mesangial cells (MC). However, the ligand responsible for activating high glucose (HG)-induced csGRP78 had not yet been determined. We have shown thus far that α2M is endogenously produced, secreted, and activated (denoted α2M*) in HG by MC, which leads to its binding to and activation thereof csGRP78. Further, α2M knockdown or α2M* neutralization attenuated Akt activation, the production of the profibrotic cytokine connective growth tissue factor (CTGF) and ECM proteins fibronectin and collagen IV. We have also shown that integrin β1 (Intβ1), a transmembrane receptor, associated with csGRP78 under HG conditions and likely acts as a tether to present csGRP78 completely extracellularly on MC. Interestingly, Intβ1 activation, even in the absence of HG, was sufficient to induce csGRP78 translocation. Further, inhibition of either csGRP78 or Intβ1 prevented synthesis, secretion and signaling of TGFβ1. This data implicates a role for Intβ1 as a required signaling partner for csGRP78-mediated profibrotic signaling. To further our understanding of csGRP78/ α2M*’s role in DKD, we investigated their ability to mediate TGFβ1 signaling through its non-proteolytic activator thrombospondin-1 (TSP1). Here, HG-induced TSP1 expression, ECM deposition, and activation of TGFβ1 was regulated by the PI3k/Akt pathway via csGRP78/α2M* in MC. Furthermore, we assessed whether this csGRP78/ α2M* axis is relevant to promoting profibrotic signaling in other renal cell types, including proximal tubule epithelial cells (PTEC) and renal fibroblasts (RF), that contribute to the pathogenesis of both later stage DKD and non-diabetic CKD. We show evidence here that HG and direct treatment with TGFβ1, a key pathologic regulator of kidney fibrosis, induce GRP78 surface translocation as well as the endogenous production and activation of α2M in both PTEC and RF. Inhibition of either csGRP78 or α2M* prevented TGFβ1 signaling measured as Smad3 activation as well as downstream ECM production. Interestingly, inhibition of this pathway under direct TGFβ1 treatment did not prevent Smad3 activation, implicating a role for Smad-independent TGFβ1 signaling through this axis. We identified the known noncanonical TGFβ1 signaling partners, yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are mediated by csGRP78 and α2M*. Lastly, we evaluated the potential therapeutic benefit of inhibiting csGRP78/α2M* interaction in the kidney fibrosis model, unilateral ureteral obstruction (UUO). Here, we show evidence that inhibition of this signaling axis using an inhibitory peptide can prevent renal fibrosis. Whether this peptide also prevents fibrosis in DKD is currently being assessed. Together, these studies strongly implicate targeting csGRP78/α2M* interaction as a novel anti-fibrotic therapeutic intervention for early and late stage DKD, as well as a potential role in non-diabetic CKD. / Thesis / Doctor of Philosophy (Medical Science) / Diabetic kidney disease is the leading cause of kidney failure in developed nations. This progressive disease leads to the loss of kidney function due to an accumulation of scar proteins in the kidney over time. High glucose is a major factor that causes this to occur. Our lab studies specific kidney cells called mesangial cells, proximal tubule epithelial cells, and fibroblasts that produce scar proteins in the presence of high glucose. We have shown that when these cells are treated with high glucose, this causes the movement of a protein called GRP78 that normally resides inside the cell to move to the cell’s surface where it can interact with other proteins. My research has established that the proteins alpha 2-macroglobulin (ɑ2M), integrin β1 (Intβ1), and thrombospondin-1 (TSP1) can bind to GRP78 on the cell surface and cause cells to make scar proteins. Preventing ɑ2M or Intβ1 from binding to GRP78 or preventing TSP1 production prevents mesangial cells from making scar proteins when exposed to high glucose. In a mouse model that overproduces these scar proteins, we showed that preventing cell surface GRP78 and α2M interaction prevents scar protein production and is thus a novel potential treatment option for kidney disease.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in α2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression. / EPSRC and DTA grant

No description available.

No / This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Syngeneic graft-versus-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic BM cells followed by a 21 day treatment with the immunosuppressive agent cyclosporine A (CsA). Clinical symptoms of the disease appear 2-3 weeks following cessation of CsA therapy and disease-associated inflammation occurs primarily in the colon and liver. The development of SGVHD is a complex process resulting from the cooperative interaction of multiple effector cell populations including NK cells, T cells and macrophages. TH1 cytokines (IL-12, TNF-α, IFN- γ), produced by these effector cells, serve as inflammatory mediators contributing to the pathogenesis of SGVHD. The SGVHD conditioning agents, irradiation and CsA, are both required for the development of disease and contribute to the production of oxidative stress. Time course studies revealed increased reactive oxygen and nitrogen species (ROS/RNS), as well as, increased colon mRNA levels for TNF-α and iNOS in CsA-treated versus control BMT animals. Since ROS/RNS are known to mediate CsA toxicity, studies were undertaken to determine the effect of oxidative stress on the induction of SGVHD. In vivo treatment with the antioxidant MnTBAP caused a reduction in colon mRNA levels for iNOS and TNF-α, as well as delayed disease development, suggesting a role for oxidative stress in the development of SGVHD. In addition, CD4+ T cells have been shown to play an important role in the inflammatory response observed in the gut of SGVHD mice. Time course studies revealed significant increases in the migration of CD4+ T cells as early as day 14 post- BMT into the colon of CsA mice as well as significant elevated mRNA levels of cell adhesion molecules. Homing studies revealed that a labeled CD4+ T cell line, generated from SGVHD mice, migrated in larger numbers into the gut of CsA-treated mice compared to control animals. This study demonstrated that CD4+ T cells responsible for the pathogenesis observed in murine SGVHD are present early after BMT in colons of CsA-treated mice, suggesting that during the 21 days of immunosuppression therapy functional mechanisms are in place that result in increased homing of effector cells to colons of CsA-treated mice.

L’endostatine est le fragment C-terminal du collagène XVIII libéré dans la matrice extracellulaire par clivage enzymatique. C'est un inhibiteur endogène de l’angiogenèse et de la croissance tumorale. L'endostatine inhibe la prolifération et la migration des cellules endothéliales induite par le Fibroblast Growth Factor-2 ou le Vascular Endothelial Growth Factor et elle inhibe la croissance de 65 types de cellules tumorales. L’endostatine fait actuellement l’objet d’essais cliniques pour le traitement de différents cancers son mécanisme d’action est encore mal connu. Nous avons caractérisé par résonance plasmonique de surface (SPR) les interactions établies par l'endostatine avec les intégrines αvβ3 et α5β1 qui sont surexprimées à la surface des cellules endothéliales activée. Nous avons identifié le site de fixation l'endostatine sur les intégrines, proposé un modèle de structure du complexe formé par l'endostatine et l'intégrine αvβ3 et montré que l'endostatine ne peut pas se lier simultanément aux intégrines et aux chaînes d'héparane sulfate présentes à la surface cellulaire. Pour identifier des partenaires supplémentaires de l'endostatine, nous avons développé des puces à protéines et à glycosaminoglycanes basées sur la SPR et capables de suivre jusqu'à 400 interactions simultanément. Nous avons identifié neuf partenaires de l'endostatine (le dermatane sulfate, la transglutaminase-2, les collagènes I, IV et VI, le peptide amyloïde β1-42, et des protéines matricellulaires dont SPARC et thrombospondine-1). Nous avons montré que l’endostatine se fixe avec une forte affinité (KD ~ 6 nM) sur la transglutaminase-2 et que cette interaction nécessite la présence de calcium mais que l'endostatine n'est pas un substrat donneur d'acyle de l'enzyme. Nous avons montré que le réseau d'interactions de l'endostatine est enrichi en protéines contenant des modules EGF (Epidermal Growth Factor). Cela offre de nouvelles perspectives pour l'identification d'autres partenaires et donc de nouvelles fonctions de l'endostatine. Des protéines contenant des modules EGF comme la fibrilline-1, composant des fibres élastiques, et des protéines de l'immunité innée par exemple sont des partenaires potentiels de l'endostatine / Endostatin is the carboxyl-terminal fragment of collagen XVIII released in the extracellular matrix by proteolytic cleavage. It inhibits angiogenesis and tumor growth. Endostatin inhibits the proliferation and migration of endothelial cells induced by Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor and it inhibits 65 different tumor types. Endostatin is currently under clinical trials for several tumors. We have used surface plasmon resonance (SPR) binding assays to characterize interactions between endostatin and α5β1 or αvβ3 integrins which are over-expressed at cell surface of actived endothelial cell. We have identified the binding site of endostatin on those integrins, and we have built a molecular modeling of the endostatin/integrin αvβ3 complex. We have shown that endostatin can not bind simultaneously to integrins and to heparan sulfate. In order to identify new partners of endostatin we have developed glycosaminoglycan and protein arrays based on SPR detection. We have found nine new partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV and VI), the amyloid peptide Aβ(1-42), and transglutaminase-2 (TG-2). We have shown that endostatin binds to transglutaminase-2 with an high affinity (KD ~ 6 nM) in a calcium-dependent manner. Enzymatic assays indicated that, in contrast to other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2, but would rather be an acyl acceptor. The endostatin network comprises a number of extracellular proteins containing EGF domains (Epidermal Growth Factor), and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases

La matrice extracellulaire est constituée d'un réseau tridimensionnel de protéines et de polysaccharides complexes, les glycosaminoglycanes. Elle apporte un support structural aux tissus et aux cellules dont elle est capable de moduler la prolifération, la migration et la différenciation. Nous avons créé une base de données d'interactions extracellulaires protéine-protéine et protéine-glycosaminoglycane, MatrixDB, qui est disponible sur le Web (http://matrixdb.ibcp.fr). Nous avons intégré des données expérimentales, des données issues de l’analyse de la littérature et des données issues de bases de données d'interactions publiquement disponibles. Nous avons respecté les standards de curation et d’échange de données du consortium IMEx dont fait partie MatrixDB. MatrixDB permet la construction et la visualisation de l'interactome extracellulaire entier et de plusieurs types de réseaux d'interactions, spécifiques d'une molécule, d'un tissu, d'une pathologie ou d'un processus biologique. Nous avons ainsi caractérisé le réseau d’interactions extracellulaire associé au vieillissement et mis en évidence le rôle important des glycosaminoglycanes et du calcium dans ce réseau. Nous avons construit le réseau d'interactions d'une matricryptine anti-angiogénique et anti-tumorale, l'endostatine, qui est issue du collagène XVIII. Les analyses structurales et fonctionnelles de ce réseau ont montré que les partenaires de l’endostatine sont majoritairement impliqués dans l’adhésion cellulaire et que les domaines EGF (Epidermal Growth Factor) sont surreprésentés. Cette propriété nous a permis d'identifier expérimentalement d'autres partenaires de l'endostatine possédant un ou plusieurs domaines EGF et de nouvelles fonctions de l’endostatine. Nous avons modélisé les complexes formés par l'endostatine avec deux de ses partenaires pour identifier les sites d'interactions. Ces prédictions, associées aux données expérimentales, ont permis de déterminer des interactions susceptibles d'être établies simultanément par l'endostatine. L'intégration de ces données et des paramètres cinétiques et d'affinité dans le réseau d'interactions de l'endostatine sera utilisée pour proposer un modèle de son mécanisme d'action qui reste mal connu / The extracellular matrix is composed of a tridimensional network of proteins and complex polysaccharides called glycosaminoglycans. It provides a structural support to tissues and modulates cell proliferation, migration and differenciation. We have created a database of protein-protein and proteinglycosaminoglycan extracellular interactions, MatrixDB (http://matrixdb.ibcp.fr). We have integrated experimental data, data issued of the literature curation and data from interaction databases publicly available. We have respected the curation and exchange standards of the IMEx consortium that includes MatrixDB. MatrixDB allows the construction and the visualization of the entire extracellular network and other types of interaction networks specific of a molecule, a tissue, a disease or a biological process. We have characterized the aging-related extracellular interaction network and underlined the important role of glycosaminoglycans and calcium in this network. We have constructed the interaction network of an antitumoral and anti-angiogenic matricryptin, endostatin, issued from collagen XVIII. Functional and structural analysis of their network showed that partners of endostatin are mostly involved in cell adhesion and that EGF domains are overrepresented. This has allowed us to to identify experimentally other partners of endostatin possessing one or more EGF domains and to propose new functions of endostatin. We have modelled complexes formed by endostatin with two of its partners to identify the binding sites.These predictions, associated with experimental data, allowed us to determine interactions able to be established simultaneously by endostatin. Integration of these data and of kinetics and affinity parameters in the interaction network of endostatin will be used to build a model of its mechanism of action that is not fully elucidated

Les cellules musculaires lisses (CML) vasculaires les composants cellulaires principaux de la paroi artérielle, sont exposées constamment aux contraintes mécaniques. Les contraintes mécaniques cycliques régulent de nombreuses fonctions des CML vasculaires via les intégrines. Parmi les intégrines, l'[alpha]v[gamma]3 est non seulement un mécano-transducteur mais aussi le récepteur de la prothrombine à la surface des CML. L?activation de l'intégrine [alpha]v[gamma]3 par les contraintes mécaniques pourrait favoriser l'adhésion des CML à la prothrombine et aussi accélérer la génération de thrombine à la surface des CML. Pour vérifier cette hypothèse, nous avons étudié l'effet des contraintes mécaniques sur la génération de thrombine par les CML et identifié les voies de la signalisation impliquées. Nous avons utilisé un modèle de Flexcell utilisant les CML aortiques de rat, soumises à un étirement cyclique (10%, 1Hz). L'exposition à l'étirement cyclique pendant 1h et 6h induit un phénotype de différenciation et non-apoptotique des CML et une augmentation de l'expression de l'intégrine [alpha]v[gamma]3. Il y a aussi une augmentation de la phosphorylation de Src, FAK, AKT de façon temps dépendant et une augmentation de la phosphorylation de l'ILK à 15 min et du clivage de taline de 5 à 60 min. L'étirement cyclique augmente l'adhésion des CML à la prothrombine et la génération de thrombine avec un effet maximum à 6h de 67% et 30% respectivement. Le peptide mimétique de l'intégrine [alpha]v[gamma]3 (cRGDPV) et le siARN [alpha]v bloquent tous les effets de l'étirement cyclique sur les CML. Le siARN taline inhibe l'expression de la sous-unité [alpha]v et également la phosphorylation de Src, AKT et ILK. Le siARN ILK n'a pas d'effet sur l'expression de l'[alpha]v mais inhibe la phosphorylation d'AKT et le clivage de taline à 6h de l'étirement cyclique. Ainsi, l'étirement cyclique induit une plus forte génération de thrombine par les CML vasculaires via l'activation des voies de signalisation dépendante de l'[alpha]v[gamma]3. Cette étude suggère que la génération de thrombine intravasculaire peut être régulée par des antagonistes de l'intégrine [alpha]v[gamma]3 et peut devenir une nouvelle cible thérapeutique chez les patients avec une pression pulsée élevée / Vascular smooth muscle cells (SMC), the main cellular components of the arterial wall, are constantly exposed to mechanical stretch. Cyclic mechanical stress regulates many functions of vascular SMC via integrins. Among the integrins, [alpha]v[gamma]3 is not only a mechanotransducer but also the receptor of prothrombin in the vascular SMC. Activation of integrin [alpha]v[gamma]3 by mechanical stretch may promote SMC adhesion to prothrombin and also accelerate thrombin generation on the surface of SMC. To test this hypothesis, we have studied the effect of mechanical stretch on the generation of thrombin by SMC and identified possible signaling pathway involved. We used a Flexcell model using rat aortic SMC subjected to cyclic stretch (10%, 1Hz). Exposure to cyclic stretch for 1h and 6h induced a phenotype of differentiation and non-apoptosis of SMC and an increased expression of integrin [alpha]v[gamma]3. There was also an increase in phosphorylation of Src, FAK, and AKT in a time dependent manner, increased phosphorylation of ILK at 15min and the cleavage of talin from 5 to 60min. Cyclic stretch increased the adhesion of prothrombin to the SMC, and thrombin generation with a maximum effect of 67% and 30% respectively. A peptide mimetic of integrin [alpha]v[gamma]3 (cRGDPV) and [alpha]v siRNA both blocked all the effects of cyclic stretch on SMC. A talin siRNA inhibited the expression of [alpha]v and the phosphorylation of Src, AKT and ILK. An ILK siRNA has no effect on the expression of [alpha]v but inhibited the phosphorylation of AKT and the cleavage of talin at 6h of stretch. Thus, cyclic stretch induced a higher thrombin generation by vascular SMC via activation of signaling pathways dependant on [alpha]v[gamma]3. This study suggests that intravascular thrombin generation can be regulated by antagonists of integrin [alpha]v[gamma]3 and can become a new therapeutic target for the patients with a high pulse pressure