Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy in man with stromal fibrosis as one of the main features. Carcinoma cells synthesized no or little collagen I protein. Pro-α1(I) collagen mRNA was expressed by stromal cells throughout the tumor, but expression of procollagen type I protein was restricted to stromal cells situated close to nests of carcinoma cells. These data suggest that the carcinoma cells stimulated collagen type I deposition by increasing pro-α1(1) collagen mRNA translation. Cocultures, of the human ATC cell line KAT-4, with fibroblasts under conditions that allow the study of stimulatory factors on collagen mRNA translation, showed that the KAT-4 cells stimulated collagen type I protein synthesis in fibroblasts. Specific inhibitors of PDGF and TGF-β1 and -β3 were able to inhibit this carcinoma cell-induced stimulation of collagen type I synthesis. These findings suggest that tumor cells were able to stimulate collagen mRNA translation in stromal fibroblasts by, at least in part, transferring PDGF and/or TGF-β1 and -β3. Xenograft transplantation of different ATC cell lines into athymic mice demonstrated that the low collagen producing carcinoma cell lines were less tumorigenic compared to non-collagen producing carcinoma cell lines. The morphology of tumors derived from non-collagen producing ATC cell lines showed a well demarked stroma surrounding carcinoma cell nests. TGF-β1 and -β3 were found to play a role in generating a high tumor interstitial fluid pressure (TIPF) in experimental KAT-4 tumors. A specific inhibitor of TGF-β1 and -β3 was able to lower TIPF and reduce tumor growth after a prolonged period of treatment, suggesting that TGF-β1 and -β3 have a role in maintaining a stroma that support tumor growth.

TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes. The cell cycle inhibitor p21Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect. The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal p21 promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as Id2 that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene Meox2, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur. This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

La progression du cancer colorectal procède selon une série de transitions, de la crypte épithéliale normale vers l'adénome conduisant au carcinome primaire in situ et aux métastases généralement localisées au niveau du foie. Ces événements séquentiels sont orchestrés par un ensemble d'altérations géniques et moléculaires (syndromes familiaux HNPCC, FAP et cancers sporadiques CIN-LOH et MSI) qui se traduisent de manière générale par l'activation constitutive de (proto)oncogènes ou par la perte de gènes suppresseurs de tumeurs ou de métastases. Si les récepteurs du TGF-β et leurs réseaux de signalisation associés ont été tout particulièrement incriminés quant à leur rôle péjoratif pendant les phases tardives de la progression des tumeurs solides et des cancers du côlon chez l'homme, les informations concernant le rôle des cytokines BMP apparentées au TGF-β dans ce domaine ne sont que très fragmentaires. Quand ce projet a été initié, une étude attribuait à BMP-7 un rôle anti-inflammatoire dans l'intestin chez le rat, suggérant ainsi que cette cytokine pouvait exercer un rôle direct et bénéfique sur la muqueuse digestive et les cellules épithéliales intestinales en particulier. Les BMP agissent par l'intermédiaire de leurs récepteurs de type II (BMPRII, ActRII, ActRIIB) , de type I (ALK-2, ALK-3, ALK-6), et des protéines SMADs (SMAD1, SMAD4, SMAD5, SMAD8). Cependant, 50% des cancers du côlon métastatiques présentent une forme mutée de SMAD4. Des mutations germinales dans le gène codant le récepteur ALK-3 sont observées chez 38% des patients atteints de polypose juvénile (JPS). Enfin, 83% des cancers colorectaux présentant une instabilité des séquences microsatellites (MSI) montrent une mutation dans le gène codant le récepteur de l'activine ActR-II. Dans ce contexte, mon projet de thèse a été centré sur l'expression et le rôle de BMP-7 sur la progression des cellules cancéreuses colorectales humaines et dans les tumeurs associées. Nous avons démontré par RT-PCR, immunohistochimie, et en ELISA que BMP-7 et ses récepteurs sont présents dans des cryptes coliques histologiquement normales, les foci de cryptes aberrantes dans la sigmoïdite, les tumeurs colorectales humaines et plusieurs lignées de cellules cancéreuses coliques. Nous avons aussi démontré que BMP-7 est un facteur de dissémination inducteur du " scattering " et de l'invasion cellulaire dans le collagène de type I. Le pouvoir invasif de BMP-7 est indépendant de SMAD4 et de l'oncogène src, mais associé à l'activation différentielle et cyclique des GTPases (Rac1 et RhoA), de la tyrosine kinase FAK (phosphorylation de la tyr925 impliquée dans la signalisation invasive et l'angiogenèse), et des MAPK /SAPK (JNK et ERK1/2). L'ensemble de ces travaux suggère que BMP-7 se comporte comme un facteur de dissémination proinvasif, agissant par un mécanisme autocrine et paracrine au niveau des cellules cancéreuses du côlon et du stroma tumoral. Cette cytokine exerce donc des actions divergentes sur la progression des tumeurs coliques humaines, en s'opposant aux processus inflammatoires transitoires (rôle bénéfique), mais en favorisant la néoplasie lors des étapes plus tardives associées à l'acquisition du pouvoir invasif à la transition adénome- carcinome pendant la cancérogenèse (rôle péjoratif). Parallèlement, dans cette thèse, nous avons démontré que l'intégrine α1 fait partie de l'échafaudage moléculaire impliqué dans l'invasion cellulaire dépendant de l'oncogène src. D'une autre part, nous démontrons que le VEGF est un inducteur autocrine de l'invasion cellulaire par les cellules cancéreuses du côlon. Selon ce modèle, le VEGF sécrété par les cellules tumorales au sein de la tumeur primaire agit à la fois sur les cellules cancéreuses et les cellules endothéliales en induisant des signaux de survie, de prolifération et d'invasion nécessaires à la croissance des tumeurs primaires et à la génération des métastases.

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

Η χρόνια νεφρική ανεπάρκεια οφείλεται σε διάφορα αίτια βλάβης του σπειράματος και του διαμεσοσωληναριακού χώρου. Χαρακτηρίζεται ιστολογικά από την παρουσία σοβαρού βαθμού σπειραματικής σκλήρυνσης, ίνωσης του διάμεσου ιστού, ατροφίας των ουροφόρων σωληναρίων και υαλίνωσης των αρτηριδίων. Η αρχική βλάβη του σπειράματος που προκαλείται από διάφορα είδη ερεθισμάτων μπορεί να ακολουθήσει την οδό της αποκατάστασης ή να εξελιχθεί προς σκλήρυνση, διαδικασίες στις οποίες συμμετέχουν κυτταροκίνες και αυξητικοί παράγοντες που προέρχονται από ενδοθηλιακά, μεσαγγειακά, επιθηλιακά σωληναριακά κύτταρα, μονοκύτταρα και ινοβλάστες1 . Ο Transforming Growth Factor-β1 (TGF-β1) είναι ο κυριότερος αυξητικός παράγοντας που μέσω πολλαπλών μηχανισμών συμμετέχει στην ανάπτυξη σκληρυντικών αλλοιώσεων2. Προάγει την παραγωγή συστατικών της εξωκυττάριας θεμέλιας ουσίας και μειώνει την αποικοδόμησή τους, προκαλεί ενεργοποίηση των μυοϊνοβλαστών, δηλαδή κυττάρων με μεταναστευτικές ιδιότητες που παράγουν κολλαγόνο, συμμετέχει στη διαφοροποίηση των επιθηλιακών σωληναριακών κυττάρων προς ινοβλάστες και ευοδώνει την κυτταρική απόπτωση η οποία οδηγεί στην απογύμνωση του νεφρικού ιστού από τα φυσιολογικά του κύτταρα. Η χρήση αντισωμάτων κατά του TGF-β1 και αναστολέων του μετατρεπτικού ενζύμου της αγγειοτενσίνης σε διάφορα πειραματικά μοντέλα νεφρικής βλάβης έχει συμβάλλει στην κατανόηση μηχανισμών που συμμετέχουν στη διαδικασία εξέλιξής της. Σε πειραματικά μοντέλα έχει διαπιστωθεί ότι ο TGF–β ευθύνεται για την αυξημένη παρουσία μυοινοβλαστών, την εναπόθεση κολλαγόνου και την απώλεια του σωληναριακού επιθηλίου. Πιο πρόσφατα, ένα μέλος της υπερ-οικογένειας των ΤGF–β, η ΒΜΡ7, φάνηκε να εξουδετερώνει την ίνωση που προκαλείται μέσω του TGF–β. Οι δραστηριότητα των παραγόντων αυτών ελέγχεται από άλλες πρωτείνες οι οποίες μπορούν να αυξήσουν ή να καταστείλουν τη διέγερση των υποδοχέων των παραγόντων αυτών. Τα BMPs είναι ενδογενή μόρια που προστατεύουν το νεφρό από διάφορα είδη βλάβης όπως γενετικές βλάβες, ανοσολογικές αντιδράσεις, περιβαλλοντικοί παράγοντες, μεταβολικά αίτια και καταστάσεις οξείας ή χρόνιας νεφρικής βλάβης. / -

La Fibrose Pulmonaire idiopathique (FPI) est une maladie fibroproliférative pour laquelle il n'existe aucun traitement efficace. Les mécanismes à l'origine de cette pathologie sont méconnus et impliquent plusieurs types cellulaires et facteurs de croissance, comme le TGF-β responsable de la différenciation de fibroblastes en myofibroblastes. Pour mieux comprendre ces mécanismes physiopathologiques, nous nous sommes intéressés à l'implication des miARN dans ce processus. Une analyse par puces à ADN de l'ensemble des miARN modulés dans des échantillons pulmonaires de souris, résistantes ou sensibles à la fibrose pulmonaire induite par la bléomycine, nous a permis d'identifier miR-199a-5p comme le meilleur candidat associé à la fibrose pulmonaire mais aussi fibrose rénale et hépatique. J'ai ensuite démontré que l'expression de miR-199a-5p était induite par le TGF-β in vitro, et que sa surexpression ectopique induisait la différenciation des fibroblastes. Une combinaison d'approche in silico et expérimentale, m'a permis d'identifier la Cavéoline-1 (CAV-1) comme cible de ce miARN. La CAV-1 est impliquée dans la dégradation du récepteur TGF-β. Ainsi, l'inhibition de CAV-1 par miR-199a-5p constitue une boucle de rétrocontrôle positif exacerbant la voie TGF-β. De manière intéressante, l'inhibition de miR-199a-5p in vitro régule la différenciation, la prolifération et la migration des fibroblastes pulmonaires par le TGF-β. Par ailleurs, nos résultats précliniques indiquent que l'inhibition de ce miARN diminue les marqueurs de fibrose, permettant d'envisager le développement de nouvelles approches thérapeutiques dans le traitement de la FPI et d'autres maladies fibroprolifératives.