Η πρωτεΐνη YKL-40 είναι μια εκκρινόμενη πρωτεΐνη, μέλος της οικογένειας των γλυκοπρωτεϊνών που ομοιάζουν με χιτινάση. Περιέχει καλά συντηρημένες περιοχές που προσδένουν χιτίνη, στερείται όμως της ενζυματικής δράσης της χιτινάσης εξαιτίας μιας σημειακής μετάλλαξης στην καταλυτική περιοχή. Από την υπάρχουσα βιβλιογραφία φαίνεται να εμπλέκεται στη διαδικασία της φλεγμονής αλλά και στη δημιουργία ίνωσης και νεοαγγειογένεσης. Στην παρούσα μελέτη, θελήσαμε να διερευνήσουμε την έκφραση της YKL-40 με την τεχνική της ανοσοιστοχημείας σε μιτροειδείς βαλβίδες του ανθρώπου με ιστοπαθολογικές αλλοιώσεις ( βλεννώδη εκφύλιση ,ινωση ,νεοαγγειογένεση ασβέστωση ) και να συσχετίσουμε την πιθανή έκφραση της πρωτεϊνης με την παρουσία και την σοβαρότητα αυτών των αλλοιώσεων καθώς και με άλλες κλινικοπαθολογοανατομικές παραμέτρους της νόσου Χρησιμοποιήθηκαν συνολικά δείγματα από 71 περιστατικά μιτροειδών βαλβίδων μονιμοποιημένα σε ουδέτερη φορμόλη 10% και εγκλεισμένα σε παραφίνη. Εκ των οποίων τα 52 ήταν παθολογικά και τα 19 ήταν φυσιολογικά. Εφαρμόσθηκε η τεχνική της ανοσοιστοχημείας και τα αντισώματα YKL-40, VEGFA, CD31, CD68 και A-SMA. Η στατιστική ανάλυση των αποτελεσμάτων έγινε με τη χρήση του στατιστικού πακέτου SPSS 10.0 for Windows. Τα αποτελέσματα της μελέτης έδειξαν ότι η πρωτεΐνη YKL-40 υπερεκφράζεται στις παθολογικές βαλβίδες (στα μακροφάγα, στα ενδοθηλιακά και στα διάμεσα βαλβιδικά κύτταρ ) και ότι τα υψηλά επίπεδα έκφρασης της σχετίζονται με τη σοβαρότητα των ιστοπαθολογικών αλλοιώσεων. Μόνο σε 2 από τις 19 φυσιολογικές βαλβίδες παρατηρήθηκε θετική ανοσοϊστοχημική χρώση, στις υπόλοιπες 17 ήταν αρνητική. Η ανοσοϊστοχημική χρώση της πρωτεΐνης YKL-40 στις μιτροειδείς βαλβίδες είχε στατιστικά σημαντική σχέση με την ανοσοϊστοχημική έκφραση της πρωτεΐνης A-SMA, της πρωτεΐνης VEGFA, με την παρουσία μακροφάγων και την μέση αγγειακή πυκνότητα.Τα επίπεδα έκφρασης της πρωτεΐνης YKL-40 ήταν μεγαλύτερα στις γυναίκες με νόσο μιτροειδούς βαλβίδας και σχετίζονται στατιστικά σημαντικά με την ηλικία. Με βάση τα ανωτέρω αποτελέσματα, μπορούμε να εισηγηθούμε ένα πιθανό ρόλο της πρωτεΐνης YKL-40 στην παθογένεια της νόσου της μιτροειδούς βαλβίδας , καθώς φάνηκε να εκφράζεται από διάφορους κυτταρικούς τύπους στις παθολογικές βαλβίδες σε σχέση με τις φυσιολογικές όπου δεν παρατηρήθηκε ανοσοθετικότητα του μορίου. Η έκφραση της πρωτεΐνης YKL-40 είχε θετική συσχέτιση με την έκφραση της a-SMA στα διάμεσα κύτταρα της βαλβίδας γεγονός που πιθανολογεί το ενδεχόμενο η πρωτεϊνη YKL-40 να προκαλεί ενεργοποίηση των διάμεσων βαλβιδικών κυττάρων με αποτέλεσμα την παραγωγή πρωτεϊνών εξωκυτταρίου ουσίας με τελική κατάληξη τη δημιουργία βλεννώδους εκφύλισης και ίνωσης . Επιπροσθέτως θα μπορούσαμε να υποθέσουμε λογω της θετικής συσχέτισης της έκφρασης της πρωτεΐνης YKL-40 με την έκφραση του VEGFA από τα βαλβιδικα κύτταρα και τα μακροφαγα ότι η πρωτεΐνη YKL-40 πιθανόν να επάγει την έκφραση του VEGFA από τα κύτταρα αυτά. Επιπρόσθετα, όπως η υπάρχουσα βιβλιογραφία υποστηρίζει, τόσο ξεχωριστά όσο και συνεργικά ο VEGFA και η YKL-40 είναι δυνατον να προκαλούν ενεργοποίηση, χημειοταξία και μετανάστευση ενδοθηλιακών κυττάρων εντός της γλωχίνας και σχηματισμό νέων αγγείων. Τα νέα αυτά αγγεία θα μπορούσαν να λειτουργήσουν θετικά ως προς την φλεγμονώδη διαδικασία προσκομίζοντας στις ήδη παχυσμένες βαλβίδες θρεπτικά συστατικά καθώς και μόρια και κύτταρα του ανοσοποιητικού συστήματος. Η παρουσία των μακροφάγων στις παθολογικές βαλβίδες φαίνεται να παίζει κεντρικό ρόλο στην ανάπτυξη των φλεγμονωδών αλλοιώσεων. Η στατιστικά σημαντική σχέση των μακροφάγων με την πρωτεΐνη YKL-40, σε συνδυασμό με την υπάρχουσα βιβλιογραφία που υποστηρίζει τόσο την έκκριση της πρωτεΐνης από τα μακροφάγα, όσο και την επίδραση της σε αυτά, μας επιτρέπει να υποθέσουμε και ένα παρόμοιο μηχανισμό στη νόσο της μιτροειδούς βαλβίδας. Αν και τα αποτελεσματα της παρουσας μελέτης είναι ενδεικτικά πιθανης συμμετοχής της YKL-40 στην νόσο της μιτροειδούς βαλβίδας χρειάζονται πολύ περισσοτερες μελέτες για να αποσαφηνισθεί ο ακριβής ρόλος της πρωτεΐνης YKL-40 στη παθογενεια της συγκεκριμένης νόσου / The protein YKL-40 is a secreted protein, a member of the family of chitinase-like glycoprotein. The protein YKL-40 contains well conserved regions that bind chitin, but lacks the enzymatic chitinase activity, because of a point mutation in the catalytic region. From the existing scientific literature it seems to be involved in the immigration and macrophage activation process in the site of inflammation. It is also possible to participate in the process of neoangiogenesis and fibrosis. Scientists argue that the protein YKL-40 participates in many diseases which are characterized by development of inflammation and has been proposed to use the protein YKL-40 as an indicator of prognosis, recurrence and treatment response in serum of patients with inflammatory diseases and malignancies. In this study, as the disease of the mitral valve is characterized by development of inflammation and because of existing studies that suggest that the protein YKL-40 induces the expression of VEGF-A protein and also has angiogenetic activity itself, we wanted to investigate the possible positive immunohistochemical staining of protein YKL-40 comparing physiological and pathological histological sections of mitral valve. A total sample of 71 cases was used. The 19 of them were normal and the 52 had lesions. The material was fixed tissue samples in 10% neutral formalin and embedded in paraffin. The 36 came from autopsy material and the remaining 35 of mitral valve replacement surgery. The kind of methodology that was followed was the indirect immunohistochemical method of streptavidin-DAB. The antibodies used were against the protein YKL-40, VEGF-A, CD31 (vascular endothelium), CD68 (macrophages) and A-SMA (activated valve interstitial cells). The statistical analysis was performed using the statistical package SPSS 10.0 for Windows. The study gave the following results: The protein YKL-40 is overexpressed in the pathological valves and its levels was related to the severity of the lesions. Cytoplasmic immunopositivity was present in macrophages, endothelial cells of the outer layers of the valves and the valves’ vessels and valve interstitial cells. Only 2 of the 19 normal valves had positive immunohistochemical stain of the protein YKL-40. Sufficient number of CD68 -positive macrophage was observed in the valves which presented histopathological lesions. The number of macrophages was significantly correlated with the severity of lesions. It was demonstrated the presence of vessels in pathological valves by immunohistochemical staining against the protein CD31. The average microvessel density was correlated with the severity of histological lesions. Pathological valves observed strong immunohistochemical expression of the protein A-SMA in the cells of the matrix. The expression of protein A-SMA was correlated significantly with the severity of lesions. The VEGF-A protein was overexpressed in pathological valves and was related to the severity of histopathological lesions. The cells which had positive immunostaining for the VEGF-A protein were macrophages, endothelial cells of valve blood vessels and valve interstitial cells. Immunohistochemical staining of the protein YKL-40 in the mitral valve was statistically significant compared to the immunohistochemical expression of the protein A-SMA, the protein VEGF-A, with the presence of macrophages and average microvessel density. The expression levels of the protein YKL-40 was higher in women with mitral valve disease and significantly associated with age. According to the previous comments, we propose the possible role of the protein YKL-40 in the pathogenesis of mitral valve disease. The protein YKL-40 seems to be expressed from multiple cellular types in pathological valves. As a different, the normal valves had non immunopositivity of the molecule. The YKL-40 protein was immunohistochemical localization in cells with morphological characteristics similar to those that were stained positive for a-SMA. Therefore, there is possibility that the presence of the protein YKL-40 in the valve interstitial cells is associated with increased cellularity of diseased valves, via the possible increase of the proliferation and activation of valve interstitial cells. As a result, the produce of ECM proteins causes mucous degeneration and fibrosis. Additionally we will assume that the protein YKL-40 is likely to induce the expression of VEGF-A from valve interstitial cells and/or macrophages during the pathogenesis of mitral valve disease. As the present bibliography consider, both separately and synergistically the protein YKL-40 is able to cause activation, chemo taxis and migration of endothelial cells within the cusp and neovascularization. These new vessels could operate positively to the inflammatory process by providing the thickened valves nutrients and molecules and cells of the immune system. The presence of macrophages at the valve lesions seems to play a major role at the inflammation procedure. Both of the statistically significant colleration between macrophages and protein YKL_40 and the current bibliography that suggests the expression of the protein YKL-40 from macrophages and the effect of the protein on them, make us to hypothesize that there is a similar mechanism at the mitral valve disease to. More research is required in order to make clear the exact role of protein YKL-40 in the mitral valve disease.

Le déclin de la fonction respiratoire dans l’asthme est associé à une augmentation de la massedu muscle lisse bronchique. La cellule musculaire lisse (CML) bronchique joue un rôle central dans la physiopathologie de l'asthme. Elle participe à l'inflammation et constitue une composante importante du remodelage des voies aériennes. Récemment, le rôle de la chitinaseYKL-40 comme bio marqueur de ce remodelage dans l'asthme a été évoqué. Dans ce contexte, la question centrale ayant motivé une partie de ce travail de thèse, a été d'étudier les effets d'YKL-40 sur différentes propriétés des CML. Nous nous sommes également intéressés au récepteur activé par les protéases, de type 2 (PAR-2), potentiel récepteur d'YKL-40, mais aussi acteur principal de l’inflammation bronchique.Ce travail a permis d'établir qu'YKL-40 est plus qu'un simple bio marqueur dans l'asthme puisqu’elle altère les propriétés physiologiques de la CML, et semble jouer un rôle dans le remodelage musculaire lisse bronchique. Par ailleurs, ce travail a également permis de mettre en évidence une surexpression du PAR-2 dans les CML d’asthmatiques ainsi qu’une augmentation de son expression et de la prolifération cellulaire, en réponse à une stimulation chronique. Ce travail a enfin permis d’optimiser la technique de l’interférence ARN lentivirale dans les CML bronchiques humaines. / The decline in lung function in asthma is associated with an increased bronchial smooth muscle (BSM) mass. BSM cells play a central role in the pathophysiology of asthma. They are involved in inflammation and are an important component of airway remodelling. Recently, the role of the chitinase YKL-40 as biomarker of this remodelling in asthma has been evoked. In this context, the central question that motivated part of this thesis was to study the effects of YKL-40 on various properties of BSM cells. We also studied the protease activated receptor 2 (PAR-2), as a potential receptor of YKL-40, as well as an important actor of airway inflammation.This work has established that YKL-40 is more than just a biomarker for asthma since YKL-40 alters physiological properties of BSM cells and appears to play a role in BSM remodelling. Moreover, this work has also highlighted an overexpression of PAR-2 in asthmatic BSM cells, as well as an increase of both PAR-2 expression and BSM cell proliferation in response to chronic stimulation. This work has finally allowed us to optimize lentiviral RNA interference in human BSM cells.

This thesis elucidates the crucial role of YKL-40 in enhancing glioma cell migration and invasion in vitro. Increased levels of YKL-40 are specifically associated with the increased invasive capacity of glioma multiforme (GBM) tumors and lower survival rate of GBM patients. In order to examine the effects of YKL-40 on the migration and invasion of GBM cells, we overexpressed YKL-40 in three different glioma cell lines. The overexpression of YKL-40 significantly enhanced glioma cells migration and invasion in vitro and also increased ERK phosphorylation, which is believed to enhance glioma cell survival, and invasiveness. Although receptors for YKL-40 are still unknown, YKL-40 induces interactions between integrin αvβ3 and syndecan-1 in endothelial cells. However, syndecan-1 does not mediate YKL-40-induced migration and invasion of glioma cells since it is expressed at very low levels, in comparison to other syndecans. In contrast, we found that syndecan-4 is expressed at high levels in all glioma cells we tested. Importantly, down-regulation of syndecan-4 dramatically reduced YKL-40-induced migration of U373 cells, suggesting that syndecan-4 may mediate the effect of YKL-40. Since inflammation has been associated with the progression of many cancers, including GBM, we studied the effect of major pro-inflammatory cytokines on the expression of both YKL-40 and syndecans. Interestingly, OSM and IL-1 synergistically enhanced both YKL-40 and syndecan-4 expression in glioma cells. This suggests that this synchronous induction of YKL-40 and syndecan-4 by OSM and IL-1 may enhance invasion of GBM in-vivo. In summary, we propose a mechanism through which YKL-40 may function under pro-inflammatory conditions. Increased expression of YKL-40 and syndecan-4 in glioma cells leads to the subsequent activation of the MAPK/ERK pathway and results in glioma cell invasion.

YKL-40 is a secreted protein that is highly up-regulated in malignant glioblastoma (GBM). Its expression is correlated with the invasive nature of GBMs and poor diagnosis of patients (Nigro et al., 2005). Previous research has shown that in astrocytes and GBM cells, YKL-40 expression is regulated by two transcription factors, NFI-X3 and STAT3, which form a complex with each other (Singh et al., 2011). Here, we show that the N-terminal domain of NFI-X3 is sufficient and required for its interaction with STAT3. We also show that the DNA-binding domain of NFI-X3 is required to induce YKL-40 expression. Thus, the interaction of NFI-X3 with STAT3 may play a role in stabilizing the otherwise weak binding of NFI-X3 to the YKL-40 promoter. Collectively, the observations made in this study shed light on the mechanisms by which NFI-X3, in concert with STAT3 regulate YKL-40 expression.

A new concept quickly gaining ground in the field of cancer research is that the inflammatory process plays a key role in cancer development and metastasis; however, the molecular mechanisms of such an involvement in cancer progression remain largely unspecified. YKL-40, also known as human cartilage glycoprotein 39, is a secreted heparin-binding protein with ties to both cancers and inflammatory disease. In these diseases, YKL-40 has been suggested to play a role in regulating tissue and extracellular matrix remodeling. It has been found that in certain cancers, including breast, colorectal and brain, that high YKL-40 serum levels correlate with poor outcome, and consequently it may serve as a biomarker. Our recent study has shown that tumor-derived YKL-40 acts as an angiogenic factor due to its ability to up-regulate vessel formation and metastasis during tumor development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. The goal of this project was to better understand the importance of tumor-derived YKL-40 in angiogenesis through both functional and structural studies. By establishing a monoclonal YKL-40 antibody for blocking YKL-40, the function of tumor-derived YKL-40 in inducing endothelial cell angiogenesis and tumor cell survival was uncovered, confirming YKL-40's importance in tumor signaling as well as offering evidence in the benefit of its neutralization. Additionally, a postulated heparin-binding domain on YKL-40 was mutated in hopes of revealing the relevance of this binding ability on YKL-40's function and whether this could serve as a target in inhibiting YKL-40 signaling.

Glioblastoma Multiforme (GBM) is the most common brain cancer and one of the most fatal forms of cancer overall. The average survival time is 10-14 months, and less than 10% of patients survive more than 5 years after diagnosis. It is characterized by extreme vasculature, chemo/radioresistance, and invasiveness into the normal brain. The current standard of care, which includes surgical removal of tumor, radiation, and the chemotherapeutic agent temozolomide, initially stunt tumor growth. Nevertheless, the tumor invariably rebounds and the patient succumbs to the disease. Therefore, there is an urgent need to develop new therapies for this devastating disease. YKL-40 is one of the most over-expressed proteins by GBM cells, and is elevated in the serum of patients with GBM. YKL-40 is implicated in a host of inflammatory diseases and has been shown to play a major role in the maturation of some cells of the immune system, especially macrophages. Thus, it has been suggested that YKL-40 may act as a prognostic biomarker for cancer and inflammatory disease. However, little is known about the role of YKL-40 in relation to cancer development and progression, and more work needs to be done to validate it as a biomarker or as a therapeutic target. It was the goal of the work described herein to uncover some of the key molecular mechanisms of GBM development and progression in the hopes of offering new therapeutic targets. Using a wide variety of in vitro and in vivo techniques, the role of a secreted glycoprotein YKL-40 in GBM was probed. It was demonstrated that YKL-40 enhanced angiogenesis, radioresistance, and progression of GBM cells. Moreover, inhibition of YKL-40 in mouse models markedly arrested tumor growth and vascularization, lending support to the idea of YKL-40 as a therapeutic target. Finally, YKL-40 drove GBM cells into a mesenchymal phenotype, where the tumor cells act as mural-like cells, supporting tumor vasculature networks. Hopefully, the results from these studies will offer the rationale to develop drugs against YKL-40 and potentially extend the lives of patients with this terrible disease.

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