Heparan sulfate proteoglycan (HSPG) or heparan sulfate (HS) degradation may contribute to endothelial cell (EC) dysfunction in diabetes. HSPGs, syndecan and perlecan, contain a protein core with mainly HS glycosaminoglycans (GAGs) attached. HSPGs modulate growth factors and function in membrane filtering. Heparanase induction is likely responsible for diabetic HS degradation. Heparin protects endothelium and insulin regulates glucose metabolism. Our objectives were to observe HSPG changes by studying EC GAG content and gene expression of syndecan, perlecan and heparanase under hyperglycemic conditions with insulin and/or heparin treatment.<p> GAGs, including HS, were determined by the carbazole assay and visualized by agarose gel electrophoresis in porcine aortic EC cultures treated with high glucose (30 mM) and/or insulin (0.01 U/ml) for 24, 48 and 72 hours and/or heparin (0.5 µg/ml) for 72 hours. High glucose decreased cell GAGs and increased medium GAGs. GAGs increased with time in control cultures and in high glucose plus insulin treated medium. GAGs were decreased with insulin but increased with insulin or heparin plus high glucose.<p> Confluent cultured human aortic ECs were incubated with control medium, high glucose and/or insulin and/or heparin for 24 hours. Real time PCR determination showed that: high glucose increased heparanase, decreased syndecan and had no effect on perlecan mRNA; insulin or heparin with/without high glucose decreased and insulin and heparin with high glucose increased heparanase mRNA; heparin and insulin with high glucose increased but insulin decreased syndecan mRNA. Actinomycin D (10 µg/ml) inhibited heparanase and syndecan mRNA with high glucose plus insulin plus heparin and inhibited heparanase mRNA with high glucose compared to time 0 but not â-actin after addition for 0, 2, 4, 8 and 24 hours. Bioinformatic studies revealed that transcription factor Sp1 activates heparanase promoter by high glucose and may play a role in regulation of perlecan and syndecan promoters.<p> Insulin or heparin inhibited the reduction in EC GAGs and syndecan mRNA and induction in heparanase by high glucose, indicating their protective effect. Decreased GAGs by insulin may relate to the pathology of hyperinsulinemia. Transcriptional regulation by heparin and/or insulin may cause variation in gene expression of heparanase, syndecan and perlecan.

Inflammation is an integral part of the body defense mechanism that occurs in vascularized tissue in response to harmful stimuli that is perceived as being a threat to tissue homeostasis. It is a complex physiological host response that is designed to neutralize and eliminate harmful agents, initiate tissue healing, and orchestrate a return to tissue homeostasis. While inflammation is designed to be an acute event that resolves following the elimination of harmful stimuli and tissue healing, there are instances where inflammation fails to resolve and instead evolves into chronic inflammation. It is now well understood that ongoing inflammation can serve as the underlying cause of many chronic inflammatory diseases, including atherosclerosis. In fact, one of the most pressing issues that is currently faced in the field of inflammation research, one that has also become the focus of numerous ongoing investigations, is how to turn this excessive, unwarranted and undesirable inflammation response off. Once thought to be a passive and simple process, resolution is now understood to be an active and complex process that is orchestrated by various inflammatory mediators, signaling pathways and biophysical processes. The discovery of novel biosynthetic pathways that turn on the pro-resolution signals has lead to a surge in research aimed at taking a closer look at processes that can stimulate the resolution of inflammation. While major advances in the field have resulted in a better understanding of the proactive nature of resolution, many of the mechanisms involved are still unknown. To date, the repertoire of chemokine receptors that participate in macrophage clearance during resolution, for the most part, remain unidentified. Overall, there is a growing appreciation that the discovery of mechanisms involved in the resolution responses can lead to the development of novel therapeutic approaches to resolve many chronic inflammatory diseases. Syndecan-1 (Sdc-1), a member of a family of cell surface proteoglycans, has been previously shown to regulate events relevant to tissue repair and chronic injury responses. Macrophage Sdc-1 expression during inflammation has been reported to be protective in various inflammatory models. Given these observations, we hypothesize that Sdc-1 expression on macrophages is a critical component of an anti-inflammatory, pro resolution program necessary for the successful resolution of inflammatory response. In this dissertation, we report the presence of a unique population of macrophages expressing Sdc-1 that are present within the vascular wall of mice undergoing atherosclerosis. Consistent with previous publications, the presence of Sdc-1 expressing macrophages was found to limit atherosclerosis progression. In addition, Sdc-1 expression on macrophages was associated with anti-inflammatory M2 polarization state and high intrinsic motility. Macrophage Sdc-1 expression was also linked with efferocytosis and enhanced macrophage egress from the site of inflammation to the draining lymphatic network. Moreover, we discovered that the chemokine receptor CXCR4, which was found on Sdc-1 expressing macrophages, was also involved in macrophage egress during inflammation resolution. In summary, while the overall mechanism regulating resolution processes is still unknown, our work has managed to identify two components that are involved in the process: macrophage Sdc-1 and CXCR4. Collectively, these results reinforce the physiological significance of macrophage efferocytosis and macrophage motility as endogenous modulators of the inflammatory response.

Η HARP (Heparin Affin Regulatory Peptide) είναι ένας αυξητικός παράγοντας ο οποίος εμφανίζει πλειάδα βιολογικών δράσεων εμπλεκόμενος στη διαφοροποίηση, τον πολλαπλασιασμό και τη μετανάστευση πολλών τύπων κυττάρων, καθώς και στην αγγειογένεση και την ανάπτυξη όγκων. Η HARP έχει χρονοειδικό και ιστοειδικό πρότυπο έκφρασης, υπερεκφράζεται όμως σε καρκινικές κυτταρικές σειρές, σε ανθρώπινους καρκινικούς όγκους και βρίσκεται σε υψηλή συγκέντρωση στον ορό του αίματος ασθενών με διάφορες μορφές καρκίνου. Η HARP ασκεί τις βιολογικές της δράσεις μετά από δέσμευση στους διαμεμβρανικούς υποδοχείς, SDC3, ALK και RPTPβ/ζ. Οι βιολογικές της δράσεις προσδιορίζονται από τη συνισταμένη των δράσεων που έχει κάθε υποδοχέας της, αντικατοπτρίζοντας τον περίπλοκο μηχανισμό δράσης της. Στη συγκεκριμένη εργασία μελετήσαμε τον τρόπο με τον οποίο η SDC3 διαμεσολαβεί τις βιολογικές δράσεις της HARP σε κύτταρα DU145 και PC3, κυτταρικές σειρές από καρκίνο ανθρώπινου προστάτη. Χρησιμοποιώντας την RNAi τεχνολογία, διαμολύναμε παροδικά τα κύτταρα με siRNA ειδικά σχεδιασμένο έναντι της SDC3, μειώνοντας τα επίπεδα έκφρασης της. Καλλιεργήσαμε κύτταρα, φυσιολογικά και μετασχηματισμένα, επιδράσαμε εξωγενώς με HARP και τα αποτελέσματα έδειξαν ότι και στις δύο καρκινικές κυτταρικές σειρές, η SDC3 είναι θετικός ρυθμιστής της επαγόμενης από τη HARP κυτταρικής προσκόλλησης και μετανάστευσης. Παράλληλα, μελετήσαμε την ενεργοποίηση μορίων που εμπλέκονται στο μονοπάτι μεταγωγής σήματος της SDC3 όπως της Src, Fak, Akt, Pten και Erk1/2. Τα αποτελέσματα έδειξαν ότι η ενεργοποίησή της SDC3 από τη HARP οδηγεί στην αύξηση των επιπέδων των pSrc, pFak, pAkt και p Erk1/2, ενώ, βρέθηκε ότι μειώνεται η φωσφορυλίωση της Pten. Συμπερασματικά, στην παρούσα εργασία μελετήθηκε η συμμετοχή της συνδεκάνης 3 στις βιολογικές δράσεις της HARP, καθώς και μόρια του σηματοδοτικού μονοπατιού μεταγωγής σήματος του υποδοχέα αυτού. Βρέθηκε ότι η HARP προσδενόμενη στον υποδοχέα αυτό, επάγει την προσκόλληση και μετανάστευση των κυττάρων, δράσεις που συνδέονται με την ανάπτυξη όγκων και τη μετάσταση καρκινικών κυττάρων. / HARP (Heparin Affin Regulatory Peptide), also known as Pleiotrophin, is a growth factor involved in several biological actions such as induction of cellular proliferation, migration and angiogenesis. Elevated concentrations of this growth factor are found in many tumours, as well as in the plasma of patients with different types of cancer. HARP exerts its actions after binding to the transmembrane receptors RPTP β/ζ, ALK and N-Syndecan (SDC3). In the present work, we studied the role of the transmembrane receptor SDC3 in the biological actions of HARP. We used DU145 and PC3 transiently transfected with specific siRNA to downregulate the accumulation of SDC3. Our results show that HARP binds to SDC3 and induces the cell adhesion and migration of DU145 and PC3 cells. We also studied the signal transduction through SDC3 receptor and the activation of signaling molecules such as Src, Fak, Akt, Pten and Erk 1/2. Our results revealed that HARP induces the phosphorylation of Src kinase, Fak and Erk1/2 after binding to SDC3 in both DU145 and PC3 cells. Also, HARP increases Akt signaling cascade in PC3 cells, while it suppresses the signaling cascade induced by PTEN in DU145 cells. Consequently, HARP interaction with SDC3, results in the activation of SDC3, which in turn triggers a signal transduction pathway that leads to specific biological cell responses activates other cytoplasmic effectors. Therefore, there starts a signaling cascade that targets specific genes and cell response. In conclusion, our results indicate that SDC3 contributes, as a positive regulator, to HARP-dependent cell adhesion and migration in both DU145 and PC3 cells.

Orientador: Rebeca de Souza Azevedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T01:17:16Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_M.pdf: 2889311 bytes, checksum: 3530f20f2b5d10e200ba02ec5d6d58b2 (MD5) Previous issue date: 2012 / Resumo: O ameloblastoma (AME) é um dos tumores odontogênicos (TO) mais comuns e, apesar de ser uma lesão benigna, pode apresentar comportamento clínico agressivo, localmente destrutivo e invasivo, enquanto o tumor odontogênico cístico calcificante (TOCC) é um TO benigno cístico que apresenta similaridades microscópicas com o AME, mas, geralmente, exibe comportamento clínico de menor agressividade. A via de sinalização Wnt/ß-catenina está envolvida no desenvolvimento e progressão tumoral de neoplasias benignas e malignas, e a expressão alterada de suas proteínas já foi identificada em alguns TO, provavelmente contribuindo para suas biologias tumorais. Dessa forma, o objetivo deste estudo foi avaliar e comparar a expressão imunoistoquímica de marcadores associados a via de sinalização Wnt/ß-catenina - Wnt1, Wnt5a, ß--catenina e syndecan-1(SDC-1) - em 17 casos de AME sólido e de 6 casos de TOCC. Os resultados revelaram que nos AME, a maioria dos casos foram focalmente positivos para Wnt1 e Wnt5a, a ß--catenina foi positiva no citoplasma isoladamente ou em associação a positividade de membrana, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 40% dos casos. Nos casos de TOCC, a positividade para Wnt1 e Wnt5a foi de 100% e incluiu também as células fantasmas. A ß--catenina foi positiva no citoplasma e no núcleo das células epiteliais da maioria dos casos, e SDC-1 exibiu positividade epitelial em 100% dos casos e positividade estromal em 16,7% dos casos. Em conclusão, este estudo evidenciou que a expressão de Wnt1 e Wnt5a é mais proeminente nos casos de TOCC, a expressão de ß--catenina em AME é citoplasmática e em TOCC é nuclear, e que a expressão estromal de SDC-1 é mais proeminente nos casos de AME, reforçando, assim, o papel de Wnt1 e Wnt5 no desenvolvimento do TOCC, de ß--catenina no desenvolvimento do TOCC e do AME, e de SDC-1 estromal no fenótipo invasivo do AME. Além disso, a expressão de Wnt1 e de Wnt5a nas células fantasmas do TOCC pode contribuir para esta histogênese. Por conseguinte, a via de sinalização Wnt/ß--catenina parece contribuir para o desenvolvimento das lesões de AME sólido e de TOCC / Abstract: Ameloblastoma (SMA) is one of the most common odontogenic tumors (OT), and although benign, it may have aggressive clinical behavior, be locally destructive and invasive, while calcifying cystic odontogenic tumor (CCOT) is a benign cystic OT that presents microscopic similarities with AME, but, in general, it has a less aggressive clinical behavior. Wnt/ß--catenin signaling pathway is involved in benign and malignant tumor development and progression, and changes in expression of its proteins have been identified in some OT, probably contributing to their tumoral biologies. Thus, the aim of this study was to evaluate and compare the immunohistochemical expression of markers associated with Wnt/ß--catenin signaling pathway - Wnt1, Wnt5a, ß--catenin and syndecan-1 (SDC-1) - in 17 cases of solid SMA and 6 cases of CCOT. The results showed that in AME, most fo the cases were focally positive for Wnt1 and Wnt5a, ß--catenin was positive in the cytoplasm only or in association with membrane positivity, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 40% of the cases. In CCOT cases, Wnt1 and Wnt5a was of 100% and also included ghost cells. ß--catenin was positive in the cytoplasm and in the nucleus of epithelial cells in most of the cases, and SDC-1 had epithelial positivity in 100% of the cases and stromal positivity in 16.7% of the cases. In conclusion, this study highlighted that Wnt1 and Wnt5a expression was most prominent in CCOT, ß--catenin expression was cytoplasmatic in AME and nuclear in CCOT, and that stromal expression of SDC-1 was most prominent in AME, so, reinforcing Wnt1 and Wnt5 role in CCOT development, ß--catenin role in CCOT and AME development, and stromal SDC-1 role in the AME invasive phenotype. In addition, Wnt1 and Wnt5a expression in ghost cells of CCOT may contribute to this histogenesis. Therefore, Wnt/ß--catenin signaling pathway seems to contribute to solid AME and CCOT development / Mestrado / Estomatologia / Mestre em Estomatopatologia

BackgroundAll cells carry a transmembrane proteoglycan calledsyndecan. Syndecans influence many functions like cell migration, cell adhesionand cell proliferation and it is involved in cellular signaling andtumourigenesis. The common features of differentiation in twomesenchymal tumor cell types, malignant mesothelioma cells and fibrosarcoma cells,are connected to the synthesis of syndecans. By studying the overexpression ofsyndecan-1 we hope to discover new features of the syndecan-1 molecule that wecan add to the puzzle of mesenchymal tumors. Methods and findingsMalignant mesothelioma cells and fibrosarcoma cellswere cultured and transfected with full-length- and truncated syndecan-1 constructs.To detect the expression of syndecan-1 on RNA level Rt-Q-PCR was conductedfollowed by immunocytochemical analysis to establish the syndecan-1 expressionon protein level. The result showed a 2-7 fold increase of syndecan-1 in thetransfectants comparing to the control. The proliferation of transfectants was analyzedby cell proliferation assay and cell cycle analysis. All transfectants showed alower proliferation rate comparing to the controls and a slight increase inG0/G1 phase. Because of the high structural similarities ofsyndecan family members, I studied how overexpression of syndecan-1 affected theother syndecans using Rt-Q-PCR. Syndecan-2 and -4 were downregulated in thetransfectants carrying syndecan-1 ectodomain, whereas the truncated versionshad the opposite effect. The expression of syndecan-bound heparan sulfate wasstudied by FACS and indicated an upregulation for heparan sulfate whenmeasuring internal- and membrane bound syndecans simultanesly. ConclusionsIn this study I haveshown that overexpression of full-length syndecan-1 and the different truncatedvariants, had similar profound effects on mesenchymal cell proliferation. Syndecan-1also influences the other members of the syndecan family suggesting a complexregulation.

Le cancer du sein est le cancer le plus fréquent chez la femme. Sa croissance et sa progression sont dépendantes de signaux hormonaux, tels que les œstrogènes, et de l’interaction des cellules cancéreuses avec le microenvironnement matriciel. La perte d’expression du récepteur aux estrogènes ERα est un marqueur de mauvais pronostic et de non réponse à l’hormonothérapie. Dans ces tumeurs agressives, l’expression du SDC-1, un protéoglycane transmembranaire impliqué dans l’angiogenèse, la prolifération et l’invasion, est augmentée suggérant un antagonisme entre la signalisation œstrogénique et le SDC-1 dans ces tumeurs. En utilisant les cellules de carcinome mammaire ER(+) MCF7, nous avons montré que les œstrogènes via le récepteur ERα sont capables d’inhiber l’expression du SDC 1 par un mécanisme nécessitant une néosynthèse protéique et la phosphorylation d’ERα par la kinase IKK. Parallèlement, nos résultats montrent que la dérégulation de l’expression du SDC-1 affecte la réponse proliférative des œstrogènes dans les cellules MCF7 en modifiant la localisation subcellulaire d’ERα. Nos résultats montrent à la fois l’inhibition tonique et E2-induite de l’expression du SDC-1 par ERα dans les cellules MCF7 et la potentialité du SDC-1 à réduire la réponse œstrogénique de ces cellules. Ainsi, ces résultats sont autant d’arguments qui renforcent l'hypothèse d'un antagonisme entre la signalisation médiée par ERα et le SDC-1 lequel influencerait l'orientation phénotypique des cellules de carcinome mammaire. / Breast cancer is the most common cancer in women. Its growth and progression depend on hormone signals, such as estrogens, and on interactions with the matrix microenvironment. The loss of the estrogen receptor ERα expression is a poor prognosis marker and predicts a resistance to antihormonal therapies. In these aggressive tumors, SDC-1 expression, a transmembrane proteoglycan involved in angiogenesis, proliferation and invasiveness is increased suggesting an antagonism between estrogenic signaling and SDC-1 in breast tumors. Using ER (+) MCF7 mammary carcinoma cells, we have shown that estrogens via the ERα are able to inhibit the expression of SDC-1 by a mechanism requiring protein synthesis and phosphorylation of ERα by the IKK kinase. Additionally, our results show that dysregulation of SDC-1 expression affects the proliferative response to estrogens in MCF7 cells by altering the subcellular localization of ERα. Our results show both a tonic and E2-induced inhibition of SDC-1 expression by ERα in MCF7 cells and the potentiality of SDC-1 to reduce the estrogenic response of these cells. Thus, these results support the hypothesis that an antagonism between ERα signaling and SDC-1 which could influence the phenotypic orientation of mammary carcinoma cells.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19664号 / 薬科博第52号 / 新制||薬科||6(附属図書館) / 32700 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 二木 史朗, 教授 松﨑 勝巳, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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