A esclerodermia sistêmica (ES) é caracterizada por fibrose da pele e órgãos internos, ativação imunológica e vasculopatia. Os modelos animais de ES sofrem com a falta de uma prova definitiva para o envolvimento vascular observado na doença humana. Um novo modelo induzido por colágeno do tipo V (COLV) reproduz muitas características da ES como fibrose e fenômenos imunológicos. No entanto, o estudo da vasculopatia ainda não foi abordado. O objetivo desse estudo foi investigar as alterações estruturais e funcionais das células endoteliais das artérias pulmonares do modelo de ES induzido pelo COLV, assim como determinar a doença pulmonar com ênfase especial no depósito de colágeno e síntese de RNAm de colágenos. Coelhos fêmeas Nova Zelândia (n = 8) foram imunizados com COLV humano emulsificado em adjuvante de Freund ou apenas com adjuvante de Freund (controle; n = 8). Após 7, 75 e 210 dias, os animais foram sacrificados e os pulmões foram analisados por microscopia eletrônica, hematoxilina e eosina, e marcações especiais incluindo imunomarcação para neovascularização (CD31), apoptose de células endoteliais (induzida pela caspase-3) e atividade endotelial (endotelina-1 e VEGF). A resposta vascular a acetilcolina (Ach) foi estudada em anéis de artéria pulmonar isolada. Para determinar o conteúdo de colágeno, expressão RNAm dos colágenos I, III e V e quantidade de colágeno (hidroxiprolina aminoácido específico), os pulmões foram submetidos a imunofluorescência, PCR em tempo real e análise bioquímica. Foi observado que os coelhos imunizados com COLV apresentaram um aumento progressivo de apoptose e atividade das células endoteliais a partir de 7 dias quando comparados aos coelhos controle (p 0,01). Em contrapartida, apenas aos 210 dias os coelhos imunizados com COLV apresentaram aumento significativo na neovascularização quando comparados com o grupo controle (p < 0,001). Coincidente com esses achados, a microscopia eletrônica mostrou alterações das células endoteliais caracterizada por apoptose, alterações degenerativas das organelas e tumefação citoplasmática. As células endoteliais pareciam estar destacadas da membrana basal. Os coelhos imunizados com COLV de 210 dias necessitaram de uma dose maior de Ach do que os controles para alcançar o relaxamento máximo de 50% (EC50) dos anéis de artéria pulmonar (p = 0,02). Por fim, foi observado que os coelhos de 210 dias imunizados com COLV apresentaram uma intensa expressão de COLV no interstício broncovascular, acompanhado de um aumento na expressão do RNAm (p < 0,001) quando comparado ao grupo controle. Os coelhos imunizados com COLV apresentaram uma maior quantidade de conteúdo de colágeno, quando comparados com os controles, nos diferentes tempos estudados (p < 0,01). Este estudo fornece evidência para a disfunção pulmonar do endotélio vascular em coelhos com ES induzida pelo COLV. As alterações observadas, nesse estudo, reforçam o papel do endotélio como evento patogênico primário na ES. Desta forma, o modelo ES induzido pelo COLV pode fornecer informações importantes sobre a patogênese da doença humana / The hallmarks of systemic sclerosis (SSc) are fibrosis of skin and internal organs, immune activity and vasculopathy. Animal models of SSc suffer from lack of a definitive evidence for vascular involvement observed in human disease. A novel model induced by collagen type V (COLV) reproduces many features of SSc, however vascular study has not been addressed. The aim of the present study was therefore evaluate pulmonary arteries for structural and functional alterations in endothelial cells in the COLV-induced SSc model, as well as determine the lung disease with special emphasis on collagen deposition and mRNA collagen synthesis. Female New Zealand rabbits (n = 8) were immunized with human COLV/Freund´s adjuvant or Freund´s adjuvant alone (control; n = 8). After 7, 75 and 210 days, the animals were sacrificed and the lungs were examined by electron microscopy, hematoxylin&eosin and special stains including immunostaining for neovascularization (CD31), endothelial cells apoptosis (caspase-3 induced) and endothelial activity (endothelin-1 and VEGF). Vascular response to acetylcholine (Ach) on isolated pulmonary artery rings was evaluated. To determine collagen content, mRNA expressions of COL I, III and V, and the quantity of collagen-specific amino acid hydroxyproline, the lungs were submitted to immunofluorescence, real-time qPCR and biochemical examination. The COLV rabbits demonstrated an endothelial cells apoptosis and activity compared to control rabbits starting at day-7 (p 0.01). A significant increase in neovascularization was observed only in the COLV-rabbits at day-210 (p < 0,001). Coincident with these findings, the electron microscopy revealed extensive endothelial cells abnormalities characterized by apoptosis, degenerative organelle changes and cytoplasmic tumefaction. Endothelial cells appeared to be detached from the basement membrane. Ach dose required to achieve 50% maximum relaxation (EC50) of pulmonary artery rings was increased in COLV rabbits at day-210 (p = 0.02). The content of hydroxyproline was increased in COLV rabbits compared with that observed in control rabbits, at the different days studied (p < 0,01). Ultimately, the COLV rabbits at 210-day showed an intense expression of COLV in the bronchovascular interstitium, followed by a markedly up-regulation of COLV mRNA (p < 0.001) as compared to controls. This study provides evidence for pulmonary vascular endothelial cell dysfunction in rabbits with COLV-induced SSc. The findings of this study reinforce the role of endothelial cells as a primary pathogenic event in SSc. The COLV model may provide insight into the pathogenesis of human disease

Contexte : l'aldostérone (Aldo) est impliquée dans la rigidité artérielle et l'insuffisance cardiaque (IC), mais les mécanismes sous-jacents restent méconnus. La galectine-3 (Gal-3), une lectine se liant aux bêta-galactoside, joue un rôle important dans la fibrose et l'IC. Dans cette étude, nous avons recherché si la Gal-3 était impliquée dans la fibrose vasculaire induite par l'Aldo. Méthodes et résultats : Des cellules musculaires lisses vasculaires de rat (CMLVs) ont été stimulées avec de l'Aldo en combinaison avec des antagonistes du récepteur minéralocorticoïde (MR) ou des inhibiteurs de la Gal-3. L'Aldo régule l'expression de la Gal-3 via le MR dans les CMLVs. De plus, la surexpression de la Gal-3 augmente spécifiquement la synthèse de collagène de type I. Les inhibiteurs de la Gal-3 ou sa sous-expression (siRNA) bloquent la synthèse de collagène de type I induite par l'Aldo. Des rats ont été traités avec de l'Aldo + sel combiné avec du spironolactone ou de la pectine de citron modifiée (MCP) pendant 3 semaines. Les rats hypertensifs traités à l'Aldo ont présenté une hypertrophie vasculaire, une fibrose et une augmentation de l'expression aortique de Gal-3. Les traitements avec le spironolactone ou le MCP préviennent tous ces effets. Des souris sauvages (WT) et mutées pour la Gal-3 (KO) ont été traitées avec de l'Aldo pendant 6 heures. Le bolus d'Aldo augmente l'expression de la Gal-3 et du collagène de type I dans l'aorte des souris WT alors qu'aucun changement ne se produit dans les souris KO pour la Gal-3. Conclusions : Nos donnés indiquent que la Gal-3 est indispensable à la réponse fibrotique de l'Aldo dans les CMLVs in vitro et in vivo, suggérant un rôle clef pour la Gal-3 dans la fibrose vasculaire / Background. Aldosterone (Aldo) is involved in arterial stiffness and heart failure (HF), but the mechanisms have remained unclear. Galectin-3 (Gal-3), a beta-galactoside-binding lectin, plays an important role in fibrosis and HF. We here investigated whether Gal-3 is involved in Aldo-induced vascular fibrosis. Methods and Results. Rat vascular smooth muscle cells (VSMCs) were stimulated with Aldo combined with mineralocorticoid receptor (MR) antagonists and Gal-3 inhibitors. Aldo upregulated Gal-3 expression via MR in VSMCs. Moreover, Gal-3 over-expression specifically enhanced collagen type I synthesis. Gal-3 inhibitors or Gal-3 silencing (siRNA) blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin (MCP) for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, fibrosis and increased aortic Gal-3 expression. Spironolactone or MCP treatment reversed all the above effects. Wild type (WT) and Gal-3 knock-out (KO) mice were treated with Aldo for 6 hours. Aldo bolus increased aortic Gal-3 and collagen type I expression in WT mice whereas no changes occurred in Gal-3 KO mice. Conclusions. Our data indicate that Gal-3 is required for the fibrotic response to Aldo in VSMCs in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis

Le microenvironnement tumoral via les macromolécules matricielles est connu pour jouer un rôle clé dans la réponse des cellules cancéreuses à la chimiothérapie en favorisant leur survie et leur prolifération. L'impact du collagène de type I, protéine matricielle majeure du microenvironnement, a été évalué au niveau des capacités migratoires des cellules tumorales et de leur réponse aux agents anticancéreux, doxorubicine et metformine. Cette approche a étémenée chez des cellules humaines HT1080 hautement invasives au moyen de systèmes de culture par coating 2D ou en matrice 3D. Les effets de modifications post-traductionnelles du collagène comme la carbamylation et de la glycation ont été également étudiées. Les résultats montrent que le collagène 3D inhibe l'activité anti-migratoire de la doxorubicine. Cette protection met en jeuune préservation des niveaux d'activation de FAK et RhoA impliquées dans la formation des fibres de stress d'actine et des plaques d'adhésion focales. Le collagène glyqué 2D et dans une moindre mesure le carbamylé inhibent l'adhésion, la migration des cellules tumorales et désorganisent leur cytosquelette d'actine via des modifications de distribution de la vinculine, de FAK et des intégrines 1. Cet impact de la glycation a été aussi mis en évidence en matrice 3D après modification du processus de glycation. Enfin, la glycation exerce un effet protecteur vis-àvis des capacités anti-prolifératives et anti-migratoires de la doxorubicine et de la metformine. En conclusion, nous mettons en évidence une nouvelle forme de résistance CAM-DR dirigée contre l'activité anti-invasive de médicaments ; cet effet pouvant être généré par une protéine matricielle native ou modifiée lors de situations physiopathologiques associées au cancer. / The tumor microenvironment via the extracellular matrix plays an important role in cancer cell response to chemotherapy by promoting their survival and proliferation. In this work, we studied the impact of collagen type I, a major matrix protein of tumor microenvironment, on the migration capacities of tumor cells and on their response to anticancer drugs such as doxorubicin and metformin. This approach was performed with the highly invasive human cell line HT1080,by means of 2D coating or 3D matrix cell culture systems. The effects of collagen posttranslational modifications such as carbamylation and glycation were also assessed. The results show that the 3D collagen inhibits the anti-migratory effect of doxorubicin. This protection is carried out through the preservation of the activation states of FAK and RhoA, which are involved in the formation of actin stress fibers and focal adhesions. On 2D coating, the glycated collagen and at a lesser extent the carbamylated one decrease the adhesion, the migration oftumor cells and, disorganize the actin cytoskeleton via a modified distribution of vinculine, FAK and beta1 integrins. This impact is also demonstrated by using 3D matrices, after adaptation of the glycation process. In addition, we reported that the glycated collagen protects against the antiproliferative and the anti-migratory effects of doxorubicin and metformin. In conclusion, we highlighted a new form of CAM-DR resistance that targets the drugs anti-invasive activity. This impact could be induced by the native form of matrix proteins or the modified one found inpathological situations which are associated to cancer.

A esclerodermia sistêmica (ES) é caracterizada por fibrose da pele e órgãos internos, ativação imunológica e vasculopatia. Os modelos animais de ES sofrem com a falta de uma prova definitiva para o envolvimento vascular observado na doença humana. Um novo modelo induzido por colágeno do tipo V (COLV) reproduz muitas características da ES como fibrose e fenômenos imunológicos. No entanto, o estudo da vasculopatia ainda não foi abordado. O objetivo desse estudo foi investigar as alterações estruturais e funcionais das células endoteliais das artérias pulmonares do modelo de ES induzido pelo COLV, assim como determinar a doença pulmonar com ênfase especial no depósito de colágeno e síntese de RNAm de colágenos. Coelhos fêmeas Nova Zelândia (n = 8) foram imunizados com COLV humano emulsificado em adjuvante de Freund ou apenas com adjuvante de Freund (controle; n = 8). Após 7, 75 e 210 dias, os animais foram sacrificados e os pulmões foram analisados por microscopia eletrônica, hematoxilina e eosina, e marcações especiais incluindo imunomarcação para neovascularização (CD31), apoptose de células endoteliais (induzida pela caspase-3) e atividade endotelial (endotelina-1 e VEGF). A resposta vascular a acetilcolina (Ach) foi estudada em anéis de artéria pulmonar isolada. Para determinar o conteúdo de colágeno, expressão RNAm dos colágenos I, III e V e quantidade de colágeno (hidroxiprolina aminoácido específico), os pulmões foram submetidos a imunofluorescência, PCR em tempo real e análise bioquímica. Foi observado que os coelhos imunizados com COLV apresentaram um aumento progressivo de apoptose e atividade das células endoteliais a partir de 7 dias quando comparados aos coelhos controle (p 0,01). Em contrapartida, apenas aos 210 dias os coelhos imunizados com COLV apresentaram aumento significativo na neovascularização quando comparados com o grupo controle (p < 0,001). Coincidente com esses achados, a microscopia eletrônica mostrou alterações das células endoteliais caracterizada por apoptose, alterações degenerativas das organelas e tumefação citoplasmática. As células endoteliais pareciam estar destacadas da membrana basal. Os coelhos imunizados com COLV de 210 dias necessitaram de uma dose maior de Ach do que os controles para alcançar o relaxamento máximo de 50% (EC50) dos anéis de artéria pulmonar (p = 0,02). Por fim, foi observado que os coelhos de 210 dias imunizados com COLV apresentaram uma intensa expressão de COLV no interstício broncovascular, acompanhado de um aumento na expressão do RNAm (p < 0,001) quando comparado ao grupo controle. Os coelhos imunizados com COLV apresentaram uma maior quantidade de conteúdo de colágeno, quando comparados com os controles, nos diferentes tempos estudados (p < 0,01). Este estudo fornece evidência para a disfunção pulmonar do endotélio vascular em coelhos com ES induzida pelo COLV. As alterações observadas, nesse estudo, reforçam o papel do endotélio como evento patogênico primário na ES. Desta forma, o modelo ES induzido pelo COLV pode fornecer informações importantes sobre a patogênese da doença humana / The hallmarks of systemic sclerosis (SSc) are fibrosis of skin and internal organs, immune activity and vasculopathy. Animal models of SSc suffer from lack of a definitive evidence for vascular involvement observed in human disease. A novel model induced by collagen type V (COLV) reproduces many features of SSc, however vascular study has not been addressed. The aim of the present study was therefore evaluate pulmonary arteries for structural and functional alterations in endothelial cells in the COLV-induced SSc model, as well as determine the lung disease with special emphasis on collagen deposition and mRNA collagen synthesis. Female New Zealand rabbits (n = 8) were immunized with human COLV/Freund´s adjuvant or Freund´s adjuvant alone (control; n = 8). After 7, 75 and 210 days, the animals were sacrificed and the lungs were examined by electron microscopy, hematoxylin&eosin and special stains including immunostaining for neovascularization (CD31), endothelial cells apoptosis (caspase-3 induced) and endothelial activity (endothelin-1 and VEGF). Vascular response to acetylcholine (Ach) on isolated pulmonary artery rings was evaluated. To determine collagen content, mRNA expressions of COL I, III and V, and the quantity of collagen-specific amino acid hydroxyproline, the lungs were submitted to immunofluorescence, real-time qPCR and biochemical examination. The COLV rabbits demonstrated an endothelial cells apoptosis and activity compared to control rabbits starting at day-7 (p 0.01). A significant increase in neovascularization was observed only in the COLV-rabbits at day-210 (p < 0,001). Coincident with these findings, the electron microscopy revealed extensive endothelial cells abnormalities characterized by apoptosis, degenerative organelle changes and cytoplasmic tumefaction. Endothelial cells appeared to be detached from the basement membrane. Ach dose required to achieve 50% maximum relaxation (EC50) of pulmonary artery rings was increased in COLV rabbits at day-210 (p = 0.02). The content of hydroxyproline was increased in COLV rabbits compared with that observed in control rabbits, at the different days studied (p < 0,01). Ultimately, the COLV rabbits at 210-day showed an intense expression of COLV in the bronchovascular interstitium, followed by a markedly up-regulation of COLV mRNA (p < 0.001) as compared to controls. This study provides evidence for pulmonary vascular endothelial cell dysfunction in rabbits with COLV-induced SSc. The findings of this study reinforce the role of endothelial cells as a primary pathogenic event in SSc. The COLV model may provide insight into the pathogenesis of human disease

<p>Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.</p>

Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

Η σεργλυκίνη (SG) είναι μια πρωτεογλυκάνη που εκφράζεται και εκκρίνεται από το σύνολο σχεδόν των αιμοποιητικών κυττάρων, ενώ αποτελεί την κύρια πρωτεογλυκάνη η οποία εκκρίνεται από τις κυτταρικές σειρές πολλαπλού μυελώματος (ΠΜ). Έχει βρεθεί ότι η SG συμμετέχει στη ρύθμιση πληθώρας παραγόντων που εμπλέκονται σε αντιδράσεις φλεγμονής. Τα αποτελέσματά μας δείχνουν ότι η SG που εκκρίνεται από τα μυελωματικά κύτταρα έχει την ικανότητα να αναστέλλει τόσο την κλασσική όσο και τη λεκτινική οδό του συστήματος του συμπληρώματος, ενώ δε φαίνεται να έχει καμία επίδραση στο εναλλακτικό μονοπάτι. Επιπρόσθετα η SG δεν έχει την ικανότητα να προκαλεί την ενεργοποίηση κάποιου από τα τρία μονοπάτια του συμπληρώματος. Βρέθηκε ότι η ανασταλτική δράση της SG εκδηλώνεται μέσω της αλληλεπίδρασής της με τους παράγοντες C1q και MBL. Οι γλυκοζαμινογλυκανικές (GAGs) αλυσίδες της SG είναι υπεύθυνες για τη δέσμευση με την κολλαγονούχα ουρά του παράγοντα C1q, ενώ στη δέσμευση με την MBL πρωτεΐνη πέρα από τη συμμετοχή των GAG αλυσίδων απαιτείται και ο πρωτεϊνικός κορμός της SG. Επιπλέον βρέθηκε ότι αλυσίδες CS-E ελαττώνουν την ικανότητα δέσμευσης της SG με τα μόρια C1q και MBL. Οι αλληλεπιδράσεις της SG με τον C1q και την MBL βρέθηκε ότι είναι ιοντικού χαρακτήρα και σε αντίθεση με τη δέσμευση της SG με την MBL, που εξαρτάται από την παρουσία των ιόντων Ca2+, η αλληλεπίδραση της SG με τον C1q είναι ανεξάρτητη των ιόντων αυτών. Αν και τα επίπεδα της SG στον ορό των ασθενών με ΠΜ εμφανίζονται αυξημένα σε σχέση με τους φυσιολογικούς μάρτυρες, δεν εντοπίστηκαν στατιστικά σημαντικές διαφορές στην δραστικότητα της κλασσικής και της εναλλακτικής οδού του συμπληρώματος στους ασθενείς με ΠΜ και στους φυσιολογικούς δότες. Ωστόσο ενδιαφέρον αποτελεί το γεγονός ότι στους ασθενείς με ΠΜ τα υψηλά επίπεδα SG στον ορό εμφάνισαν τάση συσχέτισης με ελαττωμένη δραστικότητα της κλασσικής οδού του συμπληρώματος. Επιπρόσθετα, η SG που εκκρίνεται απ τα μυελωματικά πλασματοκύτταρα έχει την ικανότητα να προστατεύει τα κύτταρα αυτά από την επίδραση του συμπληρώματος, μετά την ενεργοποίησή του με τη χρήση φαρμάκων. Φαίνεται ότι και η SG της μεμβράνης των μυελωματικών κυττάρων παρουσιάζει προστατευτική δράση έναντι του συμπληρώματος, μιας και κύτταρα που δεν εκφράζουν SG στην επιφάνειά τους είναι δύο με τρείς φορές περισσότερο ευαίσθητα στη δράση του συμπληρώματος, σε σχέση με κύτταρα που την εκφράζουν. Προτείνουμε ότι τόσο η εκκρινόμενη όσο και η δεσμευμένη στην κυτταρική επιφάνεια SG προστατεύουν τα μυελωματικά πλασματοκύτταρα κατά την ανοσοθεραπεία και συμβάλουν στην επιβίωσή τους. Στην παρούσα μελέτη δείξαμε ότι η SG αλληλεπιδρά με το κολλαγόνο τύπου Ι, την πιο άφθονη μορφή κολλαγόνου στο οστό. Επιπλέον βρέθηκε ότι η SG της κυτταρικής επιφάνειας συμμετέχει στην προσκόλληση των μυελωματικών κυττάρων στο κολλαγόνο τύπου Ι. Η αλληλεπίδραση αυτή φαίνεται ότι επάγει την έκφραση από τα μυελωματικά πλασματοκύτταρα των ΜΜΡ-2 και ΜΜΡ-9. Επιπρόσθετα, υπολογίστηκαν τα επίπεδα των ΜΜΡ-2 και ΜΜΡ-9 στον ορό και στο μυελό ασθενών με ΠΜ. Βρέθηκε ότι στον ορό των ασθενών με ΠΜ τα επίπεδα των δύο ενζύμων εμφανίζονται ελαττωμένα σε σχέση με τα φυσιολογικά δείγματα, ενώ αντίθετα στον μυελό των ασθενών η ΜΜΡ-2 βρέθηκε σημαντικά αυξημένη σε σχέση με τους φυσιολογικούς δότες. Καμία μεταβολή δεν παρατηρήθηκε στα επίπεδα της ΜΜΡ-9. Τέλος τα επίπεδα της ΜΜΡ-2 του μυελού ασθενών με ΠΜ βρέθηκε ότι σχετίζονται τόσο με τα επίπεδα του ενζύμου στον ορό, όσο και με τον δείκτη οστικής απορρόφησης ΝΤx, υποδηλώνοντας τη συμμετοχή της στην παθοβιοχημεία της οστικής νόσου που εμφανίζεται στο ΠΜ. Αντιθέτως καμία συσχέτιση δεν βρέθηκε για την ΜΜΡ-9, δηλώνοντας ότι ίσως το ένζυμο αυτό να εμπλέκεται σε άλλες διεργασίες που επιτελούνται στο ΠΜ. / Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. It was also shown that SG could not initiate any activation of the complement system. The inhibitory effect of SG is due to direct interactions with C1q and mannose binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas binding to MBL requires the presence of SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SGbinding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. Although we found the serum levels of SG to be elevated in patients with MM compared to healthy controls, no statistical significant differences were observed for classical and alternative pathway activity in sera between MM patients and healthy donors. Despite that, it is proved that increase levels of SG show a tendency to correlate with decreased levels of classical pathway activity in serum of MM patients. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. It is also demonstrated that SG on the surface of MM cells inhibits complement deposition on the membrane of these cells. Cells which do not express SG on its’ surface are 2-3 times more sensitive to complement attack compared with cells expressing high levels of SG. This might protect myeloma cells during immunotherapy and promote survival of malignant plasma cells. Moreover, it is shown that SG from MM cells is capable to interact with collagen type I, the most abundant collagen in bone. Furthermore, SG is present on the surface of myeloma plasma cells and it is involved in the adhesion of MM cells to collagen type I in bone marrow microenvironment. In addition, it is shown that the interaction of myeloma plasma cells to collagen type I, mediated by cell surface SG, induces expression and secretion of both MMP-2 and MMP-9 from MM cells. Along the process, we have investigated levels of MMP-2 and MMP-9 in serum and marrow of patients with MM. Decreased levels of both MMP-2 and MMP- 9 in serum of MM patients have been observed compared to healthy donors. Instead, MMP-2 was found to be increased in bone marrow of MM patients compared to control samples, while no differences observed for MMP-9. Finally, marrow levels of MMP-2 seem to correlate with serum levels of the enzyme along with a marker of bone resorption, NTx. This might indicate the implication of MMP-2 in the pathogenesis of bone disease in MM. Even though, no correlation of MMP-9 with NTx was observed, proving that MMP-9 may play a significant role in different pathogenetic mechanism occurring in MM.

Made available in DSpace on 2014-06-11T19:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-04Bitstream added on 2014-06-13T20:02:12Z : No. of bitstreams: 1 paulo_ao_dr_arafo.pdf: 1640675 bytes, checksum: 92907baf8888322bc6931c68024509f2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did´t have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn’t enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It’s possible to conclude that APDC don’t increase bone formation if associate to HA/Col.

Resumo: O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / Abstract: The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did't have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn't enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It's possible to conclude that APDC don't increase bone formation if associate to HA/Col. / Orientador: Idomeo Bonetti Filho / Coorientador: José Mauro Granjeiro / Banca: Mário Tanomaru Filho / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Manoel Eduardo de Lima Machado / Banca: Arlindo Di Spagna Souza / Doutor