Keloids are a fibroproliferative disorder of unknown etiology developing in the skin after injury or spontaneously. The aim of this thesis is to gain deeper insight into the role of TGF-β and its signaling pathway proteins, SMADs, in the pathogenesis of keloids and describe the gene expression profile in different keloid sites in the search for potential target genes for future treatment. Further aim is to develop an instrument to describe the quality of life of patients with keloids. We find cultured keloid fibroblasts to express an increased level of TGF-β1 mRNA and a decreased level of TGF-β3 mRNA compared to control skin. Keloid derived fibroblasts exhibit significantly decreased mRNA levels of TGF-β receptor type II (TβRII) and the ratio of TβRI/TβRII mRNA expression is increased. This suggests that a certain expression pattern of TGF-β subtypes and receptors may be important in keloid pathogenesis. Analysis of keloid derived fibroblasts reveal decreased SMAD3 mRNA expression and decreased ratio of SMAD2/SMAD3 mRNA implicating a disturbed SMAD signaling. Keloid fibroblasts up-regulate SMAD4 protein after stimulation with TGF-β1 and display diminished levels of the inhibitory proteins SMAD6 and 7. This may contribute to unlimited and deregulated TGF-β signaling leading to increased extracellular matrix production (ECM). The gene expression pattern is described in fibroblasts from different keloid sites using microarrays covering the whole human genome. This study reveals 105 regulated genes (79 genes are up- and 26 down-regulated) resulting in a unique gene expression profile in different sites of keloids, where progression or regression of the keloid process took place. In cells from the central part of keloids with clinical signs of regression, an up-regulation of apoptosis inducing genes as ADAM12 and ECM degrading genes as MMP19 is found. These genes may contribute to regression of keloids and might be possible future target genes for treatment. Overexpression of apoptosis inhibitors as AVEN and down-regulation of angiogenesis inhibiting genes as PTX3 found at the active margin of keloids may be responsible for the invasive character of the keloid margin. We develop a disease specific questionnaire to measure the quality of life of patients with keloids. We find two scales, psychological and physical impairment, describing the dimensions of quality of life in patients with scars. These two scales are independent of each other and show a high test-retest reliability. Single items which clinically characterize the disease show correlations to these scales. The results of this study demonstrate for the first time a severe impairment of quality of life of patients suffering from keloids and hypertrophic scars. In conclusion the described alteration in TGF-β expression and its receptors, the disrupted SMAD signaling pathway and the unique gene expression patterns in different keloid sites provide new knowledge on ECM formation and degradation in keloids. Regulatory genes in ECM homeostasis may be future target genes for keloid prevention, regression and treatment. The disease specific quality of life instrument of patients with keloids and scars is a useful tool to estimate success in future therapeutic efforts over time.

Ulm, Univ. Diss., 2006.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18895号 / 医博第4006号 / 新制||医||1009(附属図書館) / 31846 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 武藤 学, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) coordinate tissue development and postnatal remodelling by regulating proliferation, differentiation and apoptosis. However, their role in wound healing remains unclear. To study this, transgenic mice overexpressing Smad1 (K14-caSmad1) or the BMP antagonist Noggin (K14-Noggin) were utilised, together with human and mouse ex vivo wound healing models and in vitro keratinocyte culture. In wild-type mice, transcripts for Bmpr-1A, Bmpr-II, Bmp ligands and Smad proteins were decreased following tissue injury, whilst Bmpr-1B expression was up-regulated. Furthermore, immunohistochemistry revealed a down-regulation of BMPR-1A in hair follicles adjacent to the wound in murine skin, whilst in murine and human wounds, BMPR-1B and phospho-Smad-1/5/8 expression was pronounced in the wound epithelial tongue. K14-caSmad1 mice displayed retarded wound healing, associated with reduced keratinocyte proliferation and increased apoptosis, whilst K14-Noggin mice exhibited accelerated wound healing. Furthermore, microarray analysis of K14-caSmad1 epidermis revealed decreased expression of distinct cytoskeletal and cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a versus controls. Human and mouse keratinocyte proliferation and migration were suppressed by BMP-4/7 both in vitro and ex vivo, whilst they were stimulated by Noggin. Additionally, K14-caSmad1 keratinocytes showed retarded migration compared to controls when studied in vitro. Furthermore, Bmpr-1B silencing accelerated migration and was associated with increased expression of Krt16, Krt17 and Myo5a versus controls. Thus, this study demonstrates that BMPs inhibit proliferation, migration and cytoskeletal re-organization in epidermal keratinocytes during wound healing, and raises a possibility that BMP antagonists may be used for the future management of chronic wounds.

No description available.

The emergence of pesticide-resistant mosquitoes and drug-resistant parasites in the last twenty years has made control of malaria more difficult. One novel strategy to better control malaria is the development and release of transgenic mosquitoes whose enhanced immunity prevents transmission of the parasite to the mammalian host. One candidate effector gene is Anopheles stephensi nitric oxide synthase (AsNOS), whose inducible expression and subsequent synthesis of nitric oxide (NO) limits Plasmodium development in A. stephensi. In mammals, one of the most potent physiological regulators of NOS gene expression and catalytic activity is transforming growth factor-β (TGF-β). Moreover, human TGF-β can activate Drosophila melanogaster Smads, the proteins responsible for TGF-β signal transduction. We have determined that following a bloodmeal, active human TGF-β1 (hTGF-β1) persists in the midgut of A. stephensi for up to 48 hours. My data demonstrate that the midgut epithelium recognizes hTGF-β1 as an immunomodulatory cytokine. Specifically, induction of AsNOS by hTGF-β1 occurs in the midgut within minutes of bloodfeeding. Moreover, hTGF-β1 limits development of the human malaria parasite Plasmodium falciparum in the midgut. In other experiments, provision of the AsNOS catalytic inhibitor L-NAME partially reverses the effect of hTGF-β1 on Plasmodium development. These results suggest that AsNOS is a target of hTGF-β1 signaling and additional effectors that impact parasite development may be regulated by hTGF-β1 as well. The fact that hTGF-β1 signals mosquito cells to limit malaria parasite development suggests that there is an endogenous TGF-β signaling network in place. An analysis of the A. gambiae genome database revealed the presence of six TGF-β ligands, including gene duplication in the 60A gene, the first evidence of ligand gene duplication outside of chordates. In addition to five receptors, three Smads were identified in the A. gambiae genome predicted to support TGF-β/Activin- and BMP-like signaling. Midgut epithelial cells and an immunocompetent A. stephensi cell line express all three Smads, confirming that a signaling pathway is in place to support signaling by divergent exogenous and endogenous TGF-β superfamily proteins. The results presented here provide the first evidence of immunological crosstalk between divergent free living hosts of a single parasite. Further, these results imply that the interface between mammals and the mosquitoes that feed on them provide a unique opportunity for circulating molecules in the blood, including TGF-β and other cytokines, to alter the mosquito immune response. / Master of Science

No intuito de avaliar o papel do receptor b2-adrenérgico no processo de regeneração muscular, os músculos tibialis anterior de camundongos knockout para o adrenoceptor b2 (b2KO) foram criolesados e analisados após 1, 3, 10 e 21 dias. Análises de aspectos morfológicos e contráteis, atuação de macrófagos M1 e M2, conteúdo de AMPc e ativação de elementos da via de sinalização TGF-b/smad foram realizadas. Os músculos em regeneração dos animais b2KO apresentaram redução do calibre das fibras musculares, redução na função contrátil em 10 dias após criolesão, atenuado aumento do conteúdo de AMPc nos músculos em 10 dias após criolesão, aumento da inflamação e do número de macrófagos nos músculos em regeneração em 3 e 10 dias após lesão, predominância de macrófagos M1, diminuição da ativação de TbR-I e smad2/3 e da expressão de smad4 em 3 dias após lesão, e aumento na expressão de akirina1 em 10 dias após lesão. Nossos resultados sugerem que o adrenoceptor b2 contribui para a regulação das fases iniciais da regeneração muscular. / In this study, we investigated the role of the b2-adrenoceptor in skeletal muscle regeneration. Tibialis anterior muscles from b2-adrenoceptor knockout (b2KO) mice were cryolesioned and analysed after 1, 3, 10, and 21 days. Analysis of structural and contractile aspects, M1 and M2 macrophage profile, cAMP content, and activation of TGF-b/smad signalling elements. Regenerating muscles from b2KO mice showed diminished calibre of regenerating myofibres, decreased muscle contractile function at 10 days when compared with those from wild-type, attenuated augment in cAMP content in muscles at 10 days post-injury, increase in inflammatory process and in the number of macrophages at 3 and 10 days, prevalence of M1 macrophage phenotype, reduction in TbR-I and smad2/3 activation, and in the smad4 expression at 3 days, and an increase in akirin1 expression at 10 days in muscles from b2KO mice when compared to those from wild-type. Our data suggest that the b2-adrenoceptor contributes to the control of the initial stages of muscle regeneration.

In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.

No intuito de avaliar o papel do receptor b2-adrenérgico no processo de regeneração muscular, os músculos tibialis anterior de camundongos knockout para o adrenoceptor b2 (b2KO) foram criolesados e analisados após 1, 3, 10 e 21 dias. Análises de aspectos morfológicos e contráteis, atuação de macrófagos M1 e M2, conteúdo de AMPc e ativação de elementos da via de sinalização TGF-b/smad foram realizadas. Os músculos em regeneração dos animais b2KO apresentaram redução do calibre das fibras musculares, redução na função contrátil em 10 dias após criolesão, atenuado aumento do conteúdo de AMPc nos músculos em 10 dias após criolesão, aumento da inflamação e do número de macrófagos nos músculos em regeneração em 3 e 10 dias após lesão, predominância de macrófagos M1, diminuição da ativação de TbR-I e smad2/3 e da expressão de smad4 em 3 dias após lesão, e aumento na expressão de akirina1 em 10 dias após lesão. Nossos resultados sugerem que o adrenoceptor b2 contribui para a regulação das fases iniciais da regeneração muscular. / In this study, we investigated the role of the b2-adrenoceptor in skeletal muscle regeneration. Tibialis anterior muscles from b2-adrenoceptor knockout (b2KO) mice were cryolesioned and analysed after 1, 3, 10, and 21 days. Analysis of structural and contractile aspects, M1 and M2 macrophage profile, cAMP content, and activation of TGF-b/smad signalling elements. Regenerating muscles from b2KO mice showed diminished calibre of regenerating myofibres, decreased muscle contractile function at 10 days when compared with those from wild-type, attenuated augment in cAMP content in muscles at 10 days post-injury, increase in inflammatory process and in the number of macrophages at 3 and 10 days, prevalence of M1 macrophage phenotype, reduction in TbR-I and smad2/3 activation, and in the smad4 expression at 3 days, and an increase in akirin1 expression at 10 days in muscles from b2KO mice when compared to those from wild-type. Our data suggest that the b2-adrenoceptor contributes to the control of the initial stages of muscle regeneration.

Orientador: Ricardo Della Coletta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T17:48:45Z (GMT). No. of bitstreams: 1 Bersaneti_JoseliAssem_D.pdf: 811147 bytes, checksum: f8bb5e097207f57ec74bd4e214234d0b (MD5) Previous issue date: 2011 / Resumo: O aumento gengival induzido por ciclosporina A (CsA) é causado por um acúmulo excessivo de matriz extracelular, resultado da ação do fator de crescimento transformante-beta 1 (TGF-ß1) sobre a atividade dos fibroblastos gengivais. O presente estudo investigou in vitro os efeitos da superexpressão de Smad7, um inibidor da sinalização de TGF-ß1, nos eventos biológicos associados ao acúmulo de matriz extracelular induzido por CsA. Para verificar os efeitos inibitórios de Smad7, nós superexpressamos Smad7 em fibroblastos de gengiva normal (GN) de uma maneira estável. Células superexpressando Smad7 e células controle foram incubadas com CsA e a síntese de colágeno tipo I, produção e atividade de metaloproteinase de matriz 2 (MMP-2) e proliferação celular foram analisadas por ELISA, zimografia, curva de crescimento, método de incorporação de bromodeoxiuridina (BrdU) e análise do ciclo celular. Os efeitos de CsA na viabilidade celular e apoptose dos fibroblastos de GN também foram avaliados. Análises de western blot e imunofluorescência para fosforo-Smad2 foram realizados para verificar a ativação da cascata de sinalização de TGF-ß1. Apesar do tratamento com CsA estimular a produção de TGF-ß1 tanto nas células controle quanto nos fibroblastos superexpressando Smad7, a via de sinalização de TGF-ß1 foi marcantemente inibida nas células superexpressando Smad7, como revelado pelos níveis reduzidos de fosforilação de Smad2. Nas células superexpressando Smad7, os efeitos de CsA na proliferação, síntese de colageno tipo I e produção e atividade de MMP-2 foram significativamente bloqueados. A superexpressão de Smad7 bloqueou a proliferação dos fibroblastos gengivais induzida por CsA via regulação de p27. CsA e a superexpressão de Smad7 não induziram morte celular. Os resultados deste estudo confirmaram que a expressão de TGF-ß1 é correlacionada com os eventos moleculares associados ao aumento gengival induzido por CsA, e sugerem que a superexpressão de Smad7 é efetiva no bloqueio de tais eventos, incluindo proliferação, síntese de colágeno tipo I e atividade de MMP-2 / Abstract: Cyclosporin A (CsA)-induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor-beta1 (TGF-ß1). Herein the in vitro investigation of effects of overexpression of Smad7, a TGF-ß1 signaling inhibitor, in the events associated with CsA-induced extracellular matrix accumulation was performed. To assess the inhibitory effects of Smad7, we stably overexpressed Smad7 in fibroblasts from normal gingiva (NG). Smad7 overexpressing cells and controls were incubated with CsA and type I collagen synthesis, matrix metalloproteinase 2 (MMP-2) production and activity, and cellular proliferation were evaluated by ELISA, zymography, growth curve, BrdUincorporation assay and cell cycle analysis. CsA effects on cell viability and apoptosis of NG fibroblasts were also evaluated. Western blot and immunofluorescence for phospho-Smad2 were performed to measure the activation of TGF-ß1 signaling. Although the treatment with CsA stimulated TGF-ß1 production in both control and Smad7 overexpressing fibroblasts, its signaling was marked inhibited in Smad7 overexpressing cells as revealed by low levels of Smad2 phosphorylation. In Smad7 overexpressed cells, the effects of CsA on proliferation, synthesis of type I collagen and MMP-2 production and activity were significantly blocked. Smad7 overexpression blocked CsA-induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. The data presented here confirm that TGF-ß1 expression is related to the molecular events associated with CsA-induced gingival overgrowth, and suggest that Smad7 overexpression is effective in the blockage of those events, including proliferation, type I collagen synthesis and MMP-2 activity / Doutorado / Estomatologia / Doutor em Estomatopatologia