Background: Different types of cardiomyopathies are associated with variable hypertrophic response. A number of growth factors are thought to play a role in pathologic cardiac remodeling. Aims: We compared the modulation of the TGF-ƓÒ superfamily and IGF-1 signaling pathways and their target genes, the cell cycle regulatory proteins in tachycardia-induced dilated cardiomyopathy, a model with no detectable hypertrophy and in ischemic cardiomyopathy, a model with a marked hypertrophic reaction. Methods: In the first study, endomyocardial biopsies were obtained weekly in 15 dogs, during the development of tachycardiomyopaty. Genes involved in the myostatin-TGF-ƓÒ-Activin-A/Smad signaling pathway, p21 and cyclin D were quantified and correlated to echocardiographic measures of hypertrophy. In the second study, myocardial tissue samples were obtained in 8 dogs with a healed myocardial infarction, in 8 dogs with heart failure induced by overpacing and in 7 healthy dogs. We measured gene expression of IGF-1, its receptor (IGF-1R) and cyclins A, B, D1, D2, D3 and E and correlated them to the level of hypertrophy. Results: Tachycardiomyopathy was characterized by chambers dilation with no identifiable hypertrophy. Ischemic cardiomyopathy was characterized by eccentric hypertrophy. In tachycardiomyopathy, Activin-A mRNA was 4-fold higher than at baseline. Smad7 was overexpressed in severe heart failure; p21, a direct target gene of the Smad pathway was upregulated 8-fold and cyclin D1 was down-regulated. In that model, IGF-1 was overexpressed but neither IGF-1R nor any of the cyclins studied. In ischemic cardiomyopathy, IGF-1, IGF-R, and cyclins B, D1, D3 and E gene expression were upregulated. In tachycardiomyopathy, Activin-A and p21 were inversely correlated to the thickness of the interventricular septum. In normal dogs and in the both models of cardiomyopathy, IGF-1R was correlated to the thickness of the interventricular septum and to cyclins. Conclusions: Taken together, these results agree with the notion that Activin-A, IGF and cyclins are involved in the modulation of hypertrophic response observed in cardiomyopathies.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Ventral body wall closure (VBW) defects are amongst the most common human congenital anomalies. They represent a wide and heterogeneous group of phenotypic defects that can present in isolation or as a component in a larger syndromic anomaly. In addition, the incidence of associated anomalies is high and reaches 75% of fetuses in some types of VBW closure defects. Nevertheless, the embryonic origin and the underlying cellular and molecular mechanisms between ventral closure defects and their associated congenital anomalies remain poorly characterised. This is in part due to the poor understanding of the physiological mechanisms that regulate the development of ventral organs and the lack of representative transgenic animal models allowing detailed in vivo analysis of defect formation. Transforming growth factor beta (TGF-ÃŽÂ2) signalling is essential for VBW closure and vascular and cardiac development. Yet, its mechanism of action and the responding cell(s) in the body wall remain largely unknown. In addition, in various cells TGF-B can induce the expression of Tagln, encoding for a cytoskeleton associated protein that enhances cell migration. No function has been ascribed to TAGLN in body wall development. I define here a role of TGF-B during a critical time window in embryonic development to fashion the ventral body wall, anterior diaphragm and parts of the circulatory system. I identify a population of TAGLN+ myofibroblasts that respond to a temporally regulated TGF-B signalling originating from the epithelium of the primary body wall. Deletion of TGF-B receptor in TAGLN+ cells leads to failure of ventral body wall closure, anterior diaphragmatic hernia, cardiac and outflow tract anomaly. Nevertheless, the descending aorta and the large aortic branches are spared. By using advanced transgenic methodology, I generated novel transgenic mouse lines that enabled me to fate map the cells that initiate the formation of important mesenchymal tissues. These studies revealed that the origin of aortic vascular smooth muscle cells can be traced back to a group of progenitor cells that reside in the wall of the dorsal aorta before the VBW closure. My studies provide intriguing evidence for spatially restricted role for TGF-B signalling in ascending but not descending aorta morphogenesis. I used a variety of techniques to characterise, analyse and quantify important mechanisms during mesenchymal and vascular development, their response to injury and repair. This thesis has been written in an alternative format, comprising the different areas which have been investigated. Collectively, the results presented here provide new insights into the role of migratory and mechanically stabilising cells in the development and maintenance of critical structures in the body and their common role in the development of concurrent congenital anomalies. A detailed understanding of the molecular signalling pathways and cells that drive VBW closure raises the hope that the related birth defects can in the future be treated by precise gene and cell therapies.

Die Therapie des häufigsten primären Hirntumors bei Erwachsenen, des Glioblastoms, gestaltet sich aufgrund der relativen Resistenz gegen Bestrahlung und Zytostatika schwierig und resultiert in einer äußerst schlechten Prognose. Ziel dieser Arbeit war die Identifikation von zellulären Faktoren, die bei einer Behandlung mit dem neuartigen onkolytischen Virus Ad5-Delo3RGD (ΔE1A-13S, ΔE19K, ΔE3, zusätzliches RGD-Motiv) eine Bedeutung hinsichtlich des Ansprechens auf die Therapie haben. Dazu erfolgte nach der Transduktion von Gliomzellen mit adenoviralen Vektoren, mit unterschiedlichem E1A-Status, eine Analyse der Modulation der globalen Genexpression. Die E1A-Deletante, Ad5-Ad312, war replikationsinkompetent in Gliomzellen und hatte infolgedessen nur einen marginalen Einfluss auf Genebene und keine biologischen Effekte auf Proteinebene. Das Wildtypvirus (Ad5-wt) mit intakter und das onkolytische Virus (Ad5-Delo3RGD) mit mutierter E1A-Region induzierten eine starke Modifikation der zellulären Genexpression in Gliomzellen. Die Transduktion der Gliomzellen mit Wildtypvirus bzw. onkolytischen Virus resultierte in einer bis zu 60 %-igen Hemmung der Sekretion des Angiogenesefaktors VEGF. Fernerhin konnte in Glioblastomzellen gezeigt werden, dass Ad5-wt und Ad5-Delo3RGD eine 50 bis 60 %-ige Inhibition der Sekretion von TGF-β2, sowie eine bis zu 65 %-ige Hemmung der Transkriptionsaktivität des TGF-β-Signalwegs induzierten. Damit reprimieren besagte Viren zelluläre Faktoren, deren Expression im Gliom mit einer schlechten Prognose korreliert ist. Aufgrund dieser Daten konnte für maligne Gliome ein Modell von Ad5-Delo3RGD-Therapie relevanten zellulären Faktoren entwickelt werden, welches den Zusammenhang zwischen TGF-β2, VEGF bzw. MIR-181 und der Virotherapie verdeutlicht. Mit Hilfe dieses Modells steigt das Verständnis um die antineoplastische Aktivität des onkolytischen Virus, und ermöglicht damit eine Verbesserung der Ad5-Delo3RGD-basierten Therapie. / Glioblastoma is the most common type of brain tumor among adults. The therapy is very difficult, due to its relative resistance to radiation and zytostatica, and often results in fatal prognosis. The main objective of this thesis was the identification of cellular factors associated with therapy response to adenoviral treatment using the newly developed oncolytic virus Ad5-Delo3RGD. Therefore, after viral transduction of glioma cells with adenoviral vectors, using E1A-wildtype and -mutant viruses, an analysis of the modulation of mRNA expression profiles was conducted. The E1A deletion mutant Ad5-Ad312 was replication-deficient in glioma cells and therefore had only marginal influence of host gene transcription level and no biological effect on protein level. Both, wildtype adenovirus type 5 (Ad5-wt), with intact E1A, and oncolytic virus (Ad5-Delo3RGD), with mutated E1A, induced strong modifications of gene expression profiles in glioma cells. The transduction of glioma cells with wildtype or oncolytic virus resulted in a 60 % reduced secretion of the angionesis factor Vascular Endothelial Growth Factor (VEGF). In addition it was shown that Ad5-wt and Ad5-Delo3RGD induced a reduced secretion of TGF-ß2 by 50 to 60 % and a repression of the SMAD2/SMAD3/SMAD4-specific transcription activity up to 65 %. Thus the viruses inhibit cellular factors, which expression is corelated to a weak prognosis. Based on this data a new model for malign glioma was developed, which illustrates the relationship between virotherapy and the cellular factors TGF-β2, VEGF and MIR-181, which are associated with treatment response to Ad5-Delo3RGD-based therapy. The model helps to understand the basic antineoplastic activity of the oncolytic virus and therefore should help to improve Ad5-Delo3RGD-based therapy.

Made available in DSpace on 2014-07-29T15:07:41Z (GMT). No. of bitstreams: 1 Dissertacao_Denise C Toledo.pdf: 1395118 bytes, checksum: 592dff1a4d2ff80089be7e8691c93893 (MD5) Previous issue date: 2012-03-08 / The canine gland has drawn interest for research due to its similarities with the human prostate and the great incidence of lesions. Moreover, the canine prostate shows high incidence of diseases. The main lesions that affect the prostate are prostatitis, benign prostatic hyperplasia (BPH), cysts and adenocarcinoma. Recently attention has been given to lesions considered premalignant such as prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy (PIA), both studied in the human gland and also found in the canine prostate. In order to evaluate the development of prostate cancer starting as premalignant lesions, some immunohistochemical markers are employed, such as tissue inhibitors of metalloproteinases (TIMP), which have a key role in regulating the catalytic action of metalloproteinases (MMP), and transforming growth factor-β (TGF-β), that induces angiogenesis and inhibits cell proliferation and is considered a mediator of prostate growth. The objective of this study was to investigate the expression of TIMP-1, TIMP-2 and TGF-β in canine normal prostate tissue and with proliferative lesions. For this, 150 adult canine prostates were obtained from postmortem examinations. After microscopic evaluation 54 glands, compatible with normal, epithelial BPH, stromal BPH, PIA, PIN and adenocarcinoma were selected and used to make tissue microarray block (Tissue Microarray - TMA). TMA slides were subjected to immunohistochemistry with anti-TIMP-1, anti-TIMP-2 and anti-TGF-β, to assess staining intensity of epithelial cells and stromal cells.Cytoplasmatic staining of canine prostate cells by TIMP-1, TIMP-2 and TGF-β was observed, with TIMP-1 and TIMP-2 being more expressed in premalignant and malignant lesions, while TGF-β was expressed mainly by normal tissue and BPH. Furthermore, there were differences in the expression between epithelial and stromal cells. / A próstata canina, além das similaridades com a próstata humana, apresenta grande incidência de afecções. As principais lesões que acometem a próstata são as prostatites, a hiperplasia prostática benigna (HPB), os cistos e o adenocarcinoma, sendo que, recentemente, se tem dado atenção às lesões consideradas pré-malignas, como a neoplasia intraepitelial prostática (PIN) e a atrofia inflamatória proliferativa (PIA), ambas estudadas na glândula humana, e também verificadas na próstata do cão. Para avaliar o desenvolvimento de neoplasias prostáticas a partir das lesões pré-malignas, alguns marcadores imunoistoquímicos são empregados, como os inibidores teciduais de metaloproteinases (TIMP), que apresentam importante função na regulação da ação catalítica das metaloproteinases (MMP), e o fator de crescimento transformador β (TGF-β), que induz a angiogênese e inibe a proliferação celular, sendo considerado um mediador do crescimento prostático. O objetivo deste estudo foi verificar a expressão de TIMP-1, TIMP-2 e TGF-β no tecido prostático canino normal e com lesões proliferativas. Para isso foram colhidas, em exames necroscópicos, 150 próstatas de cães adultos e idosos. O material foi avaliado histologicamente e selecionadas amostras de 54 próstatas com predominância de histomorfologia normal, HPB epitelial, HPB estromal, PIA, PIN e adenocarcinoma, que foram utilizadas para a confecção de um bloco de microarranjo tecidual (Tissue Microarray - TMA). As lâminas de TMA foram submetidas à imunoistoquímica com os anticorpos anti-TIMP-1, anti-TIMP-2 e anti-TGF-β, sendo avaliada a intensidade de marcação das células epiteliais e estromais. Verificou-se que há marcação citoplasmática das células prostáticas caninas para TIMP-1, TIMP-2 e TGF-β, sendo as proteínas TIMP-1 e TIMP-2 mais expressas nas lesões proliferativas pré-malignas e malignas, enquanto TGF-β foi expresso principalmente pelo tecido normal e com HPB epitelial e estromal. Ainda, houve diferença de marcação entre células epiteliais e estromais.

Bakgrund: Sjukdomar i cirkulationsorganen är den vanligaste dödsorsaken i Sverige följt av tumörsjukdomar. År 2019 stod tumörsjukdomarna för hela 27% av dödsfallen. Epithelial-mesenchymal transition (EMT) är en process då epitelceller de-deffinerentierar till celler med en mesenkymal fenotyp. Vid denna process mister cellerna sin polaritet och cell-cell-kontakt. Cellerna får även en ändrad morfologi, från en rektangulär form till mer utsträckta och oregelbundna former. Processen möjliggör att cellerna får migrerande och invasiva egenskaper. EMT behövs för att cancercellerna ska kunna sprida sig från den primära tumören och bilda metastaser/dottertumörer på andra ställen i kroppen. Det är välkänt att Transforming Growth Factor beta (TGF-b) kan framkalla EMT. TGF-b kan stimulera tumörutvecklingen på många olika sätt, där ett av dessa sätt är genom att stimulera EMT. Andra studier har visat att kalcitrol, som är den aktiva formen av vitamin D, kan hämma TGF-b förmåga att driva tumörutveckling.   Syfte: Syftet är att undersöka om vitamin D kan motverka TGF-b orsakat EMT.    Metod: Data hämtades från databasen PubMed. För att finna relevanta studier användes sökorden Vitamin D, TGF-beta, EMT, Cancer tillsammans med sökkriterier. Inklusionskriterna var vetenskapliga orginalartiklar, artiklar i full text samt skrivna på språket engelska. Exklusionskriterierna vara artiklar äldre än 10 år.   Resultat: När humana cancerceller behandlades med vitamin D resulterade det i minskad EMT. Förändrat EMT kunde påvisas genom minskad migration och invasion samt ökning av epitela egenskaper. Vitamin D minskade cellmigrationen på ett tids- och dosberoende sätt. In vitro-testerna indikerade på att i cellkultur av cancerceller verkar vitamin D genom att motverka EMT, både i närvaro och i frånvaro av TGF-b. När EMT-programmet däremot redan initerats m.h.a. TGF-b kan inte längre vitamin D motverka att cellerna genomgår EMT.   Slutsats: Intag av vitamin D före och under TGF-b-signalering har ett positivt resultat på EMT-programmet.

Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive and severe interstitial lung diseases (ILDs) for which there is no cure. IPF is characterized by an excessive accumulation of fibroblasts which secrete an abundance of extracellular proteins such as collagen. These processes lead to repetitive tissue scarring and fibrosis in the lung parenchyma. As a result, lungs become rigid limiting oxygen intake and gas exchange. Once diagnosed, IPF is fatal within 2-3 years. There is no known cause or proven treatment that significantly improves outcomes. Although the cause is unknown, the current model of IPF suggests that an overactive epithelial repair mechanism caused by genetic and epigenetic factors as well as environmental exposures is responsible for the chronic fibrosis and scarring characteristic of IPF. The transforming growth factor beta (TGF-B) signaling pathway has been implicated as a major contributor in activating this chronic fibrosis. An upstream activator of the TGF-B pathway, avB6, has been identified as a potential therapeutic target. My collaborators in Dr. David Baker's lab at the University of Washington have created a novel avB6 integrin inhibitor (BP2_disulf) whose efficacy in improving IPF outcomes has yet to be tested. In my study, I test the ability of BP2_disulf to combat IPF through the use of the standard IPF murine model and translatable end points like non-invasive uCT scans, pulmonary function tests, bronchoalveolar lavage fluid (BALF) profiles, and histology. With these methods, I demonstrate that intraperitoneal injection of BP2_disulf in bleomycin-injured mice has the ability to decrease rate of fibrotic progression and pulmonary function decline compared to mice treated with bleomycin alone. These results prove that BP2_disulf is a promising therapeutic not only for IPF but other ILDs as well. Further efficacy validation and investigation into an aerosolized delivery method will advance this drug to clinical trials and make it accessible to those in need.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19396号 / 医博第4047号 / 新制||医||1012(附属図書館) / 32421 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 野田 亮, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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