Este trabalho avaliou o efeito da dieta hiperlipídica em camundongos para o estudo da inflamação cardiovascular. Camundongos C57Bl/6 machos com 8 semanas de vida foram utilizados nos ensaios, sendo divididos nos grupos dieta controle e dieta hiperlipídica. Após 8 semanas foram avaliados: o ganho de peso, a porcentagem de tecido adiposo, pressão arterial sistólica, frequência cardíaca, perfil lipídico e glicêmico séricos. A partir de cortes histológicos de aortas e corações corados com picrossirius foram feitas análises morfométricas. Em cortes histológicos de aorta foram realizadas a análise fibras elásticas e colágenas usando a coloração de Weigert-Van Gieson. Também foram realizadas a quantificação de fibras colágenas em aortas, usando a coloração de picrossirius. Nos tecidos aórticos e cardíacos foram feitos: 1) Ensaios de atividade enzimática para ECA e MPO. 2) Ensaios de Immunoblotting para a detecção proteíca para ECA e TGF-b. Também foram feitos ensaios de imuno-histoquímica para marcação e localização de ECA e TGF-b no tecido aórtico. / This work evaluated the effect of high-fat diet in mice for the study of cardiovascular inflammation. C57BL / 6 mice at 8 weeks of age were used in the tests were divided in groups control diet and high fat diet. After 8 weeks were evaluated: weight gain, percentage of fat, systolic blood pressure, heart rate, serum lipids and glucose levels. From histological aortas and hearts stained with picrosirius morphometric analyzes. Histological sections of the aorta were performed to analyze elastic and collagen fibers using Weigert-Van Gieson staining. Also the quantification of collagen fibers in aortas using picrosirius staining. In aortic and cardiac tissues were made: 1) Enzymatic activity assays for ACE and MPO. 2) Immunoblotting assays to detect proteinous for ACE and TGF-b. Also were peformed Immunohistochemistry assays for marking and localization of ACE and TGF- b in the aortic tissue.

Blood vessels are dynamic structures whose properties are continuously adapted by resident vascular cells. Existing mechanobiological models tend to ignore regulatory signalling and cell population dynamics, both key determinants of arterial growth and remodelling (G&R). In this D.Phil., a combined theoretical, experimental and computational approach is used to formulate, refine and implement a novel model of the arterial wall that includes vascular mechanics, microstructure, biochemical metabolism and signalling, and cell phenotype and population dynamics. A mathematical chemo-mechano-biological (CMB) model is formulated by coupling a biomechanical model of the arterial wall as a cylindrical nonlinear elastic membrane to a system of biologically-informed evolution laws governing fibroblast cell-mediated, transforming growth factor (TGF)-β-regulated collagen metabolism. Model simulation of inflammatory aneurysm development suggests that increasing TGF-β levels promotes a cell-driven profibrotic response leading to aneurysm stabilisation, illustrating the model's ability to couple chemo-biological processes to tissue-level mechanical evolution. To inform the theoretical framework experimentally, a recent mouse model of post-developmental disruption of medial smooth muscle TGF-β signalling is for the first time subjected to hypertension, and characterised by biaxial mechanical testing and (immuno)histological staining. Increased adventitial TGF-β levels following perturbation are associated with strong profibrotic responses (increased cellularity, collagen deposition, thicker walls) altering tissue mechanics (lower biaxial stress, higher structural stiffness). Simulation of realistic arterial geometries is enabled by coupling the 1D CMB model to a three-dimensional structural solver. Heterogeneous spatial distributions of mechanical, microstructural and chemo-biological variables determining the evolution of complex saccular aneurysm geometries can be simulated with this 3D implementation. A novel chemo-mechano-biological model of vascular cell dynamics and regulatory signalling governing arterial G&R is formulated, informed by specifically-generated experimental data, and implemented in an advanced 3D computational framework. This will allow for virtual investigation of therapies acting on chemo-biological agents of arterial G&R, with potential benefits for vascular disease patients.

Introdução: Proteínas da Superfamília do fator transformador de crescimento β (TGF-β) estão implicadas na regulação de diversas funções biológicas. Embora alguns estudos revelaram a sua presença no endométrio ectópico de portadoras de adenomiose, a sua função na etiopatogenia da doença permanece pouco conhecida. Objetivo: o estudo visa comparar a expressão imuno-histoquímica de TGF-β1 no endométrio ectópico de portadoras de adenomiose com o endométrio tópico de pacientes sem essa condição. Método: Estudo de caso-controle utilizando imuno-histoquímica em amostras uterinas (blocos de parafina) do Hospital de Clínicas de Porto Alegre. A amostra contém 28 casos de adenomiose e 21 controles. Resultados: Não encontramos associação entre tabagismo e adenomiose (P = 0,75), abortos e adenomiose (P = 0,29), gestações e adenomiose (P = 0,85), curetagens e adenomiose (P = 0,81), dor pélvica e adenomiose (P = 0,72) e presença de mioma e adenomiose (P = 0,15). Além disso encontramos relação entre sangramento uterino anormal (SUA) e adenomiose (P = 0,02) e cesarianas prévias e adenomiose (P = 0,02) . A expressão imuno-histoquímica de TGF-β1 no endométrio ectópico de portadoras de adenomiose não teve diferença significativa quando comparado com a expressão dessa proteína no endométrio tópico de pacientes sem adenomiose (P = 0,86). Conclusão: Nosso estudo foi um dos primeiros a comparar a expressão de TGF-β1 no endométrio de pacientes com e sem adenomiose. Em nossa análise não obtivemos diferença significativa entre os grupos, resultado diferente do encontrado em outros dois estudos. Mais estudos são necessários para investigar o papel da superfamília TGF no desenvolvimento e manutenção da adenomiose. / Background: Proteins of transforming growth factor β superfamily (TGF-β) are implicated in the regulation of various biological functions. Although some studies have revealed their presence in ectopic endometrium of women with adenomyosis, their role in the pathogenesis of the disease remains largely unknown. Objective: The study aims to compare the immunohistochemical expression of TGF- β1 in ectopic endometrium of women with adenomyosis with the topic endometrium of patients without this condition. Methods: Casecontrol study using immunohistochemistry in uterine samples (paraffin blocks) obteined from Hospital de Clínicas de Porto Alegre. The sample contained 28 adenomyosis cases and 21 controls. Results: We found no significant difference between smoking and adenomyosis (P = 0.75), abortions and adenomyosis (P = 0.29), pregnancies and adenomyosis (P = 0.85), curettage and adenomyosis (P = 0.81), pelvic pain and adenomyosis (P = 0.72) and presence of myoma and adenomyosis (P = 0.15). We did find a relationship between adenomyosis and abnormal uterine bleeding (AUB) (P = 0.02) and previous cesarean section and adenomyosis (P = 0.02). Immunohistochemical expression of TGF-β1 in ectopic endometrium of women with adenomyosis did not have significant difference when compared with the expression of this protein in the topic endometrium of patients without adenomyosis (P = 0.86). Conclusion: Our study was one of the first to compare the TGF-β1 expression in the endometrium of patients with and without adenomyosis. In our analysis we have not had significant difference between the groups, unlike observed in two other studies. More studies are needed to investigate the role of TGF superfamily in the development and maintenance of adenomyosis.

Antagonism of the activin receptor signaling pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous research has shown that antagonism of activin signaling promotes muscle growth, attenuates muscle wasting, and restores function in both wild type and diseased animals. Our laboratory has recently developed a novel small molecule (SGI-1252) that inhibits activin downstream (i.e., Smad2/3 phosphorylation) signaling. Purpose: In this study we determined how eight weeks of orally administered SGI-1252 affected TGF-ß signaling, whole body mass, individual limb muscle mass, and muscle fiber cross sectional area (CSA). Methods: Wild-type (WT) mice were treated with SGI-1252 or a vehicle control (VC) via oral gavage (400 mg/kg 3 times per week) for 8 weeks. Body mass was measured twice per week during the 8-week treatment period. At the end of the treatment period, gastrocnemius and tibialis anterior (TA) muscles were excised, weighed, and prepared for histological and biochemical analyses. Results: Following 8 weeks of treatment, there was no difference in weight gain between SGI-1252 (24.8 ± 1.8g) and VC treated mice (23.2 ± 1.5g) (p = 0.06). Gastrocnemius whole muscle mass was significantly greater in the SGI-1252 treated group relative to the VC treated mice (139.6 ± 12.8 mg vs 128.8 ± 14.9 mg) (p = 0.04), although when normalized with body mass there was no difference in gastrocnemius mass. For the TA muscle, there were no significant differences in whole muscle mass between SGI-1252 and VC groups, yet TA muscles in the SGI-1252 treated group had a reduced muscle fiber CSA compared to controls (621 ± 44 µm2 vs 749 ± 36 µm2) (p = 0.0005). There was a statistical trend of decreasing Smad2 phosphorylation in the SGI-1252 treated TA muscles (mean SGI-1252 = 0.668 vs VC = 0.848) (p = 0.06), and no significant differences in Smad2 phosphorylation in the gastrocnemius. Conclusions: Contrary to our hypothesis, 8 weeks of orally administered SGI-1252 was not effective in promoting increases in whole body mass, limb whole muscle mass, or myofiber cross sectional area. This may be due to the inability of SGI-1252, at the administered dose, to effectively decrease signaling downstream of the activin receptor. Clearly, studies using a wider range of doses and delivery methods will be needed to ascertain the efficacy of SGI-1252 as a potential therapeutic.

No description available.

Tgf-β, Wnt, and Fgf signaling pathways are required for many developmental processes. Here, I investigated the requirement of these signaling pathways during tail regeneration and limb development in the Mexican axolotl (Ambystoma mexicanum). Using small chemical inhibitors during tail regeneration, I found that the Tgf-β signaling pathway was required from 0-24 and 48-72 hours post tail amputation (hpa), the Wnt signaling pathway was required from 0-120 hpa, and the Fgf signaling pathway was required from 0-12hpa. Tgf-β1 was upregulated after amputation and thus may mediate Tgf-β signaling pathway during tail regeneration. Both Smad-mediated and non-Smad mediated Tgf-β signaling were activated as early as 1hpa. Smad-mediated Tgf-β signaling via activated pSmad2 and pSmad3, and via phosphorylated Erk and Akt. Two different Tgf-β signaling pathway inhibitors, SB505124 and Naringenin, differentially regulated pSmad2, pSmad3, p-Erk, and p-Akt, while SB505124 and Naringenin both inhibited tail regeneration; only SB505124 reduced cell proliferation. Wnt/β-Catenin signaling was increased and was enhanced by Wnt-C59. Disruption of the Wnt signaling pathway directly or indirectly activated Erk and Akt signaling. Disruption of the Fgf signaling pathway decreased p-Erk and increased p-Akt. All three signaling pathways affected cell proliferation and mitosis during tail regeneration. The Wnt pathway inhibitor Wnt-C59 prevented forelimb bud outgrowth. The critical window for Wnt signaling regulating forelimb bud outgrowth was approximately developmental stage 40-42. Wnt signaling ligand Wnt3a and tight junction protein Zo-1 were expressed in the epidermis of the forelimb bud and both were down-regulated by Wnt-C59. Moreover, both Wnt and Fgf signaling pathways affected cell proliferation and mitosis of mesodermal cells during forelimb bud outgrowth. Overall, my results show that Tgf-β, Wnt, and Fgf signaling pathways are required for axolotl tail regeneration. All three pathways affect Erk and Akt signaling and guide cell proliferation and mitosis. The Wnt signaling pathway is required for forelimb bud outgrowth, and it appears to regulate expression of Wnt3a and Zo1, and control cell proliferation and mitosis of mesodermal cells underlying the forelimb epidermis. These data enrich understanding of signaling network dynamics that underlie tissue regeneration and vertebrate limb development.

<p>Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an <i>in vitro </i>and <i>in vivo </i>perspective<i>.</i></p><p>The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration. </p><p>Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity.</p><p>Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs. </p>

Articular cartilage lines the surfaces of load bearing joints and has limited capabilities for self-repair due to its alymphatic and avascular structure. Attempts at making repairs to this tissue has resulted in substandard materials and/or causing further injury to the patient making this tissue a prime candidate for tissue engineering studies incorporating adult stem cells. These studies have given rise to some answers and many more questions including a search for alternative stem cell sources and what biochemical changes the cells undergo during the differentiation of these stem cells into chondrocytes, the cells which make up articular cartilage. Recently, periodontal dental ligament stem cells (PDLs) have come to the forefront as a practical alternative to other adult stem cells as well as the involvement of the mitogen-activated protein kinases (MAPKs) in stem cell differentiation via mechanical stimulation. During dynamic unconfined compression, levels of p42/44 MAPK increased by 50% (p<0.05). Additionally, the expression of the chondrogenic differentiation factor SRY (sex determining region Y)-box 9 (SOX-9) increased by 3-fold (p<0.05) as well as the chondrocyte marker aggrecan by over 2-fold after 4h of dynamic unconfined compression. Addition of the p42/44 phosphorylation inhibitor PD98059, along with compression, yielded no change in SOX-9 or aggrecan expression levels from basal levels in uncompressed controls. Inhibition of p38 MAPK or JNK phosphorylation during unconfined compression had no effect on the elevated expression of SOX-9 and aggrecan as compared to compressed cells without the addition of an inhibitor. It is therefore the overall findings of this study that PDLs possess the ability to differentiate into chondrocytes by mechanical compression and this differentiation is mediated by the p42/44 MAPK cascade.

Transforming growth factor (TGF) β is a tumor suppressor during early tumor development, by inhibiting proliferation and inducing apoptosis. At later stages of cancer, it becomes a tumor promoter, and promotes tumor cell migration and invasion. TGFβ signals via its type II and type I receptors to several downstream signaling pathways. In the present work we have focused on the TRAF6 (tumor necrosis factor receptor-associated factor 6)/ TAK1 (TGFβ activated kinase 1) signaling pathway and the Smad7-dependent activation of p38 in prostate carcinoma cells (PC3U). We found that TGFβ-induced activation of the ubiquitin ligase TRAF6 was needed for cell invasion, by a mechanism that involves activation of the metalloproteinase TNFα converting enzyme (TACE), via protein kinase Cζ (PKCζ). TACE cleaves the TβRI, whereafter the intracellular domain (ICD) translocates to the nucleus, where it binds to the transcriptional co-activator p300 and regulates gene expression, promoting invasion. Interestingly, the translocation of the TβRI ICD was observed in several cancer cell lines and in sections of primary tumors, but not in primary prostate epithelial cells. We also found that Smad7 and adenomatous polyposis coli (APC) are important for TGFβ- and epidermal growth factor (EGF)-induced cell migration in PC3U cells. TGFβ induces the formation of a complex consisting of Smad7, p38, glycogene synthase kinase 3β (GSK-3β), APC and β-catenin, which localizes to the membrane ruffles in the leading edge of migrating cells. The complex links the TβRI to the microtubule system and promotes membrane ruffling and microtubule polarization, which are known to be important for cell migration. In the EGF signaling pathway, Smad7 was found to be important for phosphorylation of the EGF receptor at Tyr1068, for the activation of p38 and JNK, and for induction of membrane ruffles. Smad7 is required for TGFβ-induced activation of p38 and apoptosis. We found that Smad7 forms a complex with p38 and ataxia telangiectasia mutated (ATM), which is important for activation of p53 mediated apoptosis. Many tumor cells including the PC3U cells lack a functional p53, which is one of the reasons to why cancer cells can avoid the tumor suppressor effects of TGFβ.

Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an in vitro and in vivo perspective. The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration. Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity. Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs.