Extracellular matrix (ECM) microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. The focus of this PhD thesis is the study of two microfibrillar assemblies, fibrillin-1 and collagen VI. Fibrillin is a large ECM glycoprotein which facilitates the deposition of elastin in elastic tissues such aorta, skin and lung and sequesters growth factors in the matrix. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the ECM allowing for the transduction of biochemical and mechanical signals. The structures of some individual domains and short fragments of both fibrillin and collagen VI have been solved, but it is not fully understood how they are arranged into microfibrils and how these microfibrils are arranged into tissues. Therefore the aim of this project has been to determine the hierarchical organisation of fibrillin and collagen VI across multiple length scales. The nanoscale structure of the fibrillin microfibril was determined using negative stain TEM and single particle reconstruction. Microfibrils had a hollow tube-like structure with well-defined bead, arm, interbead and shoulder regions. To overcome flexibility observed in the microfibril, separate sub-models of the different fibrillin regions were modelled. The bead region had a complex double layered structure with an interwoven core and ring structures. The arm region had four separate densities which are potentially formed from dimers of fibrillin molecules. Serial block face scanning electron microscopy (SBF-SEM) and electron tomography allowed for the in situ 3D imaging of individual fibrillin microfibrils in ciliary zonule tissue. Microfibrils in ciliary zonule fibres were held together by cross bridges between microfibrils. These ciliary zonule fibres were then organised into larger fascicle-like structures which were stabilised by circumferentially arranged ciliary zonule fibres. The frozen hydrated structure of the collagen VI half-bead was reconstructed using cryo-TEM. The half-bead region had a compact hollow head, and flexible tail regions, the tail regions were linked together by the collagenous interbead region. SBF-SEM and electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM had a meshwork-like organisation formed from globular densities ~30 nm in diameter. Together a combinatorial approach to image ECM microfibrils from the sub-molecular level to intact tissue structures spanning nanometre to millimetre length scales is presented. This provides a better understanding of how fibrillin and collagen VI microfibrils are organised in tissues.

Fibrillin-1 is the main component of the 10-12 nm microfibrils, which are found in several elastic and non-elastic tissues. Human fibrillin-1 contains multiple calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with transforming growth factor β-binding protein-like (TB) domains. TB4 domain contains a flexible RGD loop which mediates cell adhesion via αVβ3, α5β1 and αVβ6 integrins. Mutations which introduce amino acid substitutions into TB4 are associated with a wide spectrum of diseases such as Marfan syndrome (MFS), ectopia lentis, Stiff skin syndrome (SSS). Amino acid substitutions such as W1570C, C1564S and C1577G in the TB4 domain have been found to cause SSS. The upstream TB5 domain has been predicted to modulate integrin binding and a deletion in the domain has been found in Weill-Marchesani syndrome (WMS), phenotype of which is similar to SSS (skin fibrosis and short stature), thereby suggesting that the underlying pathogenic mechanism might be similar. This study has used cellular, biochemical and biophysical methods to investigate the effects of SSS substitutions C1564S and W1570C on domain structure and function and compared it to a MFS substitution C1564Y in the TB4 domain and WMS deletion in the TB5 domain. Effects of the SSS mutations on structure of the domains were studied using limited proteolysis, nuclear magnetic resonance spectroscopy and calcium chelation experiments. Subsequently, the ability of human fibroblasts to secrete wild-type and mutant fibrillin-1 was examined to identify the effect of the mutations on the trafficking of the protein. Finally, cell binding assays and SPR was employed to investigate the effect of disease-causing mutations on fibrillin-1/integrin interactions. The results demonstrate that the SSS mutations affect TB4-cbEGF23 interface and calcium-binding to cbEGF23 but do not alter secretion of recombinant fibrillin-1 mutant fragments from the cell. On the other hand, intracellular retention was observed for MFS substitution C1564Y which was shown to be more susceptible to proteolysis than SSS substitution C1564S. WMS deletion also gives rise to partial retention of the recombinant fragment, suggesting a different pathogenic mechanism for these disorders. Cell binding assays and surface plasmon resonance (SPR) experiments show that SSS mutations affect binding to αvβ3 integrin, but not αvβ6 integrin suggesting that selectively impaired integrin interactions may contribute to pathogenesis of SSS.

Marfan syndrome, affecting approximately one out of every 5,000 people, is characterized by abnormal bone growth, ectopia lentis, and often-fatal aortic dilation and dissection. The root cause is a faulty extracellular matrix protein, fibrillin-1, which associates with elastin in many tissues. Common carotids from wild-type controls and mice heterozygous for the mgR mutation, the most commonly used mouse model of Marfan syndrome, were studied in a biaxial testing device. Mechanical data in the form of pressure-diameter and force-stretch tests in both the active and passive states were collected, as well data on the functional responses to phenylephrine, carbamylcholine chloride, and sodium nitroprusside. Although little significant difference was found between the heterozygous and wild-type groups in general, the in vivo stretch for both groups was significantly different from previously studied mouse vessels. Although the two groups do not exhibit significant differences, this study comprises a control group for future work with mice homozygous for mgR, which do exhibit Marfan-like symptoms. As treatment of Marfan syndrome improves, more Marfan patients will survive and age, increasing the likelihood that they will develop many of the vascular complications affecting the normal population, including hypertension and atherosclerosis. Therefore, it is imperative to gather biomechanical data from the Marfan vasculature so that clinicians may predict the effects of vascular complications in Marfan patients and develop appropriate methods of treatment.

Regensburg, Univ., Diss., 2009.

Connective tissue initially arises from embryonic mesenchymal stem cells (MSC) that originate from the mesoderm during embryogenesis and are capable of differentiating into connective tissue lineages such as adipocytes, osteoblasts, chondrocytes and fibroblasts. Connective tissue is composed of cells held together by the extracellular matrix (ECM). The fibrillins and latent transforming growth factor binding proteins form a superfamily of ECM proteins characterised by the presence of a unique domain, the 8- cysteine transforming growth factor beta binding domain (TGFß). The fibrillin proteins (fibrillin-1, fibrillin-2 and fibrillin-3 in most vertebrates, encoded by the FBN1, FBN2 and FBN3 genes respectively), are major components of the 10nM microfibrils found in ECM of many tissue types, for example mesenchyme-derived connective tissues. Fibrillin-1 and fibrillin-2 are also thought to be required for stabilization and storage of latent TGFβ complexes. Mutations in FBN1 cause Marfan syndrome, a connective tissue disorder characterised by abnormalities in the microfibrils resulting in musculoskeletal, ocular, cardiovascular and other complications. FBN2 mutations lead to congenital contractural arachnodactyly, which has a musculoskeletal phenotype similar to Marfan syndrome. There are currently no known diseases associated with FBN3 mutations. In this project, the expression of fibrillins was investigated using human cell lines during early development, mesenchymal stem cell differentiation and in further differentiated mesenchymal cell lines, for example in osteocytes (osteosarcomas), chondrocytes and fibroblast lineage. Immunocytochemistry was used to examine protein expression, real-time PCR and expression microarrays to determine mRNA synthesis and RNAi suppression of gene expression to determine possible functions of fibrillins and associated ECM proteins. In addition, a genome wide bioinformatics evaluation was performed of transcription start sites for the fibrillin gene family utilising the information obtained from the FANTOM5 consortium. The three fibrillin genes showed differing expression patterns in cell lines depending on the stage of development/differentiation. During embryogenesis, expression of FBN3, FBN2 and FBN1 increased sequentially in that order. Expression of FBN3 followed the same pattern as expression of known pluripotency markers, while expression of FBN2 correlated with expression of markers for later stages of mesoderm differentiation. FBN1 expression was associated with mesenchymal markers, and this was supported by a study of mesenchymal stem cells differentiation to the adipose lineage. Fibrillin-1 microfibrils and RNA expression were present early in primary adult human MSC differentiating to adipocytes, suggesting that a fibrillin matrix is required for initial MSC attachment. As differentiation proceeded, fibrillin -1 expression decreased, with rapid degradation of the microfibrils. Fibrillin-2 expression increased following differentiation and fibrillin-3 was not expressed. These results suggest that fibrillin-1 plays an important structural and regulatory role in the early stages of connective tissue development but is not required to maintain the differentiated state. Many genes showed the same expression pattern as FBN1. To better understand the importance of fibrillin-1 and its interaction with these coexpressing genes, fibrillin-1 was knocked down using siRNA in fibroblast, chondrocyte and osteosarcoma cell lines. There were little to no effects identified in chondrocyte and osteosarcoma cell lines, and only a few genes were altered following the reduction of fibrillin-1 mRNA in fibroblasts, suggesting that fibrillin-1 is not a central regulator but an endpoint. This was surprising given its potential role in controlling bioavailability of TGFβ, a key regulator of mesenchymal cells. In addition, the evolution of the fibrillin gene family was studied and it was found that the gene structure, amino acid sequence and genomic positions of each gene are widely conserved across vertebrates, suggesting an important role in vertebrate body structure. However, the differences in gene structure and sequence between the three fibrillin genes suggest divergent function. Fibrillin-1 mutations with the most severe phenotypes are located in regions that are highly conserved. This study shows that there is a clear developmental and evolutionary distinction between the three fibrillins. Fibrillin-3 was associated with pluripotency and its presence in differentiating foetal liver and brain may suggest that there are residual pluripotent cells in these developing tissues. Fibrillin-2 appeared to be a marker for the mesodermal stage and its role in adult cells is currently not clear. Fibrillin-1 was present in cells already predetermined to go to mesenchymal lineages, but it was minimal in the advanced stages of differentiation suggesting that it may be a marker for relatively plastic mesenchymal cells prior to commitment to a specific lineage. These results will assist in the understanding of disorders resulting from fibrillin gene mutations and have identified coexpressed proteins, potential modifiers that could be the targets of gene therapy and candidates for similar connective tissue.

Exposure of the skin to ultraviolet radiation (UVR) results clinically in the formation of deep wrinkles and mottled pigmentation and histologically, in a vast remodelling of the dermal extracellular matrix (ECM), in particular the elastic fibre network. Fibrillin microfibrils and fibulin-5 are early biomarkers of photoageing, where a loss of these fibres from the dermal epidermal junction is apparent. A study by our group showed that isolated fibrillin microfibrils and fibronectin which are rich in amino acids which absorb energy from UVR (UV-chromophores) are susceptible to UVR-induced damage, whilst UV-chromophore poor collagen type I is not. This research, with other earlier studies, indicates that acellular mechanism may work in tandem with cell-mediated up-regulation of matrix metalloproteinases (MMPs) in the progression of photoageing. This thesis aims to: i) test whether acellular mechanisms of photoageing are a result of direct photon absorption and/or the photodynamic production of reactive oxygen species (ROS); ii) assess the functional consequences of UVB degradation on the susceptibility of fibrillin microfibrils to MMPs and; iii) assay whether ECM proteins are differentially susceptible to solar simulated radiation (SSR) or UVA (315-400nm) alone using physiologically relevant doses of irradiation. Isolated proteins were exposed to UVB (280-315nm) in depleted-O2 conditions and in the presence of deuterium oxide. Depleted-O2 conditions decreased and deuterium oxide conditions increased UVR-induced degradation. Isolated proteins also show a similar pattern of degradation when exposed to H2O2 as an exogenous source of ROS. These results indicate that ROS play an important role in the differential degradation of dermal proteins. MMPs-3 and -9 are both upregulated in the skin after exposure to UVR and have the ability to degrade elastic fibre components. After exposure to UVB, damaged fibrillin microfibrils become more susceptible to degradation by both MMPs-3 and -9. Chromophore-rich fibrillin microfibrils and fibronectin are susceptible to degradation by both SSR and UVA alone, whereas chromophore-poor collagens type I and VI and tropoelastin are not. These results support our previous findings that amino acid composition of proteins is a good indicator of their relative susceptibility to UV-induced damage with a physiologically relevant irradiation system. In conclusion this work shows that ROS are an important mediator of acellular mechanisms of photoageing and that amino acid composition is a good indication of relative susceptibility of proteins to both ROS and UVR. The ability to predict ROS-susceptible proteins also has wider implications for human ageing as a whole.

No description available.

Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.

Orientador: Claudio Chrysostomo Werneck / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T14:55:14Z (GMT). No. of bitstreams: 1 Braga_GuilhermeGambogi_M.pdf: 40426167 bytes, checksum: 2843f77911936a8afd4b180bb9db3b50 (MD5) Previous issue date: 2012 / Resumo: Fibrilina-1 é um importante componente da rede de microfibrilas da matriz extracelular. As microfibrilas estão presentes nas fibras elásticas que são responsáveis pela elasticidade e resistência de tecidos dos pulmões, pele e grandes vasos. Mutações no gene da Fibrilina-1 estão associadas com a síndrome de Marfan. Pacientes com esta síndrome apresentam muitas manifestações clínicas nos pulmões, sistema cardiovascular e olhos. Modelos de síndrome de Marfan tem sido criados no sentido de obter informações sobre o desenvolvimento desta doença. Estudos recentes em modelos de camundongos tem sugerido a importância da atividade exacerbada do TGF ? promovendo a quase totalidade das alterações fenotípicas encontradas nestes camundongos, o que pode ser revertido pelo tratamento com o Losartan. O principal objetivo deste projeto foi avaliar a formação de fibras elásticas em cultura de células obtidas de animais deficientes em fibrilina-1, bem como o possível efeito do tratamento destas células com losartan. Os fibroblastos derivados de animais deficientes em fibrilina- 1 foram estudados usando imunofluorescência, western blotting, microscopia eletrônica de varredura e transmissão e real-time PCR. Assim, a deficiência em fibrilina-1 ocasionou uma redução conjunta da deposição de fibrilina-2, MAGP-1 e tropoelastina na matriz extracelular em cultura de fibroblastos de derme. Foi possível verificar aumento no níveis de expressão das metaloproteinases MMP-2 e MMP-9. O tratamento dos fibroblastos derivados de animais deficientes em fibrilina-1 com o fármaco losartan levou a recuperação parcial da deposição das proteínas Fibrilina-2, MAGP-1 e tropoelastina na matriz extracelular de cultura in vitro. Porém, o tratamento dos fibroblastos derivados de animais deficientes em fibrilina-1 com fármaco Captopril e os peptídeos de Angiotensina I e II não influenciaram na deposição das mesmas na matriz extracelular de cultura in vitro / Abstract: Fibrillin-1 is an important microfibril network component. Microfibrils are present in elastic fiber responsible for resilience and elastic properties from structures like lungs, skin and large vessels. Mutations in Fibrillin-1 gene are associated with Marfan's Syndrome. Marfan's Syndrome patients shown many different clinical manifestations in lungs, cardiovascular system and eyes tissues. Marfan's models have been created to get better insights about this disease development. Recent studies from mice models have suggested an important role to unbalanced TGF ? activity promoting almost whole alterations found in those mice which might be rescued by losartan treatment. Our main goal were evaluate elastic fiber formation in cell culture obtained from fibrillin-1 defficient mice as well as losartan's treatment effect on those cells. The fibroblasts derived from deficient animals in fibrillin-1 were studied using immunofluorescence, western blotting, scanning electron microscopy and transmission and real-time PCR. Thus, the deficiency in fibrillin-1 led to a joint reduction of the deposition of fibrillin-2, MAGP-1 and tropoelastin in the extracellular matrix in culture of fibroblasts in the dermis. It was possible to observe an increase in levels of expression of metalloproteinases MMP-2 and MMP-9 were found. The treatment of the fibroblasts derived from deficient animals in fibrillin-1 with the drug losartan has led to the partial recovery of the deposition of protein fibrillin-2, MAGP-1 and tropoelastin in the extracellular matrix of culture in vitro. However, the treatment of fibroblasts derived from deficient animals in fibrillin-1 with drug Captopril and the peptides of Angiotensin I and II not influenced in the deposition of the same in the extracellular matrix of culture in vitro / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Claudio Chrysostomo Werneck / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:06:14Z (GMT). No. of bitstreams: 1 Pereira_CatherineNatalia_M.pdf: 4403950 bytes, checksum: 7142bd3654bd7f9d99e12cda4d33aeeb (MD5) Previous issue date: 2015 / Resumo: Fibrilina-1 (FBN-1) é um importante componente da rede de microfibrilas da matriz extracelular (MEC). As microfibrilas estão presentes nas fibras elásticas que são responsáveis pela resiliência de tecidos como pulmões, pele e grandes vasos. Mutações no gene da fibrilina-1 estão associadas à Síndrome de Marfan, doença autossômica dominante, caracterizada por uma desordem do tecido conjuntivo. Pacientes com esta Síndrome apresentam anomalias no sistema esquelético e trato cardiovascular. Dados da literatura relacionam a menor quantidade de FBN-1 na MEC com a atividade exacerbada do TGF-? promovendo a quase totalidade das alterações fenotípicas encontradas. Estudos preliminares em nosso laboratório com camundongos que possuem menor quantidade de FBN-1 de um camundongo normal tem demonstrado que necessitam do dobro do tempo para a formação de trombo em um modelo de trombose arterial. As plaquetas tem fundamental importância neste processo, quando são ativadas secretam várias moléculas que determinam a formação dos trombos. Realizamos um estudo comparativo do proteoma de plaquetas e da artéria aorta de camundongos selvagens e deficientes em FBN-1, através da técnica de eletroforese bidimensional (2DE) juntamente com a espectrometria de massas a fim de encontrar diferenças no perfil proteico que justificassem tais sintomas. Diversas proteínas plaquetárias foram encontradas apenas no grupo controle, como endoplasmina, fator de von Willebrand, calpaína, dentre outras; assim como a proteína vinculina foi encontrada apenas no grupo deficiente em FBN-1. Todas as proteínas encontradas podem ser de grande interesse para o esclarecimento a respeito do maior tempo de formação de trombo e os sintomas relacionados à Síndrome de Marfan / Abstract: Fibrillin-1 (FBN-1) is an important microfibril network component of extracellular matrix (ECM). Microfibrils are present in elastic fibers responsible for tissues resiliency, such as lungs, skin and great vessels. Mutations in fibrillin-1 gene are associated with Marfan Syndrome, a dominant autosomal disease characterized by connective tissue disorder. Patients with this syndrome show abnormalities in skeletal system and cardiovascular tract, aorta dilatation and aneurysms. Literature data relate less FBN-1 in the ECM with TGF-? heightened activity, promoting almost all the phenotypic alterations. Preliminary studies in our laboratory demonstrated that mice containing half amount of FBN-1 presented prolonged thrombosis time when compared to wild-type mice submitted to an arterial thrombosis model. Platelets are important in this process, when activated they release several molecules and factors which determine thrombi formation. We conducted a comparative proteome study of platelet and aorta from wild-type against FBN-1 deficient mice by two-dimensional electrophoresis (2DE) coupled with mass spectrometry in order to find some differences in protein profile that could justify such symptoms. Several platelet proteins were found only into control group, as endoplasmin, von Willebrand factor, calpain; as well as vinculin was found only in the FBN-1 deficient group. All proteins found may have great interest for understanding the prolonged thrombus formation time, and symptoms related to Marfan Syndrome / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular