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Successful additive manufacturing involves the optimisation of numerous process parameters that significantly influence product quality and manufacturing success. One commonly used criteria based on a collection of parameters is the global energy distribution (GED). This parameter encapsulates the energy input onto the surface of a build, and is a function of the laser power, laser scanning speed and laser spot size. This study uses machine learning to develop a model for predicting manufacturing layer height and grain size based on GED constituent process parameters. For both layer height and grain size, an artificial neural network (ANN) reduced error over the data set compared with multi linear regression. Layer height predictions using ANN achieved an R2 of 0.97 and a root mean square error (RMSE) of 0.03 mm, while grain size predictions resulted in an R2 of 0.85 and an RMSE of 9.68 µm. Grain refinement was observed when reducing laser power and increasing laser scanning speed. This observation was successfully replicated in another α + ß Ti alloy. The findings and developed models show why reproducibility is difficult when solely considering GED, as each of the constituent parameters influence these individual responses to varying magnitudes.
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BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.
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Proteínas de Transporte , Matriz Extracelular , Animais , Camundongos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Matriz Extracelular/metabolismo , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Isoformas de Proteínas/metabolismo , Ligação ProteicaRESUMO
Accurate elemental quantification of materials by X-ray detection techniques in electron microscopes or microprobes can only be carried out if the appropriate mass absorption coefficients (MACs) are known. With continuous advancements in experimental techniques, databases of MACs must be expanded in order to account for new detection limits. Soft X-ray emission spectroscopy (SXES) is a characterization technique that can detect emitted X-rays whose energies are in the range of 10 eV to 2 keV by using a varied-line-spaced grating. Transitions producing soft X-rays can be detected and accurate MACs are required for use in quantification. This work uses Monte Carlo modeling coupled with multivoltage SXES measurements in an electron probe micro-analyzer (EPMA) to compute MACs for the L2,3-M and Li Kα transitions in a variety of aluminum alloys. Electron depth distribution curves obtained by the software MC X-ray are used in a parametrized fitting equation. The MACs are calculated using a least-squares regression analysis. It is shown that X-ray distribution cross-sections at such low energies need to take into account additional contributions, such as CosterKronig transitions, Auger yields, and wave function effects in order to be accurate.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Additive manufacturing, often known as three-dimensional (3D) printing, is a process in which a part is built layer-by-layer and is a promising approach for creating components close to their final (net) shape. This process is challenging the dominance of conventional manufacturing processes for products with high complexity and low material waste1. Titanium alloys made by additive manufacturing have been used in applications in various industries. However, the intrinsic high cooling rates and high thermal gradient of the fusion-based metal additive manufacturing process often leads to a very fine microstructure and a tendency towards almost exclusively columnar grains, particularly in titanium-based alloys1. (Columnar grains in additively manufactured titanium components can result in anisotropic mechanical properties and are therefore undesirable2.) Attempts to optimize the processing parameters of additive manufacturing have shown that it is difficult to alter the conditions to promote equiaxed growth of titanium grains3. In contrast with other common engineering alloys such as aluminium, there is no commercial grain refiner for titanium that is able to effectively refine the microstructure. To address this challenge, here we report on the development of titanium-copper alloys that have a high constitutional supercooling capacity as a result of partitioning of the alloying element during solidification, which can override the negative effect of a high thermal gradient in the laser-melted region during additive manufacturing. Without any special process control or additional treatment, our as-printed titanium-copper alloy specimens have a fully equiaxed fine-grained microstructure. They also display promising mechanical properties, such as high yield strength and uniform elongation, compared to conventional alloys under similar processing conditions, owing to the formation of an ultrafine eutectoid microstructure that appears as a result of exploiting the high cooling rates and multiple thermal cycles of the manufacturing process. We anticipate that this approach will be applicable to other eutectoid-forming alloy systems, and that it will have applications in the aerospace and biomedical industries.
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During an investigation of the Mg-rich end of the Mg-Al-La system, a new ternary phase with the composition of (Al,Mg)3La was identified. The crystal structure of this phase was determined by conventional X-ray powder diffraction and transmission electron microscopy analysis and refined using high-resolution X-ray powder diffraction. The (Al,Mg)3La phase is found to have an orthorhombic structure with a space group of C2221 and lattice parameters of a = 4.3365â (1)â Å, b = 18.8674â (4)â Å and c = 4.4242â (1)â Å, which is distinctly different from the binary Al3La phase (P63/mmc). The resolved structure of the (Al,Mg)3La phase is further verified by high-angle annular dark-field scanning transmission electron microscopy.
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Electron and proton microprobes, along with electron backscatter diffraction (EBSD) analysis were used to study the microstructure of the contemporary Al-Cu-Li alloy AA2099-T8. In electron probe microanalysis, wavelength and energy dispersive X-ray spectrometry were used in parallel with soft X-ray emission spectroscopy (SXES) to characterize the microstructure of AA2099-T8. The electron microprobe was able to identify five unique compositions for constituent intermetallic (IM) particles containing combinations of Al, Cu, Fe, Mn, and Zn. A sixth IM type was found to be rich in Ti and B (suggesting TiB2), and a seventh IM type contained Si. EBSD patterns for the five constituent IM particles containing Al, Cu, Fe, Mn, and Zn indicated that they were isomorphous with four phases in the 2xxx series aluminium alloys including Al6(Fe, Mn), Al13(Fe, Mn)4 (two slightly different compositions), Al37Cu2Fe12 and Al7Cu2Fe. SXES revealed that Li was present in some constituent IM particles. Al SXES mapping revealed an Al-enriched (i.e., Cu, Li-depleted) zone in the grain boundary network. From the EBSD analysis, the kernel average misorientation map showed higher levels of localized misorientation in this region, suggesting greater deformation or stored energy. Proton-induced X-ray emission revealed banding of the TiB2 IM particles and Cu inter-band enrichment.
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Several recent papers report spectacular, and unexpected, order of magnitude improvement in creep life of alloys upon adding small amounts of elements like zinc. This microalloying effect raises fundamental questions regarding creep deformation mechanisms. Here, using atomic-scale characterization and first principles calculations, we attribute the 600% increase in creep life in a prototypical Mg-rare earth (RE)-Zn alloy to multiple mechanisms caused by RE-Zn bonding-stabilization of a large volume fraction of strengthening precipitates on slip planes, increase in vacancy diffusion barrier, reduction in activated cross-slip, and enhancement of covalent character and bond strength around Zn solutes along the c-axis of Mg. We report that increased vacancy diffusion barrier, which correlates with the observed 25% increase in interplanar bond stiffness, primarily enhances the high-temperature creep life. Thus, we demonstrate that an approach of local, randomized tailoring of bond stiffness via microalloying enhances creep performance of alloys.
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Latent transforming growth factor-ß-1 binding protein-2 (LTBP-2) belongs to the LTBP-fibrillin superfamily of extracellular proteins. Unlike other LTBPs, LTBP-2 does not covalently bind transforming growth factor-ß1 (TGF-ß1) but appears to be implicated in the regulation of TGF-ß1 bioactivity, although the mechanisms are largely unknown. In experiments originally designed to study the displacement of latent TGF-ß1 complexes from matrix storage, we found that the addition of exogenous LTBP-2 to cultured human MSU-1.1 fibroblasts caused an increase in TGF-ß1 levels in the medium. However, the TGF-ß1 increase was due to an upregulation of TGF-ß1 expression and secretion rather than a displacement of matrix-stored TGF-ß1. The secreted TGF-ß1 was mainly in an inactive form, and its concentration peaked around 15 h after addition of LTBP-2. Using a series of recombinant LTBP-2 fragments, the bioactivity was identified to a small region of LTBP-2 consisting of an 8-Cys motif flanked by four epidermal growth factor (EGF)-like repeats. The LTBP-2 stimulation of TGF-ß expression involved the phosphorylation of both Akt and p38 mitogen-activated protein kinase (MAPK) signalling proteins, and specific inactivation of each protein individually blocked TGF-ß1 increase. The search for the cell surface receptor mediating this LTBP-2 activity proved inconclusive. Inhibitory antibodies to integrins ß1 and αVß5 showed no reduction of LTBP-2 stimulation of TGF-ß1. However, TGF-ß1 upregulation was partially inhibited by anti-αVß3 integrin antibodies, suggestive of a direct or indirect role for this integrin. Overall, the study indicates that LTBP-2 can directly upregulate cellular TGF-ß1 expression and secretion by interaction with cells via a short central bioactive region. This may be significant in connective tissue disorders involving aberrant TGF-ß1 signalling.
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Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , Fibrose/metabolismo , Humanos , Fosforilação , Reação em Cadeia da PolimeraseRESUMO
We have recently shown that Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) has a single high-affinity binding site for fibroblast growth factor-2 (FGF-2) and that LTBP-2 blocks FGF-2 induced cell proliferation. Both proteins showed strong co-localisation within keloid skin from a single patient. In the current study, using confocal microscopy, we have investigated the distribution of the two proteins in normal and fibrotic skin samples including normal scar tissue, hypertrophic scars and keloids from multiple patients. Consistently, little staining for either protein was detected in normal adult skin and normal scar samples but extensive co-localisation of the two proteins was observed in multiple examples of hypertrophic scars and keloids. LTBP-2 and FGF-2 were co-localised to fine fibrous elements within the extracellular matrix identified as elastic fibres by immunostaining with anti-fibrillin-1 and anti-elastin antibodies. Furthermore, qPCR analysis of RNA samples from multiple patients confirmed dramatically increased expression of LTBP-2 and FGF-2, similar TGF-beta 1, in hypertrophic scar compared to normal skin and scar tissue. Overall the results suggest that elevated LTBP-2 may bind and sequester FGF-2 on elastic fibres in fibrotic tissues and modulate FGF-2's influence on the repair and healing processes.
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Cicatriz Hipertrófica/genética , Fator 2 de Crescimento de Fibroblastos/genética , Queloide/genética , Proteínas de Ligação a TGF-beta Latente/genética , Pele/metabolismo , Adolescente , Adulto , Sítios de Ligação , Estudos de Casos e Controles , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , Tecido Elástico/lesões , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Elastina/genética , Elastina/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrilina-1 , Fibrilinas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Queloide/metabolismo , Queloide/patologia , Proteínas de Ligação a TGF-beta Latente/metabolismo , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Reepitelização/fisiologia , Transdução de Sinais , Pele/lesões , Pele/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor ß (TGF-ß), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-ß, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9-14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.
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Sítios de Ligação , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/química , Proteínas de Ligação a TGF-beta Latente/química , Proteínas de Ligação a TGF-beta Latente/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fibrilinas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Queloide/metabolismo , Proteínas de Ligação a TGF-beta Latente/farmacologia , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes , Pele/metabolismoRESUMO
The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFß family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvß3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFß signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.
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Proteínas Contráteis/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Microfibrilas/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Desenvolvimento Ósseo , Fibrilinas , Expressão Gênica , Glicoproteínas/fisiologia , Hematopoese , Humanos , Proteínas dos Microfilamentos/fisiologia , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Fatores de Processamento de RNA , CicatrizaçãoRESUMO
Using the evolutionary optimization algorithm, as implemented in the USPEX crystal predictor program, and first principles total energy calculations, the compositional phase diagrams for Al-Sc and Al-Ta alloy systems at zero temperature and pressure have been calculated. In addition to the known binary intermetallic phases, new potentially stable alloys, AlSc3 and AlTa7, have been identified in the Al-poor region of the phase diagram. The dynamic and thermal stability of their lattices has been confirmed from the calculated vibrational normal mode spectra in the harmonic approximation.
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Comparative immunolocalisations of latent transforming growth factor-beta-1 binding protein (LTBP)-2, fibrillin-1, versican and perlecan were undertaken in foetal human and wild type C57BL/6 mouse and Hspg2 exon 3 null HS deficient mouse intervertebral discs (IVDs). LTBP-2 was a prominent pericellular component of annular fibrochondrocytes in the posterior annulus fibrosus (AF), interstitial matrix adjacent to nucleus pulposus (NP) cells and to fibrillar and cell associated material in the anterior AF of the human foetal IVD and also displayed a pericellular localisation pattern in murine IVDs. Perlecan and LTBP-2 displayed strong pericellular colocalisation patterns in the posterior AF and to fibrillar material in the outer anterior AF in the foetal human IVD. Versican was a prominent fibril-associated component in the posterior and anterior AF, localised in close proximity to fibrillin-1 in fibrillar arrangements in the cartilaginous vertebral rudiments around paraspinal blood vessels, to major collagen fibre bundles in the anterior and posterior AF and shorter fibres in the NP. Fibrillin-1 was prominent in the outer anterior AF of the human foetal IVD and in fibres extending from the AF into the cartilaginous vertebral rudiments. LTBP-2 was prominently associated with annular fibrils containing fibrillin-1, versican was localised in close proximity to these but not specifically with LTBP-2. The similar deposition levels of LTBP-2 observed in the AF of the Hspg2 exon 3 null and wild type murine IVDs indicated that perlecan HS was not essential for LTBP-2 deposition but colocalisation of LTBP-2 with perlecan in the foetal human IVD was consistent with HS mediated interactions which have already been demonstrated in-vitro.
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Proteoglicanas de Heparan Sulfato/metabolismo , Disco Intervertebral/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilinas , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microfibrilas/metabolismo , Microscopia Confocal/métodosRESUMO
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd=26.47±5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd=24.66±5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.
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Proteínas da Matriz Extracelular/metabolismo , Heparina/genética , Heparitina Sulfato/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Tropoelastina/metabolismo , Animais , Células Cultivadas , Tecido Elástico/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Tropoelastina/genéticaRESUMO
The interfaces between the phase separated regions in the dendritic grains of laser-deposited samples of the high entropy alloy CoCrCuFeNiAl have been studied using aberration-corrected analytical (scanning) transmission electron microscopy ((S)TEM). The compositional variations have been determined using energy dispersive x-ray spectroscopy (EDS) in (S)TEM. It was found that between B2, consisting mainly of Al, Ni, Co, and Fe, and disordered bcc phase, consisting mainly of Cr and Fe, there is a transition region, approximately 1.5 nm in width, over which the chemical composition changes from the B2 to that of the bcc phase. The crystal structure of this interfacial region is also B2, but with very different sublattice occupancy than that of the adjacent B2 compound. The structural aspects of the interface between the ordered B2 phase and the disordered bcc phase have been characterized using high angle annular dark-field (HAADF) imaging in STEM. It has been determined that the interfaces are essentially coherent, with the lattice parameters of the two B2 regions and the disordered bcc phase being more or less the same, the uncertainty arising from possible relaxations from the proximity of the surfaces of the thin foils used in imaging of the microstructures. Direct observations show that there is a planar continuity between all three constituent phases.
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Ligas/química , Microscopia Eletrônica de Transmissão e Varredura/métodos , Espectrometria por Raios X/métodos , EntropiaRESUMO
STUDY DESIGN: A comparative immunolocalization study of elastin-associated proteins and established intervertebral disc (IVD) extracellular matrix (ECM) components. OBJECTIVE: To localize for the first time, elastic fiberassociated proteins with structural fibrillar components in the annulus fibrosus (AF) of the fetal IVD. SUMMARY OF BACKGROUND DATA: Elastin has been identified histochemically in adult bovine, human, and immature rat IVDs, and in fetal human IVDs using electron microscopy; however, no immunolocalization studies have been undertaken for associated components in human fetal IVDs. METHODS: En-bloc fixation of thoracolumbar spinal segments in formalin and Histochoice followed by standard histochemical processing, paraffin embedding, microtome sectioning, and identification of IVD ECM components using a range of specific mono- and polyclonal antibodies and bright-field and laser scanning confocal microscopy. RESULTS: The elastic fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 were prominently immunolocalized in the outer lamellar layers of the AF of the human fetal IVD. Dual localization of selected components by confocal microscopy demonstrated that versican and LTBP-2 were colocalized with fibrillin-1 microfibrils in the AF lamellae with a similar distribution to the elastin fibers. LTBP-2 was also associated with pericellular perlecan in the outer AF. These interconnections between elastin-associated proteins resulted in an elastic network, which connected the AF cells with the adjacent cartilaginous vertebral bodies. CONCLUSION: Specific immunolocalization of fibrillin-1, MAGP-1, and versican with elastin in the outer AF of the fetal human IVD has been demonstrated. We deduce from the established distributions of the elastin-associated proteins and their known interactivities with matrix components that these stabilize and aid in the integration of the elastic fibers in the annular lamellae and may be responsible for the generation of tensional forces in the outer AF, which direct the assembly of this tissue.
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Colágeno/análise , Proteínas Contráteis/análise , Tecido Elástico/química , Elastina/análise , Proteínas da Matriz Extracelular/análise , Imuno-Histoquímica , Disco Intervertebral/química , Proteínas de Ligação a TGF-beta Latente/análise , Vértebras Lombares/química , Proteínas dos Microfilamentos/análise , Proteoglicanas/análise , Vértebras Torácicas/química , Tecido Elástico/embriologia , Fibrilina-1 , Fibrilinas , Idade Gestacional , Humanos , Disco Intervertebral/embriologia , Vértebras Lombares/embriologia , Microfibrilas/química , Microscopia Confocal , Fatores de Processamento de RNA , Vértebras Torácicas/embriologia , Versicanas/análiseRESUMO
Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-ß activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-ß bioactivity in tissues by binding latent TGF-ß binding proteins. We therefore examined expression of fibrillins 1-3, latent TGF-ß binding proteins 1-4, and TGF-ß 1-3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-ß pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.
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Regulação da Expressão Gênica no Desenvolvimento , Ovário/metabolismo , Síndrome do Ovário Policístico/genética , Fator de Crescimento Transformador beta/genética , Animais , Bovinos , Feminino , Fibrilinas , Humanos , Imuno-Histoquímica , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Folículo Ovariano/embriologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Síndrome do Ovário Policístico/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca(2+) ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca(2+) dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG-LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.
Assuntos
Membrana Basal/fisiologia , Proteoglicanas de Heparan Sulfato/fisiologia , Heparina/fisiologia , Proteínas de Ligação a TGF-beta Latente/fisiologia , Microfibrilas/fisiologia , Sindecana-4/fisiologia , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Quelantes/farmacologia , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Ensaio de Imunoadsorção Enzimática , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/fisiologia , Proteínas Recombinantes/metabolismoRESUMO
Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.