RESUMEN

Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs toward mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle shape with abundant smooth muscle α-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle α-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent upregulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers platelet endothelial cell adhesion molecule 1 and vascular endothelial-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus, MSC potency and vascular induction are regulated by perturbing mesenchymal fate.

Asunto(s)

Células Endoteliales/citología , Fibronectinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mesodermo/citología , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Adulto , Inductores de la Angiogénesis/metabolismo , Animales , Colágeno/farmacología , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Fibronectinas/deficiencia , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Laminina/farmacología , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteína Homeótica Nanog , Neovascularización Fisiológica/efectos de los fármacos , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteoglicanos/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Adulto Joven

RESUMEN

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Asunto(s)

Síndrome de Marfan/genética , Receptores de Activinas Tipo I/genética , Disección Aórtica/genética , Animales , Aneurisma de la Aorta Torácica/genética , Proteínas Contráctiles/fisiología , Bases de Datos Genéticas , Proteínas de la Matriz Extracelular/fisiología , Fibrilina-1 , Fibrilinas , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Síndrome de Marfan/complicaciones , Ratones , Microfibrillas/metabolismo , Proteínas de Microfilamentos/genética , Modelos Animales , Modelos Biológicos , Desnaturalización Proteica/genética , Proteínas Serina-Treonina Quinasas , Factores de Empalme de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética

RESUMEN

The extracellular matrix is an intricate network of macromolecules which provides support for cells and a framework for tissues. The detailed structure and organisation of most matrix polymers is poorly understood. These polymers have a complex ultrastructure, and it has proved a major challenge both to define their structural organisation and to relate this to their biological function. However, new approaches using automated electron tomography are beginning to reveal important insights into the molecular assembly and structural organisation of two of the most abundant polymer systems in the extracellular matrix. We have generated three-dimensional reconstructions of collagen fibrils from bovine cornea and fibrillin microfibrils from ciliary zonules. Analysis of these data has provided new insights into the organisation and function of these large macromolecular assemblies.

Asunto(s)

Matriz Extracelular/ultraestructura , Imagenología Tridimensional/métodos , Tomografía Computarizada por Rayos X/métodos , Animales , Bovinos , Córnea/ultraestructura , Matriz Extracelular/química , Colágenos Fibrilares/química , Colágenos Fibrilares/ultraestructura , Fibrilinas , Microfibrillas/química , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/ultraestructura , Microscopía Electrónica/métodos

RESUMEN

Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic 'beads-on-a-strong' appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.

Asunto(s)

Proteínas de la Matriz Extracelular/metabolismo , Microfibrillas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Animales , Biopolímeros , Elasticidad , Proteínas de la Matriz Extracelular/ultraestructura , Fibrilina-1 , Fibrilinas , Predicción , Humanos , Microfibrillas/química , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Microscopía de Fuerza Atómica , Pliegue de Proteína

RESUMEN

Type VIII collagen is upregulated after vessel injury, and this collagen has been implicated in both smooth muscle cell migration and angiogenesis. This study examines the temporal and spatial pattern of expression of type VIII collagen in porcine coronary vessels at specific time points after balloon angioplasty. In situ hybridization studies demonstrated that collagen VIII messenger ribonucleic acid (mRNA) was markedly elevated in the neoadventitia at 3 days post-angioplasty. By 14 days, elevated collagen VIII message was seen mainly in the neointima and this expression decreased to background levels by 90 days. The distribution of collagen VIII protein, detected using immunohistochemistry, was similar but the up-regulation lagged behind the mRNA increase by a few days. Pre-treatment of sections with pepsin highlighted variations in the organization and appearance of extracellular collagen VIII containing structures in both injured and normal vessels. New vessel formation was evident in the neoadventitia after 3 days, but there was no colocalization of type VIII collagen immunostaining with that of von Willebrand factor (a marker of endothelial cells) in the neoadventitia. These data show that up-regulation of collagen VIII in the neoadventitia is an important early marker of the coronary arterial response to injury, and is not associated with new vessel formation.

Asunto(s)

Angioplastia Coronaria con Balón , Colágeno Tipo VIII/metabolismo , Músculo Liso Vascular/fisiología , Neovascularización Fisiológica/fisiología , Regulación hacia Arriba/fisiología , Animales , Movimiento Celular , Vasos Coronarios , Femenino , Hibridación in Situ , ARN Mensajero , Porcinos , Factor de von Willebrand/metabolismo

RESUMEN

OBJECTIVE: To determine if there are abnormalities in fibrillin 1-containing microfibrils in the extracellular matrix (ECM) of primary dermal fibroblasts explanted from patients with systemic sclerosis (SSc). METHODS: Explanted fibroblasts from unaffected skin of 12 SSc patients were used to examine fibrillin 1-containing microfibrils by immunofluorescence (IF) using a monoclonal antibody (mAb) to fibrillin 1. Metabolic labeling of the fibroblast cultures was used to study the synthesis, secretion, and processing of fibrillin 1, as well as to observe microfibril formation and stability. Microfibrils elaborated by the SSc cells were analyzed by electron microscopy for ultrastructural abnormalities, and the results were confirmed by immunoblotting. RESULTS: Control and SSc fibroblasts displayed a prominent meshwork of fibrillin 1-containing microfibrils when visualized by IF using a fibrillin 1 mAb. Paradoxically, metabolic studies indicated a paucity of fibrillin 1 in the ECM in the majority of the SSc fibroblast strains. Subsequent rotary-shadowed electron microscopy revealed reduced amounts of and ultrastructural abnormalities in the microfibrils elaborated by all strains of SSc cells. Immunoblots confirmed the lack of the high molecular weight form of fibrillin 1 in the SSc fibroblasts of Choctaw American Indians. Finally, in vitro studies indicated that the amount of fibrillin 1 in the ECM of SSc cells diminished at a faster rate than the amount of fibrillin 1 in the ECM of control cells with time. CONCLUSION: Although SSc fibroblasts assemble microfibrils, these microfibrils are unstable, suggesting that an inherent defect of fibrillin 1-containing microfibrils may play a role in the pathogenesis of SSc.

Asunto(s)

Dermis/citología , Fibroblastos/ultraestructura , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/metabolismo , Esclerodermia Sistémica/patología , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Proteínas de la Matriz Extracelular/inmunología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Cinética , Masculino , Microfibrillas/metabolismo , Proteínas de Microfilamentos/inmunología , Persona de Mediana Edad , Esclerodermia Sistémica/etiología , Esclerodermia Sistémica/metabolismo

RESUMEN

Photoaged skin is characterized by coarse and fine wrinkles. The mechanisms of wrinkle formation are undetermined, but appear to be due to changes within the matrix of the dermis and at the dermal-epidermal junction. Previous studies have identified marked reductions in procollagens I and III, collagen VII, and the fibrillin-rich microfibrillar apparatus in this area. Topically applied all-trans retinoic acid can repair photoaged dermal matrix, but this takes at least 6 mo of treatment. In this study, we have examined the abundance and distribution of fibrillin-1 prior to, and following, 192 wk of all-trans retinoic acid treatment. We have further developed a short-term protocol to determine the utility of potential repair agents, using fibrillin-1 as the marker for outcome. Individuals with clinically assessed severe photoaging were recruited to the study (n = 8). 0.025% all-trans retinoic acid, 5% sodium lauryl sulfate (irritant control), or vehicle were applied under occlusion to photoaged extensor forearm. A fourth control area was also occluded. After 96 h, punch biopsies were taken under local anesthesia and processed for either transmission electron microscopy or snap frozen. Frozen sections were prepared for immunohistochemistry and in situ hybridization immunohistochemistry. Electron microscopy revealed aberrant elastic fibers in the papillary dermis of photoaged forearm skin, with sparse microfibrillar apparatus and interstitial collagen. After application of 0.025% all-trans retinoic acid, there was increased deposition of both these dermal matrix components, with the aberrant elastic fibers no longer apparent. Significant increases (p < 0.05) were observed at the protein and mRNA levels for fibrillin-1 following all-trans retinoic acid and sodium lauryl sulfate treatments, with all-trans retinoic acid having a significantly greater effect than irritant control (p < 0.001); however, neither application had significant effect on the abundance of collagen VII or its mRNA. Investigation of collagen I synthesis revealed no difference following treatments. To ascertain the clinical relevance of using fibrillin-1 as a marker for photoaging, facial skin was biopsied at baseline and after long-term (192 wk) topical all-trans retinoic acid treatment (n = 5). Biopsies were wax-embedded and sections prepared for immunohistochemistry for fibrillin-1. Significant increases in the abundance of the microfibrillar apparatus was observed proximal to the dermal- epidermal junction (p < 0.001) following long-term all-trans retinoic acid application. This study indicates that all-trans retinoic acid can significantly affect fibrillin-1 content in photoaged skin. Furthermore, fibrillin-1 can be used as a "reporter" molecule in short-term protocols for testing the utility of topical agents in the repair of photoaged skin.

Asunto(s)

Proteínas de Microfilamentos/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Biomarcadores , Esquema de Medicación , Evaluación Preclínica de Medicamentos/métodos , Femenino , Fibrilina-1 , Fibrilinas , Histocitoquímica , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Piel/metabolismo , Piel/patología , Factores de Tiempo , Tretinoina/uso terapéutico

RESUMEN

Fibrillins are large, cysteine-rich glycoproteins that form microfibrils and play a central role in elastic fibrillogenesis. Fibrillin-1 and fibrillin-2, encoded by FBN1 on chromosome 15q21.1 and FBN2 on chromosome 5q23-q31, are highly similar proteins. The finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively, has highlighted their essential role in the development and homeostasis of elastic fibres. MFS is characterized by cardiovascular, skeletal and ocular abnormalities, and CCA by long, thin, flexed digits, crumpled ears and mild joint contractures. Although mutations arise throughout FBN1, those clustering within exons 24-32 are associated with the most severe form of MFS, so-called neonatal MFS. All the mutations described in CCA occur in the "neonatal region" of FBN2. Both MFS and CCA are thought to arise via a dominant negative mechanism. The analysis of mouse mutations has demonstrated that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly. In the current investigation, we have analysed the classical mouse mutant shaker-with-syndactylism using a positional candidate approach and demonstrated that loss-of-function mutations outside the "neonatal region" of Fbn2 cause syndactyly in mice. These results suggest that phenotypes distinct from CCA may result in man as a consequence of mutations outside the "neonatal region" of FBN2.

Asunto(s)

Glicoproteínas/genética , Proteínas de Microfilamentos/genética , Mutación , Sindactilia/genética , Secuencia de Aminoácidos , Animales , Exones , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Heterocigoto , Ratones , Ratones Endogámicos , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Eliminación de Secuencia , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

BACKGROUND: Several of the characteristic clinical features of photoaged skin, including wrinkling, are thought to be dependent on changes in the dermal matrix brought about by chronic sun exposure. Such changes include reductions in collagens I, III and VII, an increase in elastotic material in the reticular dermis and a marked reduction in the microfibrillar glycoprotein fibrillin. OBJECTIVES: To examine whether type VI collagen, a microfibrillar collagen necessary for cell-cell and cell-matrix communication, is affected by the photoageing process. METHODS: Six healthy volunteers with moderate to severe photoageing were enrolled into the study. Immunohistochemistry and in situ hybridization histochemistry were used to examine the levels of type VI collagen in photoprotected and photoaged sites. RESULTS: In photoprotected skin, type VI collagen was concentrated in the papillary dermis immediately below the dermal-epidermal junction, around blood vessels, hair follicles and glandular structures. The distribution of type VI collagen was unchanged in photoaged skin, although we observed an increase in the abundance of the alpha3 chain of collagen VI in the upper papillary dermis, at its junction with the dermal-epidermal junction (P < 0.05). No alterations were observed for any alpha chain at the mRNA level. CONCLUSIONS: These studies suggest that chronic sun exposure (photoageing) has little or no effect on either the distribution, abundance or levels of expression of type VI collagen in human skin. Thus, type VI collagen, unlike other matrix components so far studied, appears to be relatively unaffected by the photoageing process.

Asunto(s)

Colágeno/metabolismo , Envejecimiento de la Piel/fisiología , Piel/metabolismo , Anciano , Colágeno/genética , Femenino , Técnica del Anticuerpo Fluorescente , Antebrazo , Humanos , Sueros Inmunes , Hibridación in Situ , Masculino , Persona de Mediana Edad , ARN Mensajero/genética

RESUMEN

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Asunto(s)

Microfibrillas/química , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Automatización , Sitios de Unión de Anticuerpos , Biopolímeros/química , Biopolímeros/inmunología , Biopolímeros/metabolismo , Bovinos , Células Cultivadas , Factor de Crecimiento Epidérmico/química , Fibrilinas , Fibroblastos , Técnica del Anticuerpo Fluorescente , Oro Coloide/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Microfibrillas/inmunología , Microfibrillas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/inmunología , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica de Transmisión de Rastreo , Modelos Moleculares , Datos de Secuencia Molecular , Tono Muscular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Tomografía/métodos

RESUMEN

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.

Asunto(s)

Proteínas de la Matriz Extracelular/ultraestructura , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Desplazamiento del Cristalino/genética , Elasticidad , Proteínas de la Matriz Extracelular/genética , Fibrilinas , Humanos , Síndrome de Marfan/genética , Proteínas de Microfilamentos/genética , Modelos Estructurales

RESUMEN

Constructs of each of the three chains of type VI collagen were generated and examined in an in vitro transcription/translation assay supplemented with semipermeabilized cells. Each of the constructs when used in the in vitro system was shown to be glycosylated and to undergo intracellular assembly, the extent of which was determined by the nature of the C-terminal globular domains. All three chains containing the C1 domain formed monomers; however, the C2 domain was required for dimer and tetramer formation. In the case of the full-length alpha2(VI) chain, monomers, dimers, and tetramers formed in a time-dependent manner. Although the splice variant alpha2(VI)C2a could form monomers, it was unable to form dimers and tetramers. Similar results to the alpha2(VI) chain were found for the full-length alpha1(VI) chain, although assembly was at a slower rate. In the case of the alpha3(VI) chain containing both C1 and C2 domains only monomers were observed. Addition of the C3, C4, and C5 did not change this pattern. Homology modeling suggested that a 10-amino acid insertion in the C2 domain of the alpha3(VI) chain may interfere with dimer formation. A near full-length construct of the alpha3(VI) chain only formed monomers but was shown to facilitate tetramer formation in cotranslation experiments.

Asunto(s)

Colágeno/química , Colágeno/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , ADN Complementario/metabolismo , Dimerización , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pepsina A/metabolismo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética , Células Tumorales Cultivadas

Asunto(s)

Citoesqueleto de Actina/química , Proteínas de Microfilamentos/fisiología , Citoesqueleto de Actina/ultraestructura , Tejido Conectivo/química , Tejido Conectivo/ultraestructura , Oftalmopatías/etiología , Proteínas del Ojo/química , Proteínas del Ojo/ultraestructura , Fibrilinas , Humanos , Microscopía Electrónica de Rastreo

Asunto(s)

Colágeno/ultraestructura , Animales , Matriz Extracelular/metabolismo , Microscopía Electrónica de Rastreo/métodos

RESUMEN

The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.

Asunto(s)

Proteínas de Microfilamentos/metabolismo , Subtilisinas/metabolismo , Retículo Endoplásmico/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Furina , Glicosilación , Humanos , Proteínas de Microfilamentos/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacología , Conformación Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

The zonular filaments from the eyes of cows are rich in microfibrils containing fibrillin. Tensile tests, stress-relaxation tests and X-ray diffraction studies were used to study the relationship between the mechanical behaviour of zonular filaments and the molecular packing and structure of the fibrillin-rich microfibrils. Zonular filaments show a non-linear (J-shaped) stress-strain curve and appreciable stress-relaxation. It is proposed that the non-linear properties are due to local variations in waviness in the microfibrils or assemblies of microfibrils, which straighten out and become more regularly aligned with strain. Previous and current X-ray diffraction results consistently show a partial ordering of microfibrils in zonular filaments into staggered aggregates which become more ordered and laterally aligned on stretching. Although the removal and re-addition of Ca(2+) is known to change the molecular structure of fibrillin, no effect was observed on the tensile properties of the zonular filaments. It is hypothesised that strain-induced deformation in the supramolecular aggregate packing may not be Ca(2+)-sensitive but could dominate the mechanical behaviour of microfibrillar arrays in zonular filaments.

Asunto(s)

Bovinos/anatomía & histología , Ojo/ultraestructura , Microfibrillas/fisiología , Microfibrillas/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Animales , Fenómenos Biomecánicos , Calcio/administración & dosificación , Elasticidad , Fibrilinas , Microfibrillas/química , Difracción de Rayos X

RESUMEN

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9-11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

Asunto(s)

Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calnexina , Calreticulina , Línea Celular , Dimerización , Exones , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Glicina/análisis , Humanos , Proteínas de Microfilamentos/genética , Prolina/análisis , Biosíntesis de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/metabolismo , Transcripción Genética , Transfección

RESUMEN

Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.

Asunto(s)

Metaloendopeptidasas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , ADN Complementario , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Fibrilinas , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transcripción Genética , Transfección

RESUMEN

Chronic sun exposure results in photoaged skin with deep coarse wrinkles and loss of elasticity. We have examined the distribution and abundance of fibrillin-rich microfibrils, key structural components of the elastic fiber network, in photoaged and photoprotected skin. Punch biopsies taken from photoaged forearm and from photoprotected hip and upper inner arm of 16 subjects with a clinical range of photoaging were examined for fibrillin-1 and fibrillin-2 expression and microfibril distribution. In situ hybridization revealed decreased fibrillin-1 mRNA but unchanged fibrillin-2 mRNA levels in severely photoaged forearm biopsies relative to photoprotected dermal sites. An immunohistochemical approach demonstrated that microfibrils at the dermal-epidermal junction were significantly reduced in moderate to severely photoaged forearm skin. Confocal microscopy revealed that the papillary dermal microfibrillar network was truncated and depleted in photoaged skin. These studies highlight that the fibrillin-rich microfibrillar network associated with the upper dermis undergoes extensive remodeling following solar irradiation. These changes may contribute to the clinical features of photoaging, such as wrinkle formation and loss of elasticity.

Asunto(s)

Dermis/metabolismo , Epidermis/metabolismo , Proteínas de Microfilamentos/metabolismo , Envejecimiento de la Piel/patología , Adulto , Anciano , Anciano de 80 o más Años , Dermis/patología , Epidermis/patología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Proteínas de Microfilamentos/genética , Microscopía Confocal , Persona de Mediana Edad , ARN Mensajero/metabolismo , Luz Solar/efectos adversos

RESUMEN

Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.

Asunto(s)

Tejido Conectivo/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas de Microfilamentos/metabolismo , Envejecimiento , Sustitución de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Desplazamiento del Cristalino/genética , Retículo Endoplásmico/metabolismo , Exones/genética , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/ultraestructura , Fibrilina-1 , Fibrilinas , Humanos , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/ultraestructura , Microscopía Electrónica , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Polímeros/metabolismo , Prolina/genética , Prolina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura