Biomedical subjects

Cay M Kielty

Publications and source records attributed to Cay M Kielty.

34 records · Page 2Linked to original sources

PubMed2004

Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers.

Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of approximately 180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of approximately 700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(VIII)(3). Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of approximately 114 degrees and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences.

Cell Line↗

PubMed2004

Direct cell contact influences bone marrow mesenchymal stem cell fate.

Adult bone marrow-derived mesenchymal stem cells (MSC) can differentiate into various cell types of mesenchymal origin, but mechanisms regulating such cellular changes are unclear. We have conducted co-culture experiments to examine whether mesenchymal stem cell differentiation is influenced by indirect or direct contact with differentiated cells. Cultured adult mesenchymal stem cells showed some characteristics of synthetic state vascular smooth muscle cells (SMC). When co-cultured with vascular endothelial cells (EC) without cell contact, they exhibited abundant well-organised smooth muscle alpha-actin (alpha-actin) filaments. Direct co-culture with endothelial cells resulted in increased smooth muscle alpha-actin mRNA and protein, yet also comprehensive disruption of smooth muscle alpha-actin filament organisation. In order to assess whether these cell contact effects on mesenchymal stem cells were cell type specific, we also analysed direct co-cultures of mesenchymal stem cells with dermal fibroblasts. However, these experiments were characterised by the appearance of abundant spindle-shaped myofibroblast-like cells containing organised smooth muscle alpha-actin filaments. Thus, direct contact with distinct differentiated cells may be a critical determinant of mesenchymal stem cell fate in blood vessels and other connective tissues.

Actin Cytoskeleton↗

PubMed2004

A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts.

Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining. We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1 x 10(6) cells/cm(2), 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.

Biocompatible Materials↗

PubMed2004

Substrate-dependent morphology of supramolecular assemblies: fibrillin and type-VI collagen microfibrils.

Substrate hydrophobicity/hydrophilicity has previously been shown to affect the morphology and biological function of isolated proteins. We have employed atomic force microscopy to investigate substrate dependent morphologies of two biochemically distinct native supramolecular assemblies: fibrillin and type-VI collagen microfibrils. These morphologically heterogeneous microfibrillar systems are found in many vertebrate tissues where they perform structural and cell-signaling roles. Fibrillin microfibrils adsorbed to a hydrophilic mica substrate adopted a diffuse morphology. Fibrillin microfibrils adsorbed to mica coated with poly-L-lysine or to borosilicate glass substrates had a more compact morphology and a directional asymmetry to the bead, which was not present on mica alone. Intermediate morphologies were observed along a substrate gradient. The classical double-beaded appearance of type-VI collagen microfibrils was evident on mica coated with poly-L-lysine and on glass. On hydrophilic mica, morphology was severely disrupted and there was a major conformational reorganization along the whole collagen microfibril repeat. These observations of substrate dependent conformation have important implications for the interpretation of data from in vitro protein interaction assays and cellular signaling studies. Furthermore, conformational changes may be induced by local charge environments in vivo, revealing or hiding binding sites.

Actin Cytoskeleton↗

PubMed2003

Fibrillin microfibrils are stiff reinforcing fibres in compliant tissues.

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Aged↗

PubMed2003

Raman microscopy and X-ray diffraction, a combined study of fibrillin-rich microfibrillar elasticity.

Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfibrillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman microscopy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.

Animals↗

PubMed2003

The supramolecular organization of collagen VI microfibrils.

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.

Animals↗

PubMed2003

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins.

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.

Antibodies↗

PubMed2003

Expression of elastic components in healthy and varicose veins.

This study evaluates possible changes in the synthesis/degradation of elastic components of the vein wall in an attempt to explain the development of varicosis. Healthy and varicose saphenous veins were subjected to immunohistochemical analysis using anti-elastin, anti-fibrillin-1, anti-elastase, anti-transforming growth factor (TGF)-beta and anti-latent TGFbeta binding protein (LTBP)-2 monoclonal antibodies. In situ hybridization was performed using specific probes for tropoelastin and fibrillin-1. In healthy veins, elastin and fibrillin-1 showed even, overlapping distribution patterns indicating their particular abundance in the adventitia and at the intima/media interface. The expression of tropoelastin and fibrillin-1 was high in smooth muscle cells bordering the elastic laminae. Elastin, fibrillin-1, and cells expressing fibrillin-1 and tropoelastin mRNA showed a patchy disorganized pattern, particularly in the proximal varicose segments of patients under 50 years of age. Enhanced elastase activity was noted in both control and varicose specimens from elderly subjects. Varicose veins specimens showed greater LTBP-2 and TGF expression. Both molecules were detected in the subendothelium and the media, particularly in areas of marked injury. Our findings suggest that the development of the varicose condition involves a restructuring of the elastic component of the vein wall, perhaps as a consequence of changes in the transcription mechanisms of muscle layer cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Paxillin-associated focal adhesion involvement in perinatal pulmonary arterial remodelling.

Birth is followed by remodelling of the actin cytoskeleton of pulmonary arterial smooth muscle cells, then by extracellular matrix deposition. Hypothesising that the cell/matrix adhesions would also be remodelled, we investigated the expression, localisation and biochemical characteristics of the focal adhesion protein paxillin in vivo, in vessels from normal and pulmonary hypertensive neonatal piglets. Initially we showed that in intact porcine pulmonary arteries exposed to cytochalasin D there was a reduction filamentous actin accompanied by a reduction in paxillin-associated focal adhesions, similar to that seen in cultured pulmonary arterial smooth muscle cells. Vessels from normal and hypoxic animals were found to have two isoforms of paxillin, of 60 and 66 kDa with pI values of 6.7-4.2. Transient changes occurred during the first 14 days of life. Between birth and 6 days there was a reduction in the amount of both paxillin isoforms, a shift to more acidic pI values and an increase in paxillin phosphorylation. Simultaneously, immunostaining showed a transient reduction in paxillin expression, a change temporally and spatially associated with a previously demonstrated reduction in actin. Findings are consistent with an immediate postnatal spatial reorganisation of paxillin-associated focal adhesions. Paxillin content and remodelling was abnormal in pulmonary hypertensive arteries, the response varying according to postnatal age.

Actins↗

PubMed2002

Mutation of the signal peptide region of the bicistronic gene DSPP affects translocation to the endoplasmic reticulum and results in defective dentine biomineralization.

Dentine dysplasia type II is an autosomal dominant disorder in which mineralization of the dentine of the primary teeth is abnormal. On the basis of the phenotypic overlap between, and shared chromosomal location with, dentinogenesis imperfecta type II, a second disorder of dentine mineralization, it has been proposed that the two conditions are allelic. As recent studies have shown that dentinogenesis imperfecta type II results from mutation of the bicistronic dentine sialophosphoprotein gene (DSPP ), we have tested this hypothesis by sequencing DSPP in a family with a history of dentine dysplasia type II. Our results have shown that a missense change, which causes the substitution of a tyrosine for an aspartic acid in the hydrophobic signal peptide domain of the protein, underlies the phenotype in this family. Biochemical analysis has further demonstrated that this mutation causes a failure of translocation of the encoded proteins into the endoplasmic reticulum, and is therefore likely to lead to a loss of function of both dentine sialoprotein and dentine phosphoprotein.

Dentin↗

PubMed2002

Microfibril-associated glycoprotein-2 interacts with fibrillin-1 and fibrillin-2 suggesting a role for MAGP-2 in elastic fiber assembly.

Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils. The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2). Little is known about how microfibrillar proteins interact to support fiber assembly. We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands. Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2. Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding. This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1. The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction. Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1. Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin. These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly. The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.

Amino Acid Sequence↗

PubMed2002

Elastic fibres.

Elastic fibres are essential extracellular matrix macromolecules comprising an elastin core surrounded by a mantle of fibrillin-rich microfibrils. They endow connective tissues such as blood vessels, lungs and skin with the critical properties of elasticity and resilience. The biology of elastic fibres is complex because they have multiple components, a tightly regulated developmental deposition, a multi-step hierarchical assembly and unique biomechanical functions. However, their molecular complexity is at last being unravelled by progress in identifying interactions between component molecules, ultrastructural analyses and studies of informative mouse models.

Animals↗

PubMed2002

Fibrillin: from microfibril assembly to biomechanical function.

Fibrillins form the structural framework of a unique and essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity. Their biological importance is emphasized by the linkage of fibrillin mutations to Marfan syndrome and related connective tissue disorders, which are associated with severe cardiovascular, ocular and skeletal defects. These microfibrils have a complex ultrastructure and it has proved a major challenge both to define their structural organization and to relate it to their biological function. However, new approaches have at last begun to reveal important insights into their molecular assembly, structural organization and biomechanical properties. This paper describes the current understanding of the molecular assembly of fibrillin molecules, the alignment of fibrillin molecules within microfibrils and the unique elastomeric properties of microfibrils.

Animals↗

PubMed2002

Expression of latent TGF-beta binding proteins and association with TGF-beta 1 and fibrillin-1 following arterial injury.

OBJECTIVES: Transforming growth factor-beta (TGF-beta), a potent regulator of wound healing and scar formation, is thought to have a key role in the response to arterial injury. Latent TGF-beta binding proteins (LTBPs), members of the fibrillin superfamily, govern TGF-beta1 release, targeting and activation in vitro and also play a role as structural components of fibrillin-rich microfibrils. Despite the potential of LTBPs to modulate the response to arterial injury through either or both of these mechanisms, as yet their expression and function in the injured vasculature remain poorly defined. METHODS: In this study, a porcine model of coronary angioplasty was used to investigate LTBP-1 and LTBP-2 synthesis and their association with TGF-beta 1 and fibrillin-1. RESULTS: After angioplasty, increased LTBP-1 and LTBP-2 immunostaining was detected in a similar distribution to increased TGF-beta 1 expression in the neointima and in the neoadventitia. Overnight organ cultures revealed the formation of large latent TGF-beta 1 complexes containing LTBP-1. Increased LTBP-1 proteolysis after arterial injury correlated with increased active and latent TGF-beta levels. LTBP-2 synthesis increased in response to arterial injury but was neither present in large latent complexes nor proteolytically processed. LTBP-1 and LTBP-2 both co-localised to fibrillin-rich fibrillar structures in the neointima and adventitia. CONCLUSIONS: These data suggest that LTBP-1 may have a TGF-beta 1 binding role in the arterial response to injury, and that LTBP-1 and LTBP-2 may have a structural role in association with microfibrils within the developing neointimal lesion. LTBP-1 proteolysis is potentially an important regulatory step for TGF-beta activation in the vasculature and inhibition of proteolysis could represent a novel therapeutic modality for controlling the arterial injury response.

Angioplasty, Balloon, Coronary↗

PubMedDate not supplied

The role of transforming growth factor beta1 in the vascular system.

The transforming growth factor beta (TGFbeta) family of cytokines exert pleiotropic effects upon a wide variety of cell types. TGFbeta1 has been demonstrated to be of fundamental importance in the development, physiology and pathology of the vascular system. As the role of TGFbeta1 in these processes becomes clearer, influencing its activity for therapeutic benefit is now beginning to be investigated. This review presents an overview of the role of TGFbeta1 in the vasculature. The cellular and extracellular biology of the TGFbeta family is first addressed, followed by an overview of the function of TGFbeta1 during vascular development, atherogenesis, hypertension, and vessel injury.

Animals↗