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Biomedical subjects

C M Kielty

Publications and source records attributed to C M Kielty.

At least 37 records · Page 2Linked to original sources
PubMed1999

The supramolecular organisation of fibrillin-rich microfibrils determines the mechanical properties of bovine zonular filaments.

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.

Animals↗
PubMed1998

Calcium determines the supramolecular organization of fibrillin-rich microfibrils.

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of approximately 56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.

Animals↗
PubMed1998

The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils.

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.

Animals↗
PubMed1998

Purification of fibrillin-containing microfibrils and collagen VI microfibrils by density gradient centrifugation.

A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M(r) microfibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS-PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.

Actin Cytoskeleton↗
PubMed1998

X-Ray diffraction studies of fibrillin-rich microfibrils: effects of tissue extension on axial and lateral packing.

X-ray diffraction of hydrated fibrillin rich microfibrils, in the form of zonular filaments from bovine eyes, demonstrated meridional diffraction peaks indexing on a fundamental periodicity of approximately 56 nm in the relaxed state. The effect of sample extensions of up to 50% in length produced an increase in the axial periodicity of only 4% as judged by alteration of the diffraction peak position of the third meridional order. This effect was shown to be reversible. Further extension to 100% of the tissue rest length caused extensive deterioration in the quality of the diffraction and resulted in a more complex meridional diffraction series, where the fundamental axial periodicity also changed to a length of approximately 80 nm. The fibrillin diffraction image also contains an equatorial diffraction peak that is enhanced upon tissue extension. The measurement of the molecular spacing from the equatorial diffraction profile indicated that the closest approach of molecules gave a broad interference peak of spacing 28 nm, this is nearly twice the molecular diameter as estimated from electron microscopy of dehydrated samples.

Animals↗
PubMed1998

Cellular and extracellular biology of the latent transforming growth factor-beta binding proteins.

The latent transforming growth factor-beta binding proteins (LTBP) are a recently identified family of widely expressed multidomain glycoproteins that range in size from 125 kDa to 240 kDa. Four LTBP genes have been described, and the homology of latent transforming growth factor-beta binding proteins molecules to the fibrillins has resulted in their inclusion in the so-called 'fibrillin superfamily'. They form intracellular covalent complexes with latent transforming growth factor-beta and target these growth factors to the extracellular matrix. This review describes their structure, summarizes current understanding of their dual roles as growth factor binding proteins and components of the extracellular matrix, and highlights their significance in tissue development and disease.

Amino Acid Sequence↗
PubMed1998

Fibrillin microfibrils are reduced in skin exhibiting striae distensae.

Striae distensae (striae: stretch marks) are a common disfiguring condition associated with continuous and progressive stretching of the skin--as occurs during pregnancy. The pathogenesis of striae is unknown but probably relates to changes in those structures that provide skin with its tensile strength and elasticity. Such structures are components of the extracellular matrix, including fibrillin, elastin and collagens. Using a variety of histological techniques, we assessed the distribution of these extracellular matrix components in skin affected by striae. Pregnant women were assessed for the presence of striae, and punch biopsies were obtained from lesional striae and adjacent normal skin. Biopsies were processed for electron microscopy, light microscopy and immunohistochemistry. For histological examination, 7 microns frozen sections were stained so as to identify the elastic fibre network and glycosaminoglycans. Biopsies were also examined with a panel of polyclonal antibodies against collagens I and III, and fibrillin and elastin. Ultrastructural analysis revealed alterations in the appearance of skin affected by striae compared with that of normal skin in that the dermal matrix of striae was looser and more floccular. Light microscopy revealed an increase in glycosaminoglycan content in striae. Furthermore, the number of vertical fibrillin fibres subjacent to the dermal-epidermal junction (DEJ) and elastin fibres in the papillary dermis was significantly reduced in striae compared with normal skin. The orientation of elastin and fibrillin fibres in the deep dermis showed realignment in that the fibres ran parallel to the DEJ. However, no significant alterations were observed in any other extracellular matrix components. This study identifies a reorganization and diminution of the elastic fibre network of skin affected by striae. Continuous strain on the dermal extracellular matrix, as occurs during pregnancy, may remodel the elastic fibre network in susceptible individuals and manifest clinically as striae distensae.

Actin Cytoskeleton↗
PubMed1997

Fibrillin-rich microfibrils: an X-ray diffraction study of the fundamental axial periodicity.

Microfibrils are ubiquitous matrix polymers which are thought to provide elastic properties in all extracellular matrix structures. The major component of the elastic microfibrils is the protein fibrillin; its molecular structure is unknown. In electron microscopy, microfibrils appear as beaded structures exhibiting a variable periodicity, indicating that they may be elastomeric. The X-ray diffraction of fibrillin-rich microfibrils in the form of zonular filaments from bovine eyes exhibits meridional diffraction peaks indexing on a fundamental periodicity of 55 nm in the relaxed state. The application of a 40% extension produced a lengthening of the periodicity by 3% as judged by alteration of the D spacing of the principal peaks. This effect was shown to be reversible. Changes in the periodicity of the meridional reflections indicate changes in the fundamental structure of the microfilaments, but cannot account for all long range elastomeric properties of fibrillin-containing microfibrils.

Actin Cytoskeleton↗
PubMed1997

Microfibrillar elements of the dermal matrix.

Connective tissue microfibrils are key structural elements of the dermal matrix which play major roles in establishing and maintaining the structural and mechanical integrity of this complex tissue. Type VI collagen microfibrils form extensive microfibrillar networks which intercalate between the major collagen fibrils and are juxtaposed to cellular basement membranes, blood vessels and other interstitial structures. Fibrillin microfibrils define the continuous elastic network of skin, and are present in dermis as microfibril bundles devoid of measureable elastin extending from the dermal-epithelial junction and as components of the thick elastic fibres present in the deep reticular dermis. Electron microscopic analyses have revealed both classes of microfibrils to have complex ultrastructures. The ability to isolate intact native microfibrils from skin has enabled a combination of high resolution and biochemical techniques to be applied to elucidate their structure:function relationships. These approaches have generated new information about their molecular organisation and physiological interactions in health and disease.

Actin Cytoskeleton↗
PubMed1997

Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans.

Elasticity and resilience of the skin are determined largely by the elastin framework, whose microfibrillar scaffold is composed of fibrillin. To date, the spatial and temporal patterns of expression of human elastin and fibrillin during would healing have not been described. Ninety healthy human subjects underwent 4 mm cutaneous punch biopsy wounds from the upper inner arm, which were re-excised from day 3 to 3 months post-wounding. There were marked changes in the patterns of distribution and the amounts of elastin and fibrillin in sun-protected skin with ageing. However, there were no major age-related differences in the mRNA levels for elastin, fibrillin-1 and fibrillin-2 using in situ hybridization. Elastin and fibrillin appeared in greatest amounts in the wounds of the elderly, particularly in females. A regenerative pattern of elastin and fibrillin arcades at the dermo-epidermal junction was observed in the wounds of aged subjects. mRNA expression of elastin was greatest in the wounds of the aged (from day 3 to day 14 post-wounding) with a similar spatial and temporal pattern to fibrillin-1 expression; this suggests that fibrillin-1 is the major contributor to dermal elastic fibre construction during wound repair. Fibrillin-2 was expressed only in the wounds of the aged and expression was confined to areas proximal to dermal blood vessels. The clear-cut differences in the localization of the two members of the fibrillin family suggest that these have well-defined roles in normal skin and wound tissue. In summary, these data indicate that ageing is associated with increased expression of fibrillin and elastin during acute wound healing and concomitant restoration of the papillary dermal architecture with an improved quality of scarring.

Acute Disease↗
PubMed1997

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization.

Fibrillin-containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal-epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal-epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal-epithelial junction is not well defined. We have used fresh tissues, freshly isolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin-1 and fibrillin-2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co-ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.

Animals↗
PubMed1997

Clinical features of photodamaged human skin are associated with a reduction in collagen VII.

Chronically sun-exposed or photodamaged human skin is characterized by a number of clinical features, including wrinkles. However, little is known about the molecular mechanisms that underlie these features. We investigated the hypothesis that the mechanism of wrinkle formation may involve loss of anchoring fibrils, composed mainly of collagen VII, which are important in maintaining dermal-epidermal junction integrity. Ten volunteers with moderate to severe photodamage of dorsal forearm skin were recruited to the study. Using immunohistochemistry, transmission electron microscopy and in situ hybridization, we compared collagen VII protein and mRNA content of photodamaged forearm skin with that of sun-protected hip and upper inner arm skin from the same subjects. Numbers of anchoring fibrils per linear microns of basement membrane (mean +/- SEM) were significantly lower in photodamaged skin (1.79 +/- 0.10) as compared with sun-protected hip (2.28 +/- 0.11) and upper inner arm skin (2.21 +/- 0.10) (P < 0.01), and similarly keratinocyte expression of collagen VII mRNA, quantitated as number of positively stained keratinocytes per high power field, was significantly reduced in photodamaged skin (6.3 +/- 2.5) as compared with sun-protected hip (20.0 +/- 5.6) and upper inner arm skin (17.7 +/- 4.9) (P < 0.001). Semiquantitative assessment of immunohistochemical staining for collagen VII showed a non-significant reduction in photodamaged skin as compared with sun-protected skin. We propose that reduced content of collagen VII in photodamaged skin contributes to wrinkle formation by weakening the bond between the dermis and epidermis.

Aged↗
PubMed1996

Type VIII collagen is a product of vascular smooth-muscle cells in development and disease.

Type VIII collagen is a short-chain collagen with considerable similarity to type X collagen. We have generated chain-specific antibodies to the alpha 1 and alpha 2 chains of type VIII collagen, and used them as probes to examine the synthesis of type VIII collagen by vascular smooth muscle cells (VSMC). In addition, chain-specific oligonucleotides have been used in reverse transcriptase-PCR (RT-PCR) reactions with RNA extracted from cultured smooth muscle cells in culture and from freshly isolated vascular tissues. Radiolabelling of VSMC in culture and immunoprecipitation with chain-specific antibodies showed that both chains were expressed. Lower levels of type VIII collagen were found in adult VSMC than in neonatal VSMC. RT-PCR showed that both chains were expressed in tissues as well as cells in culture. The results indicate that type VIII collagen is a product of VSMC of normal adult vessels and is expressed at high levels by VSMC in vascular lesions.

Adult↗
PubMed1996

Fibrillin: evidence that chondroitin sulphate proteoglycans are components of microfibrils and associate with newly synthesised monomers.

We have investigated the potential association of proteoglycans with intact fibrillin-containing microfibrils from foetal bovine elastic tissues and with newly synthesised fibrillin in human and bovine cell cultures. Microfibril integrity was disrupted by chondroitinase ABC lyase and chondroitinase AC lyase, but not by keratanase or hyaluronidase. Following chondroitinase treatment, beads were disrupted but the underlying fibrillar scaffold appeared intact. Cuprolinic blue was prominently associated with beaded domains at a critical electrolyte concentration. Electron-dense rods were often associated with cuprolinic blue-treated microfibrils isolated from fixed tissues. Positive staining revealed charged foci at the beads. Newly synthesised fibrillin could be labelled with 35S TransLabel, [3H]glucosamine or 35SO4 but its electrophoretic mobility was not influenced by treatment with chondroitinase ABC or AC lyase. A diffuse 35SO4-labelled chondroitinase-sensitive component with a resistant band (Mr 35000) co-immunoprecipitated with fibrillin. These experiments indicate that chondroitin sulphate proteoglycans associate with fibrillin and contribute to microfibril assembly. This association has major implications for microfibril function in health and disease.

Actin Cytoskeleton↗
PubMed1996

The expression of types X and VI collagen and fibrillin in rat mandibular condylar cartilage. Response to mastication forces.

Types X and VI collagen and fibrillin were localized by in situ hybridization and immunohistochemical methods in the mandibular condyles of rats, and the response of these molecules to post-weaning diets of soft food, ordinary pellets, or hardened pellets was studied. Type X collagen was synthesized, particularly in conditions of soft food consistency, by cells in the perichondrium-periosteum and in the bone and by cells at the erosion front between cartilage and bone. Type X collagen synthesis diminished under higher compression forces due to mastication and with increasing age. Type VI collagen and fibrillin were synthesized by cells in the perichondrium-periosteum and by chondrocytes and by stromal osteoblasts and were not modified by higher mechanical forces. In contrast to previous findings in the growth plate of long bones, type X collagen in the mandibular condyle was not synthesized by hypertrophic chondrocytes but was associated with cells of the osteoblastic rather than the chondroblastic phenotype.

Animals↗
PubMed1995

Truncated profibrillin of a Marfan patient is of apparent similar size as fibrillin: intracellular retention leads to over-N-glycosylation.

We studied profibrillin-1 (proFib) synthesis and microfibril formation in cultured fibroblasts from an individual with severe Marfan syndrome harboring a premature stop codon (W2756ter) in one FBN1 allele. Rotary shadowing analysis of extracellular matrix produced by these cells revealed the presence of only a very few intact microfibrils which showed marked disorganisation within the interbeaded domains. Metabolic pulse-chase studies identified intracellularly a population of truncated proFib molecules which were secreted more slowly than the normal proFib derived from the normal allele. Culture media contained strikingly reduced amounts of wild-type proFib in comparison to fibrillin (Fib). Our findings imply that (1) the truncated proFib is secreted and disturbs microfibril assembly; (2) the mutation is probably close to a putative cleavage site in the proFib C terminus necessary for the conversion of proFib to Fib; (3) the truncated proFib is over-N-glycosylated due to intracellular retention rather than incomplete cleavage of proFib with persistence of N-glycosylated sites; (4) not all potential N-glycosylation sites in proFib seem to be normally used, since we could produce over-N-glycosylated proFib in normal cells by brefeldin A mediated intracellular captivation and subsequent appearance of over-glycosylated Fib in culture medium upon removal of the compound. It is conceivable that post-translational over-modification might be important for modulating the phenotype of FBN1 mutations in Marfan syndrome.

Actin Cytoskeleton↗