C. Fornieri

Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers

Solutions of tropoelastin incubated under different experimental conditions were examined by electron microscopy after negative staining and after fixation and embedding. Below 37 degrees C only polymorphous structureless elements of variable size could be found. In samples kept for a few minutes at 40 degrees C, flexible, isolated filaments of 5 nm diameter and variable length, together with a few small aggregates of filaments, were seen. No single filaments, but only bundles of filaments were detectable after incubation at 40 degrees C for longer than 5-10 min. Tropoelastin kept at 40 degrees C for longer than 10 hr formed a white precipitate, which, when fixed and embedded as in conventional electron microscopy, consisted of 0.5-2 microns thick, amorphous and branching fibers, identical to those seen in identically processed normal tissues. From these observations a model for the assembly and structure of elastic fibers is proposed.

Role of the extracellular matrix in age-related modifications of the rat aorta. Ultrastructural, morphometric, and enzymatic evaluations.

Connective tissues such as blood vessels are known to be greatly affected by age because of impaired functional properties and increased susceptibility to diseases. With the aim of providing further information on the role of the extracellular matrix in age-related modifications, we investigated the aorta in the rat model from birth to senescence by means of morphological and morphometric observations and by evaluation of lysyl oxidase activity. Results focused on the dramatic vascular rearrangements due to progressive fibrosis of the extracellular matrix and on prominent elastin modifications. The presence of lysyl oxidase activity, even in the oldest animals, might be at least partly responsible for the increased stiffness of the aging extracellular matrix. The striking age-related remodeling of the aortic architecture and the alterations of the interactions between cellular and extracellular compartments might greatly influence the functional properties of the arterial wall in senescence, at least contributing to the consequences of some apparently age-related vascular disorders.

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

Extracellular matrix modifications in rat tissues of different ages. Correlations between elastin and collagen type I mRNA expression and lysyl-oxidase activity

We used a rat model to correlate age, matrix gene expression and lysyl oxidase activity in three connective tissues, skin, aorta and lung. By in situ hybridization, we showed that intense collagen type I and elastin mRNA expression were limited to a brief postnatal period. Although there were some organ-specific differences, the mRNA abundance for these two scleroproteins drastically diminished with time. Thus, the majority of mesenchymal cells in young (60 days) and old (720 days) animals, appeared to be in a quiescent state, consistent with the slow turnover of these two scleroproteins. We also measured the activity of lysyl oxidase, an enzyme which plays a crucial role in the formation of crosslinks in both procollagen and tropoelastin molecules. In all the organs investigated, we observed a tissue-dependent pattern of activity. Moreover in this study we focused on the importance of gene matrix expression in evaluating lysyl oxidase activity of aging tissues.

The ultrastructure of elastin revealed by freeze-fracture electron microscopy

Abstract The ultrastructural organization of the elastin from beef ligamentum nuchae has been investigated by comparing fracture surfaces of stretched and relaxed specimens. When fractured both parallel and transverse to the fibre, unstretched elastin appears as a disordered granular material. In stretched material a filamentous organization in relation to the applied force becomes evident. The filamentous organization becomes more pronounced as the elongation of the fibre increases. Stretching by 180–200% produces filaments of about 5nm across which exhibit subunits along their long axes. It is concluded that elastin is organized as globules of about 5nm across arranged in long filaments which form a three-dimensional network and only become visible after stretching.

Study of elastic fiber organization by scanning force microscopy.

Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligamentum nuchae is attempted.

Structure and composition of the elastin fibre in normal and pathological conditions

Abstract Elastin fibres are present in all connective tissues of vertebrates, and are particularly abundant in those undergoing physiological mechanical stress. Elastin, the main component of the fibres, is secreted as soluble tropoelastin molecules, that are crosslinked into insoluble aggregates in the extracellular space. Tropoelastin is highly hydrophobic and is encoded by a single copy gene, localized on chromosome 7 in humans; the primary transcript undergoes alternate splicing giving rise to different tropoelastin molecules, whose distribution and physiological significance are still under investigation. Elastin shows a transient developmental expression, with barely detectable turnover during adult life. In thin sections, elastin fibres appear rather compact and consist of an amorphous material (true elastin) and of longitudinal strips, probably made of various components. By freeze-fracture and negative staining, the elastin fibre has been shown to be made of a loose irregular network of beaded filaments, which can be oriented by stretching. Cytochemical and immunocytochemical approaches have shown that elastin fibres contain proteoglycans as well as sites recognized by antibodies to lysyl oxidase and microfibrillar components. It has been suggested that microfibrils and proteoglycans might have a role in elastin fibrogenesis. Elastin fibres undergo irreversible alterations during aging and in pathological conditions. These alterations consists mainly of accumulation of ions and of abnormal matrix constituents, and/or swelling and disaggregation of the fibre itself.