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Correlations between age and rat dermis modifications. Ultrastructural- morphometric evaluations and lysyl oxidase activity
No full textThe extracellular matrix is a complex, integrated macromolecular system which plays a crucial role in the economy of each organ. In this study we focused our attention on the correlations between age and rat skin dermis. The latter was chosen as a model of the connective tissue, and was analyzed by means of electron microscopy and by measurement of the activity of lysyl oxidase, the enzyme involved in collagen and elastin crosslink formation. Ultra-structural and morphometric evaluations associated to body weight growth, showed a progressive increase in the amounts of extracellular components and a progressive reduction in the cell density. Skin from adult animals appeared characterized by a well organized matrix; by contrast, in old rats, we observed several degenerative features such as the disorganization of collagen bundles, the vacuolization of elastic fibers, and the atrophy of the mesenchimal cells. Morphometric evaluations in old animals showed a slight but significant reduction in the percentage of the total collagen measured, a fair stability in the area occupied by the elastin fibers, and an increase of the apparently non-structured matrix. The fact that lysyl oxidase activity was diminished in old rats does not corroborate the observation by several authors that increased collagen insolubility is a consequence of higher intra-and intermolecular crosslinking. This would suggest that other chemical modifications, such as crosslink oxidation or non enzymatic glycosylation, might be involved during the aging of connective tissue. The qualitative and quantitative modifications observed at all ages illustrate the correlation between connective tissue modifications and structural and/or functional properties of the skin. © 1989, Editrice Kurtis s.r.l.. All rights reserved
Extracellular Matrix Modifications in Rat Tissues of Different Ages: Correlations between elastin and collagen type I mRNA expression and Iysyl-oxidase activity
No full textWe 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
Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas
No full textHydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G.M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the exent of the enzyme inhibition. When inhibition was >65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space
Effect of DL-penicillamine on the aorta of growing chickens. Ultrastructural and biochemical studies
No full textThe effect of DL-penicillamine on the architecture of the aortic wall of growing chickens was studied, with particular attention to elastin and collagen. Penicillamine was added to the diet (0.2% and 0.4%, in the presence or not of 10 mg/kg CuSO4 and 100 mg/kg vitamin B6) from hatching, for periods from 7 days up to 2 months. The same regions of the thoracic aortas were examined and compared in all the different experimental conditions. The results showed that penicillamine induced relevant modifications in the process of elastin fibrogenesis. The alterations consisted of an increase of elastin in the extracellular space, associated with an increase in the number of elastin fibers per unit area, and a decrease of the mean profile area of the fibers. Interestingly, penicillamine induced the formation of numerous bundles of microfibrils associated or not with elastin fibers. After prolonged treatment, elastin tended to diminish and the fibers tended to fuse into polymorphic syncytia. Collagen fibrils were larger, showed more heterogeneous cross diameters, were less numerous, and were more spread out within the tissue. All the other components of the aortic wall appeared not to be altered by the chemical. Penicillamine did not modify the copper content of chick aortas, whereas it induced a 40-50% reduction of the activity of both salt and 4 M urea-soluble peptidyl lysyl oxidases in the same tissue. These data may help in understanding some of the pathologic manifestations in human beings during penicillamine treatment
Ultrastructure of elastin
No full textAlmost all structural studies on elastin have been done in higher vertebrates, in which it is organized as an extracellular network of branched fibres which vary from fractions f microns to several microns in diameter. By conventional electron microscopy, elastin appears amorphous. By both freeze-fracture and negative staining on cryosections, it can be resolved as beaded filaments 5 nm in diameter forming a 3D meshwork that, upon stretching, becomes oriented in the direction of the force applied. This filamentous aggregation of elastin molecules is confirmed in vitro by the observation that its soluble precursor, tropoelastin, shows a strong tendency to associate into short 5 nm-thick filaments that, with time, become longer and aggregate into bundles of various dimensions. If chemically fixed and embedded, these aggregates appear amorphous and identical to natural elastin fibres. The tendency of tropoelastin to aggregate into 4-5 nm-thick beaded filaments, which then associate into 12 nm-thick filaments forming a 3D network, has been observed by atomic force microscopy for recombinant human tropoelastin. Therefore, the amorphous structure of elastin seems to be a technical artefact. Apart from elastin-associated microfibrils, which are always present at the periphery of growing elastic fibres and probably have a role more complex than being a scaffold for tropoelastin aggregation in vivo, the elastic fibres seem to be composed of several matrix constituents, which are different in different organs and change with age and in pathological conditions. This is demonstrated by immunocytochemical studies on ultrathin sections
Relevance of aggregation properties of tropoelastin to the assembly and structure of elastic fibers.
No full textSolutions 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
Alterations of the connective tissue components induced by beta-aminopropionitrile
No full textThe ultrastructural and biochemical alterations induced by beta-aminopropionitrile on aorta, lung, and skin of 7-day-old chicks have been studied. The inhibition of elastin formation by beta-aminopropionitrile was associated with: (i) apposition of elastin on the old fiber in the form of button-like appendices; (ii) absence of microfibrils around the abnormally deposited elastin; (iii) presence of ruthenium red and toluidine blue O-positive material within the lathyritic elastin; (iv) increase of proteoglycan content; (v) increase of the mean diameter of collagen fibers; (vi) increased vesiculation of the endoplasmic reticulum of both fibroblast and smooth muscle cells in aorta. The role of lysysl oxidase in the assembly of elastin and collagen fibers is discussed. Particular attention has been paid to the relationship between elastin, microfibrils, and proteoglycans in the formation of the elastic fiber
Banded fibers in tropoelastin coacervates at physiological temperatures.
No full textTropoelastin 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
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