Change in elastin structure in human aortic connective tissue diseases

Summary Biochemical pathogenesis of the aortic connective tissue diseases (such as, Marfan's syndrome, dissecting aneurysm or aortic aneurysm) was examined by estimating glycoprotein, collagen and elastin contents in the aorta and the intramolecular cross-linking component (isodesmosine) and the intermolecular cross-linking components (cystine, histidinoalanine) in comparison with the control samples obtained from subjects with aortic regurgitation. The elastin content in the aorta and isodesmosine content obtained from the extract of the aortic sample found to be decreased. Ratio of cysteine residues (Cys/Cys-Cys) in the elastin fraction in disease increased. Content of histidinoalanine was found to be decreased. It may be suggested that elastin is maintained in its native nature and shape by intra- and inter-molecular cross-linking bridges, and they are readily denatured by various disease conditions. After elastin was solubilized by elastase, immunoreactive elastin content in those aortic diseases was found to be increased in the human connective tissue. Serum elastase and elastase-like activities tend to increase more than those in the control. These findings may suggest that the change in the structure of elastin would make more susceptible to elastase and other proteolytic enzymes. The reasonable hypothesis may be that molecular defect of fibillin or other constitutional structural glycoproteins produce deficient and functionally incompetent elastin associated microfibrils, and the defect of microfibrils cause to insufficient intra- and inter-molecular cross-links in elastin.     

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.     

The distribution of water in arterial elastin: Effects of mechanical stress,osmotic pressure,and temperature

Using gravimetric and radiotracer techniques, we investigated the effects of mechanical stress, osmotic pressure, and temperature on the volumes of the intra- and extrafibrillar water spaces in arterial elastin. We also investigated the effects of temperature on water flow through elastin membranes and on dynamic mechanical properties of elastin rings. Compression by mechanical or osmotic loading reduced the hydration of the elastin in an identical manner. Two distinct stages were evident; at low loads there was extensive water removal from the extrafibrillar space while high loads were required to remove water from the intrafibrillar space. Conversely, dehydration caused by mechanical extension of the matrix was associated with a much smaller loss from the extrafibrillar compartment and a large fractional decrease in the intrafibrillar space. Contraction of the matrix as a result of increased temperature had similar effects on hydration to those produced by extension. Water flux across elastin membranes, corrected for changes in viscosity, and specific hydraulic conductivity both increased as a result of temperature-induced contraction. This effect was attributed to increases in both the fractional volume of the extrafibrillar space and the fiber radius. The elastic modulus decreased with increasing temperature, but there was an increase in viscoelasticity. Previous studies have determined that viscoelasticity depends on the rate of redistribution of intrafibrillar water, so this finding provides additional evidence that heating affects primarily the volume of the intrafibrillar space. © 1995 John Wiley & Sons, Inc.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Ultrastructure and staining properties of aortic microfibrils (oxytalan)

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.     

Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta

Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing a scaffold upon which newly synthesized elastin is deposited. Ultrastructural and staining studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collagenous matrix. The most striking accumulation of microfibrils is found in the zonule of Zinn, the transparent and elastic suspensory ligament of the lens, which contains no elastin. Application of immunocytochemical staining with a peroxidase-antiperoxidase (PAP) procedure demonstrates that fibronectin is associated with the microfibrils of the zonule and aorta. Aggregates of microfibrils are identical to oxytalan ('acid enduring') fibers that have been described in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat hepatic stroma and human cardiac papillary muscle, indicating that microfibrils are a widely distributed connective tissue element with a function that extends beyond elastogenesis; their association with fibronectin and localization suggests that they serve as an elastic anchoring component of the extracellular matrix.     

Ultrastructural cytochemical properties of elastinassociated microfibrils and their relation to fibronectin

Summary The characteristics of elastin-associated microfibrils were investigated in the tunica adventitia of mouse aortas at the ultrastructural cytochemical level. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method specific for sulphate groups was used with and without prior treatment ofen bloc specimens with either monopersulphate or cupric sulphite reagent. Amorphous elastin formed a clearly identifiable central core with microfibrils located both peripherally and interstitially. Sequential oxidation with monopersulphate and HID-TCH-SP demonstrated a characteristic staining for oxytalan fibres and intensely stained the microfibrils, whereas amorphous elastin stained weakly. Sequential thiosulphation with cupric sulphite and HID-TCH-SP for the demonstration of disulphide linkages and sulphydryl groups intensely stained microfibrils and weakly to moderately stained the amorphous elastin. This reactivity of the microfibrils was not altered by digestion with chondroitinase ABC, performed prior to or after treatment with either monopersulphate or cupric sulphite. In the specimens not exposed to either monopersulphate or cupric sulphite there was no definite HID-TCH-SP staining of microfibrils and amorphous elastin. Further, immunostaining with rabbit antibody specific for mouse fibronectin localized fibronectin in the microfibrils but not in the amorphous, elastin. These results indicate that elastin-associated microfibrils in mouse aorta lack stainable sulphate complex carbohydrates but are enriched with either disulphide or sulphydryl groups, or both, and further demonstrate the close correlation between these glycoproteins and fibronectin.     

Elastogenesis in rat arterial grafts: elastin deposits on microfibrillar and non-microfibrillar structures

Summary Endothelial lesions and the subsequent migration of smooth muscle cells in the intima layer are frequently observed after vascular grafting. The expression of secretory phenotype by these cells leads to the accumulation of connective tissue and thereby provides a model for the study of elastin depositionin vivo. Rats bearing aortic grafts of auto-, iso- or homologous origin were sacrificed between 3 and 18 months after implantation. Samples were treated for routine ultrastructural observations and for post-embedding by immunoelectron microscopy using anti-human elastin and protein A-gold.Grafts showed a large intimal thickening composed of several layers of smooth muscle cells and an abundant extracellular matrix. Mature elastic fibres (amorphous elastin associated with peripheral microfibrils) were always encountered in hyperplasia, suggesting that elastin deposition may follow the classical pathway involving microfibrils, which serve as a framework for polymerization of tropoelastin molecule into the amorphous component. However, an unusual localization of elastin aggregates was observed within basement membrane-like material surrounding smooth muscle cells. When sections were stained with methanolic uranyl acetate, these areas showed small electron-dense bodies, which were also labelled with anti-elastin antibody. These structures were apparently devoid of surrounding microfibrils. These results indicate that non-microfibrillar basement membrane material might be involved in the early events of elastin deposition.     

The effect of elastin damage on the mechanics of the aortic valve

Porcine bioprosthetic heart valves degenerate and fail mechanically through a mechanism that is currently not well understood. It has been suggested that damage to the elastin component of prosthetic valve cusps could be responsible for changes in the mechanical function of the valve that would predispose it to increased damage and ultimate failure. To determine whether damage to elastin can produce the structural and mechanical changes that could initiate the process of bioprosthetic valve degeneration, we developed an elastase treatment protocol that fragments elastin and negates its mechanical contribution to the valve tissue. Valve cusps were mechanically tested before and after digestion to measure the mechanical changes resulting from elastin damage. Elastin damage produced a decrease in radial and circumferential extensibility (from 43 to 18% strain radially and 12 to 7% strain circumferentially), with a slight increase in stiffness (1.3-2.6kN/m for radial and 10.6-11.9kN/m for circumferential directions). Digestions with trypsin, which does not cleave elastin, confirmed that the changes in mechanics of the circumferential samples were likely due to the nonspecific removal of proteoglycans by elastase, while the changes in the radial samples were indeed due to elastin damage. Removing the mechanical contribution of elastin alters the mechanical behavior of the aortic valve cusp, primarily in the radial direction. This finding implies that damage to elastin will distend the cusps, reduce their extensibility, and increase their stiffness. Damage to elastin may therefore contribute to the degeneration and failure of prosthetic valves.     

Mechanical properties of elastin along the thoracic aorta in the pig

Understanding the mechanical environment of each component within the arterial wall is fundamental for understanding vascular growth and remodelling and for engineering artificial vascular conduits. We have investigated the mechanical status of arterial elastin by measuring the circumferential mechanical properties of purified elastin as function of position along the descending thoracic aorta of the pig. The tensile circumferential secant modulus, E(sec), measured in uniaxial mechanical tests, increased 30% (P<0.001), from a value of 0.88 MPa in the proximal tissue near the aortic arch to 1.14 MPa in the distal tissue near the diaphragm, indicating the stiffness of the elastin sample increased with position. Breaking stress was 54% higher in the distal tissue compared to the proximal (P<0.001), but the breaking stretch ratio did not change. E(sec) correlated with the ratio of radius to wall thickness measured in the no load state, r(nl)/h(nl), suggesting that the rise in stiffness was linked to ring morphology. The higher stiffness and strength of the distal tissue might be explained by a higher proportion of circumferentially oriented fibres in the distal tissue, which would indicate that the elastin meshwork in the thoracic aorta may become progressively anisotropic with distance from the heart. The ratio r(nl)/(h(nl)E (sec))rose only 7%, which suggests that the in vivo circumferential strain on the elastin may be constant along the pig thoracic aorta. The positional variation in elastin's properties should be taken into account in mechanical studies on purified elastin and in mathematical models of aorta mechanics.     

Identification of glycoproteins associated with elastin-associated microfibrils

The microfibrils associated with elastic tissue have been shown to be predominantly proteinaceous. On the basis of their affinity for cationic stains, including ruthenium red, they have been assumed to be glycoprotein, but more evidence to support this claim has not been adduced. Despite repeated investigation of glycoprotein materials obtained by extraction of elastic tissues with reagents that appear to remove microfibrils, the chemical composition of elastin-associated microfibrils remains obscure. An electron microscopic study of the microfibrils in two elastin-rich tissues (bovine nuchal ligament and aorta) during their development was pursued using more specific histochemical methods. The periodic acid-alkaline bismuth stain (analogous to the periodic acid-Schiff stain for glycoproteins in light microscopy) has been adapted for this study. Specific aldehyde groups (confirmed by blocking with m-aminophenol or sodium borohydride) were identified after periodate oxidation as fine granules of bismuth stain. These were shown to localize specifically along the elastin-associated microfibrils in a finely punctate form. Staining of the amorphous elastic component did not occur except for a fine rim adjacent to the microfibrils. Lectin binding with concanavalin A (with ferritin markers) confirmed that there are glucose- or mannose-containing proteins associated with the microfibrillar component of elastic tissue. This was true of these microfibrils in all layers of the aortic wall and throughout the ligament. It was also true of mature adult tissues in which there was a lesser proportion of microfibrils. It is concluded that elastin-associated microfibrils really are associated with glycoprotein(s).     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Study of the extracellular macromolecular meshwork elaborated by rat aortic cells in secondary culture]

Rat aortic smooth muscle cells isolated by digestion of the vessels by elastase and trypsin and grown in subculture, are examinated by phase, optic and electron microscopy for their ability to synthesize connective tissue components. Large amounts of extracellular material accumulates within the spaces between the cell; it consists of amorphous substance identified histochemically as elastin, of 110 A microfibrils and of periodic fibrils (430-490 A); the chemical nature of these two last components is discussed.     

The infrastructure of aortic elastic fibers

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.     

Electron microscopic observations of elastic fibres in the lung and aorta of tight-skin and beta-aminopropionitrile-fed mice.

The lung of the tight-skin (TSK) mouse was characterized by enlargement of the air spaces. Elastin in the alveolar walls of the TSK mouse exhibited fragmentation. The aorta of the TSK mouse was characterized by marked hyperplasia of loose connective tissue in the adventitia. Collagen fibres and ruthenium red-positive materials were markedly increased. Microfibrils surrounding elastin in the adventitia of the aorta were not clear in the TSK mouse. In the lung of the beta-aminopropionitrile (BAPN)-fed mouse, enlargement of the alveolar air spaces was not prominent compared with the TSK mouse. Elastic fibres in the alveolar walls did not show the fragmentation observed in the TSK mouse, and microfibrils surrounding elastin were clearly observed. However, elastic laminae in the media of the BAPN-fed mouse aorta were swollen and fragmented. Elastic fibres in the adventitia exhibited a normal appearance and microfibrils surrounding elastin in the adventitia were clearly observed. The results suggest that the mechanism of the connective tissue abnormality in the TSK mouse is different from that of BAPN, which inhibits the activity of lysyl oxidase. The abnormality of elastin and microfibrils surrounding elastin in the TSK mouse probably plays a role in the deformity or degradation of elastic fibres and the structural changes of the lung.     

An ionised/non-ionised dual porosity model of intervertebral disc tissue

The volume of the intrafibrillar water space – i.e. the water contained inside the collagen fibres – is a key parameter that is relevant to concepts of connective tissue structure and function. Confined compression and swelling experiments on annulus fibrosus samples are interpreted in terms of a dual porosity model that distinguishes between a non-ionised intrafibrillar porosity and an ionised extrafibrillar porosity. Both porosities intercommunicate and are saturated with a monovalent ionic solution, i.c. NaCl. The extrafibrillar fixed charge density of the samples is assessed using radiotracer techniques and the collagen content is evaluated by measurement of hydroxyproline concentration. The interpretation of the experimental data yields values for the intrafibrillar water content, the average activity coefficient of the ions, the Donnan osmotic coefficient, the fraction of intrafibrillar water, the stress-free deformation state, and an effective stress–strain relationship as a function of the radial position in the disc. A linear fit between the second Piola–Kirchhoff effective stress and Green–Lagrange strain yielded an effective stiffness: H_e=1.087 ± 0.657 MPa. The average fraction of intrafibrillar water was 1.16 g/g collagen. The results were sensitive to changes in the activity and osmotic coefficients and the fraction of intrafibrillar water. The fixed charge density increased with distance from the outer edge of the annulus, whereas the hydroxyproline decreased.The authors wish to thank Dr. Jill Urban for her advice concerning fixed charge density measurements, and Ing. Paul Willems for his assistance with the experiments. The research of Dr. J. M. Huyghe has been made possible through a fellowship of the Royal Netherlands Academy of Arts and Sciences.     

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.