A heart for fibrillin: spatial arrangement in adult wild-type murine myocardial tissue

Fibrillins are major constituents of microfibrils, which are essential components of the extracellular matrix of connective tissues where they contribute to the tissue homeostasis. Although it is known that microfibrils are abundantly expressed in the left ventricle of the heart, limited data are available about the presence of microfibrils in the other parts of the myocardial tissue and whether there are age or sex-related differences in the spatial arrangement of the microfibrils. This basic knowledge is essential to better understand the impact of fibrillin-1 pathogenic variants on the myocardial tissue as seen in Marfan related cardiomyopathy. We performed histological analyses on wild-type male and female murine myocardial tissue collected at different time-points (1, 3 and 6 months). Fibrillin-1 and -2 immunofluorescence stainings were performed on cross-sections at the level of the apex, the mid-ventricles and the atria. In addition, other myocardial matrix components such as collagen and elastin were also investigated. Fibrillin-1 presented as long fibres in the apex, mid-ventricles and atria. The spatial arrangement differed between the investigated regions, but not between age groups or sexes. Collagen had a similar broad spatial arrangement to that of fibrillin-1, whereas elastic fibres were primarily present in the atria and the vessels. In contrast to fibrillin-1, limited amounts of fibrillin-2 were observed. Fibrillin-rich fibres contribute to the architecture of the myocardial tissue in a region-dependent manner in wild-type murine hearts. This knowledge is helpful for future experimental set-ups of studies evaluating the impact of fibrillin-1 pathogenic variants on the myocardial tissue.     

Biomolecular analysis of elastic fibre molecules

Elastic fibres are macromolecular extracellular matrix assemblies that endow dynamic connective tissues such as arteries, lungs and skin with the property of elastic recoil. Here, we describe how we have purified elastic fibre molecules and then analysed them using a range of biochemical and biomolecular approaches. Such approaches have provided powerful insights into the complex hierarchical processes of extracellular matrix assembly. We outline molecular interaction and kinetics assays using Biacore, biophysical approaches such as multi-angle laser light scattering and analytical ultracentrifugation which provide information on molecular and macromolecular shape and mass in solution, the visualisation of molecules and assemblies using microscopy approaches such as atomic force microscopy and environmental scanning electron microscopy, and compositional analysis of macromolecular complexes using mass spectrometry. Data from these in vitro analytical approaches can be combined to develop powerful new models of elastic fibre assembly.     

Poisson's ratio of a crossed fibre sheath: the skin of aquatic salamanders

Many animal connective tissues are composite materials formed into sheaths containing regularly organized collagen fibres in a crossed, fabric-like array. From a few simple assumption about the interactions between fibres, we construct a model for the effect of such a fabric-like construction on the Poisson's ratio of connective tissue sheaths. Surprisingly, the model predicts high Poisson's ratios (often greater than 1.0) Mdashespecially high given the value of 0.5 that is usually used for primarily aqueous biological tissues (based on assumptions of incompressibility and anisotropy). However, virtually all empirical attempts to measure Poisson's ratio in animal connective tissue sheaths (including our own experiments on salamander skin) reveal similarly high Poisson's ratios. The model also predicts that Poisson's ratio will increase with increasing strain, at a rate dependent on the initial angle of the crossed fibres relative to the direction of strain. Since the Poisson's ratio of a material is directly correlated with the material's stiffness, such strain-dependent changes in Poisson's ratio have important implications for the stiffness properties of connective tissue sheaths. Given the structural support role of connective tissues, stiffness is assumed to be one of their most important qualities, and several examples of how our model might predict the stiffness qualities within the walls of cylinders formed from helically wound crossed fibre sheaths (such as mammalian annular ligament and nematode cuticle) are given.     

Occurrence of "elastic" fibres in the invertebrates

The connective tissues of a wide range of invertebrates have been investigated using a potassium permanganate/spirit blue staining technique. The method demonstrates fibres which are distinct from collagen, vertebrate elastic fibres, and reticular fibres. The fibres are variously associated with subepidermal collagen, muscles, basement membrances, the walls of blood vessels and the sheaths around nerve cords; the method also gives a positive reaction with the arborescent mesogloeal fibres of medusoid coelenterates. Their electron microscope appearance is distinctive and they exhibit a physical elasticity. Chemical tests indicate that following pretreatment with acidified potassium permanganate they show many, although not all, of the properties of vertebrate "elastin". It is concluded that they should be regarded as a special category of "elastic" fibre.     

The alpha 1 chain of type VIII collagen is associated with many but not all microfibrils of elastic fiber system.

The antigen of monoclonal antibodies which had labeled the hexagonal lattice of Descemet's membrane in a specific manner was shown to be the alpha 1 chain of type VIII collagen by immunoblotting followed by amino acid sequence analysis. With this antibody, the localization of alpha 1 (VIII) in various tissues was studied by several immunocytochemical methods. Under light microscopy, the alpha 1 (VIII) was found in a fine fibrillar form in various capsular tissues such as capsules of the liver, kidneys, adrenals, lungs and so on. It was also present in dense connective tissues such as the Achilles tendon, and periodontal and perivertebral ligaments. When some dense connective tissues which had been negative to the label including the intima of aorta, perimysium and Glisson's sheath of the liver, were subjected to pepsin digestion, epitopes were revealed which showed a specific immunofluorescence pattern. In many locations the pattern of localization coincided with that of elastic fiber components, and full or partial colocalization with tropoelastin or costaining with resorcin-fuchsin staining was observed. In immunoelectron microscopy, the antigen (alpha 1 (VIII)) was localized on the surface of, but not inside, elastic fibers. However, some tissues which are rich in elastic fibers or microfibrils remained unlabeled. These included elastic fibers of the aortic media and ligamentum nuchae as well as ciliary zonules. Therefore it is suggested that alpha 1 (VIII) is a collagen associated with microfibrils of some elastic fiber systems, but is not an intrinsic component of either elastic fibers or of microfibrils.     

The staining of elastic fibres with Direct Blue 152. A general hypothesis for the staining of elastic fibres

Summary Elastic fibres may be stained by a number of dyes, e.g. Direct Blue 1 (C.I. 24410), Direct Blue 10 (C.I. 24340), Direct Blue 15 (C.I. 24400), Direct Blue 152 (C.I. 24366) and Direct Violet 37 (C.I. 24370). A convenient method using Direct Blue 152 has been developed which is specific for elastic fibres. The method is simple and allows the demonstration of other connective tissue fibres. Staining of elastic fibres is unimpaired by numerous blocking procedures or by changes in dyebath pH. These properties are shared by several standard elastic fibre stains.As the Direct dyes and several of the standard elastic fibre stains possess numerous aromatic rings a wide range of dyes containing varying numbers of aromatic rings were examined for ability to stain elastic fibres. No association was observed between the ability to stain elastic fibres and dye class, formal charge or the presence of hydrogen bonding groups. Staining was, however, definitely associated with the presence in the dye molecule of 5 or more aromatic rings. This suggested that van der Waals forces of attraction may be responsible for elastic fibre staining both by Direct dyes and the standard elastic fibre stains. Staining of elastic fibres as a side-effect in many procedures is similarly explicable.     

Basic Fuchsin-Ferric Chloride: a Simplification of Weigert's Resorcin-Fuchsin Stain for Elastic Fibres

Weigert's resorcin-fuchsin stain for elastic fibres can be simplified by the omission of the resorcinol. The resulting “basic fuchsin-ferric chloride” gives results indistinguishable from those achieved with resorcin-fuchsin on tissues fixed in Bouin, Carnoy, Gendre, neutral formah, Susa or Zenker solutions. Gel filtration chroma tography on Sephadex LH20 showed that the staining components of basic fuchsin-ferric chloride had a high molecular weight, so selective staining of elastic fibres by this method may well be due to van der Waals attractions.     

Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules

Extracellular matrices (ECM) not only serve as structural scaffolds in organs and tissues, but also determine critical cellular functions through cell-matrix interactions. These are mediated by cell surface receptors that recognise specific structural features of ECMs and, hence, overall physical properties of the acellular environment. ECM structures are subject to hierarchic organisations, which are tightly adapted to the functions of tissues and organs. Only a few specialised tasks are reserved for isolated ECM macromolecules. Instead, molecular ECM components attain their prominent functions only after polymerising into insoluble suprastructural elements, i.e. fibrils, microfibrils, or networks that, in turn, are assembled into regional tissue structures, such as fibres or basement membranes. As an outstanding feature, most, if not all, ECM suprastructures are co-polymers of more than one molecular species that differ in their identity and relative abundance. Thus, ECM suprastructures are composite biological amalgamates. The analogy to metal alloys refers to structural and functional characteristics of ECM composites, which differ from those of each homo-polymeric aggregate. At the tissue level, biological alloys can themselves be assembled into conglomerates that again assume properties distinct from those of each individual alloy. Nevertheless, most studies in matrix biology solely focus on molecular features and mechanisms. Progress has however been made in identifying principles of interactions within suprastructural elements and their functional consequences. We are now only beginning to understand the impact of suprastructural organisation on the assembly and the functions of whole tissues and many fundamental issues in this almost pristine field await discovery.     

Tensile characteristics of collenchyma cell walls at different calcium contents

Collenchyma fibres from celery ( Apium graveolens L.) were extracted with detergent and phenol-acetic acid-water to leave the intact cell walls, free from active enzymes. Under a small, constant stress the cell wall fibres showed elastic and plastic extension and viscoelastic deformation, but viscous flow was observed only at high stresses close to the breaking stress. After complete removal of calcium ions with cyclohex-anediamine tetraacetic acid (CDTA) and incubation for 18 h, comparable levels of these extensibility components were observed at much lower stresses. However, partial removal of calcium ions with citrate did not increase the plastic, elastic or viscoelastic components even when the residual calcium was reduced to 3.5% of the exchange capacity. The breaking stress of the fibres was rather more sensitive to calcium removal, being reduced by 50% at 7% calcium saturation. CDTA-extracted fibres broke by cell separation at very low stress. These characteristics did not appear compatible with removal of calcium ions, or their displacement by protons, as a mechanism for auxin-induced growth in this material: however such mechanisms are not excluded in other tissues or under other conditions. Strong chelating agents which remove enough calcium to weaken cell walls should be avoided in experiments on other mechanisms of auxin-induced growth.     

Beyond the epithelium: cadherin function in fibrous connective tissues

In fibrous connective tissues, fibroblasts are organized into syncytia, cellular networks that enable matrix remodeling and that are interconnected by intercellular adherens junctions (AJs). The AJs of fibroblasts are mediated by N-cadherin, a broadly expressed classical cadherin that is critically involved in developmental processes, wound healing and several diseases of mesenchymal tissues. In contrast to E-cadherin-dependent junctions of epithelia, the formation of AJs in fibrous connective tissues is relatively uncharacterized. Work over the last several years has documented an expanding list of molecules which function to regulate N-cadherin mediated junctions such as: Fer, PTP1B, cortactin, calcium, gelsolin, PIP5KIgamma, PIP2, and the Rho family of GTPases. We present an overview on the regulation of N-cadherin-mediated junction formation that highlights recent molecular advances in the field and rationalizes the roles of N-cadherin in connective tissue function.     

Structure and Development of the Notochord ‘Elastica Externa’ and Nearby Components of the Elastic Fibre System of Agnathans

Between 15 days and 3 months in age, the ‘elastica externa’ of the notochord sheath of larval lampreys develops from patches of moderately dense and amorphous material into a thick, continuous and electron-dense layer. In both lampreys and hagfish, this layer stains strongly with Verhoeff's elastic stain and aldehyde fuchsin and is penetrated by collagen fibrils on both its outer and inner boundaries. Peroxidase labelling using an antibody raised against human elastin specifically labels both the notochord ‘elastica externa’ and the elastic fibre system of lampreys. The diameters of the microfibrils (10–13 nm) of the oxytalan, elaunin and elastic fibres of lampreys and hagfish are the same as those of higher vertebrates. The connective tissue immediately dorsal and ventral to the notochord of lampreys contains mainly oxytalan fibres in very young ammocoetes, a combination of oxytalan, elaunin and elastic fibres in older ammocoetes, and predominantly elastic fibres in adult lampreys. While the region of the endomeninx at the base of the spinal cord contains almost exclusively oxytalan fibres in young ammocoetes, it also possesses numerous elastic fibres in adult lampreys. These findings indicate that, as in higher vertebrates, the elastic fibres of lampreys develop from oxytalan fibres via elaunin fibres.     

Mechanobiological dysregulation of the epidermis and dermis in skin disorders and in degeneration

During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers–Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.     

Applying elastic fibre biology in vascular tissue engineering

For the treatment of vascular disease, the major cause of death in Western society, there is an urgent need for tissue-engineered, biocompatible, small calibre artery substitutes that restore biological function. Vascular tissue engineering of such grafts involves the development of compliant synthetic or biomaterial scaffolds that incorporate vascular cells and extracellular matrix. Elastic fibres are major structural elements of arterial walls that can enhance vascular graft design and patency. In blood vessels, they endow vessels with the critical property of elastic recoil. They also influence vascular cell behaviour through direct interactions and by regulating growth factor activation. This review addresses physiological elastic fibre assembly and contributions to vessel structure and function, and how elastic fibre biology is now being exploited in small diameter vascular graft design.     

Some histological aspects of the palmar digital pads in the vervet monkey

The structural organisation of the digital touch pads has been investigated in the vervet monkey (Cercopithecus pygerythrus) by light microscopy. It has been demonstrated that the subcutaneous tissue of the terminal digital pad consists of a highly organised septal framework containing large amounts of elastic fibres, with adipose tissue sweat gland compartments lying between the connective tissue septa. It is suggested that the presence of elastic fibres in the septal framework of the terminal digital pad is an important factor in accomplishing an even more supple response to prehensile and tactile activities of the hand.     

Dorsal hump morphology in pink salmon (Oncorhynchus gorbuscha)

Mature male Pacific salmon (Genus Oncorhynchus) develop a dorsal hump, as a secondary male sexual characteristic, during the spawning period. Previous gross anatomical studies have indicated that the dorsal humps of salmon are mainly composed of cartilaginous tissue (Davidson [1935] J Morphol 57:169–183.) However, the histological and biochemical characteristics of such humps are poorly understood. In this study, the detailed microstructures and components of the dorsal humps of pink salmon were analyzed using histochemical techniques and electrophoresis. In mature males, free interneural spines and neural spines were located in a line near to the median septum of the dorsal hump. No cartilaginous tissue was detected within the dorsal hump. Fibrous and mucous connective tissues were mainly found in three regions of the dorsal hump: i) the median septum, ii) the distal region, and iii) the crescent‐shaped region. Both the median septum and distal region consisted of connective tissue with a high water content, which contained elastic fibers and hyaluronic acid. It was also demonstrated that the lipid content of the dorsal hump connective tissue was markedly decreased in the mature males compared with the immature and maturing males. Although, the crescent‐shaped region of the hump consisted of connective tissue, it did not contain elastic fibers, hyaluronic acid, or lipids. In an ultrastructural examination, it was found that all of the connective tissues in the dorsal hump were composed of collagen fibers. Gel electrophoresis of collagen extracts from these tissues found that the collagen in the dorsal hump is composed of Type I collagen, as is the case in salmon skin. These results indicate that in male pink salmon the dorsal hump is formed as a result of an increase in the amount of connective tissue, rather than cartilage, and the growth of free interneural spines and neural spines. J. Morphol. 275:514–527, 2014. © 2013 Wiley Periodicals, Inc.     

Distribution and organization of the elastic system fibres in healthy human gingiva

Summary The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.     

Extracellular vesicles are integral and functional components of the extracellular matrix

Extracellular vesicles (EV) are small plasma membrane-derived particles released into the extracellular space by virtually all cell types. Recently, EV have received increased interest because of their capability to carry nucleic acids, proteins, lipids and signaling molecules and to transfer their cargo into the target cells. Less attention has been paid to their role in modifying the composition of the extracellular matrix (ECM), either directly or indirectly via regulating the ability of target cells to synthesize or degrade matrix molecules. Based on recent results, EV can be considered one of the structural and functional components of the ECM that participate in matrix organization, regulation of cells within it, and in determining the physical properties of soft connective tissues, bone, cartilage and dentin. This review addresses the relevance of EV as specific modulators of the ECM, such as during the assembly and disassembly of the molecular network, signaling through the ECM and formation of niches suitable for tissue regeneration, inflammation and tumor progression. Finally, we assess the potential of these aspects of EV biology to translational medicine.     

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.     

Low strain nanomechanics of collagen fibrils

The high stiffness of collagenous tissues such as tendon and ligament is derived in large part from the mechanics and geometries of the constituent collagen's hierarchical forms. The primary structural unit in connective tissues is the collagen fibril for which there exists little direct mechanical or deformational study. Therefore, the current understanding of the mechanisms involved is extrapolated from whole tissue data. To address this, the elastic response due to bending of readily extractable adult collagen fibrils was studied, and the results were compared to previously reported radial indentation experiments. A demonstration of a material anisotropy arising without loss of the assumptions of homogeneity is presented.