Tissue structure and macromolecular diffusion in umbilical cord. Immobilization of endogenous hyaluronic acid

Diffusion of endogenous hyaluronic acid and 125I-labelled albumin, monitored by desorption from umbilical cord (Wharton's jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role. The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.     

Tissue structure and macromolecular diffusion in umbilical cord immobilization of endogenous hyaluronic acid

Diffusion of endogenous hyaluronic acid and ¹²⁵I-labelled albumin, monitored by desorption from umbilical cord (Wharton's jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover. time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.     

Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly

Wharton's jelly of human umbilical cord is known to contain hyaluronic acid and sulphated glycosaminoglycans (probably as proteoglycans) immobilized in an insoluble collagen fibril network. A secondary, independent, insoluble network based on glycoprotein microfibrils of 13 nm diameter and interpenetrated with the collagen network has now been found in amounts corresponding to 9% of the weight of collagen. Elastin, however, is absent. Tissue slices placed in physiological buffer swell to two-fold their in vivo volume. This is due to the influence of the polysaccharides since treatment with either testicular hyaluronidase, Streptomyces hyaluronidase or chondroitinase ABC, causes their quantitative removal and abolishes the swelling tendency of tissue. Tissue so treated remains close to its in vivo volume indicating that for this state the fibrillar network, overall, is in its relaxed unstressed configuration. Subsequent treatment with a protease causes the degradation of the glycoprotein microfibril network and a two-fold increase in tissue volume while treatment with bacterial collagenase, resulting in the solubilization of 46% of the collagen, causes only a slight deswelling. These results suggest that the unstressed configuration of the network system at the in vivo volume of tissue is due to the collagen network being held in compression by the microfibril network. With intact tissue protease digestion with trypsin, in addition, causes a preferential release of sulphated glycosaminoglycans. Hyaluronic acid, however, remains largely immobilized.     

Evidence for the intramolecular pleating model of fibrillin microfibril organisation from single particle image analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.     

Tissue specific differences in fibrillin microfibrils analysed using single particle image analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and play a fundamental role in the deposition of elastin. The microfibrils are 57 nm periodic supramolecular protein polymers with a mass of 2.5 MDa per repeat. The organisation of molecules within a microfibril is still open to debate and structural studies are only just starting to unravel this issue. The contribution of microfibril associated proteins to microfibril ultrastructure and whether there are any tissue specific differences in microfibril structure is still unknown. Therefore, we have used low dose electron microscopy, single particle image analysis and atomic force microscopy to study the structure of fibrillin microfibrils from different tissues. EM images of microfibrils from aorta, ciliary zonules and vitreous humor were collected and more than 500 microfibril repeats from each sample were subjected to averaging. Averages from each sample were analysed using axial stain exclusion patterns and difference images to detect any variations between them. The overall morphology of fibrillin microfibrils was conserved between tissues and there were only very minor differences in the bead and shoulder region of microfibrils. These data suggest that the structure of isolated microfibrils represents the fibrillin scaffold, and either microfibril associated molecules are lost on purification or play only a minor role in microfibril structure.     

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.     

Proteoglycans contribute locally to swelling,but globally to compressive mechanics,in intact cervine medial meniscus

Loss of charged proteoglycans in the knee meniscus, which aid in the support of compressive loads by entraining water, is an effect of degeneration and is often associated with osteoarthritis. In healthy menisci, proteoglycan content is highest in the inner white zone and decreases towards the peripheral red zone. We hypothesized that loss of proteoglycans would reduce both osmotic swelling and compressive stiffness, spatially localized to the avascular white zone of the meniscus. This hypothesis was tested by targeted enzymatic digestion of proteoglycans using hyaluronidase in intact cervine medial menisci. Mechanics were quantified by creep indentation on the femoral surface. Osmotic swelling changes were assessed by measuring collagen fiber crimp period in the radial-axial plane in the lamellar layer along both the tibial and femoral contacting surfaces. All measurements were made in the inner, middle, and outer zones of the anterior, central, and posterior regions. Mechanical measurements showed variation in creep behavior with anatomical location, along with spatially uniform decreases in viscosity (average of 21%) and creep stiffness (average of 15%) with hyaluronidase treatment. Lamellar collagen crimp period was significantly decreased (average of 27%) by hyaluronidase, indicating a decrease in osmotic swelling, with the largest decreases seen in locations with the highest proteoglycan content. Taken together, these results suggest that while proteoglycans have localized effects on meniscus swelling, the resulting effect on compressive properties is distributed throughout the tissue.     

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.     

Type VI collagen microfibrils: evidence for a structural association with hyaluronan

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.     

The stability of extracellular β-glucosidase fromAspergillus niger is significantly enhanced by non-covalently attached polysaccharides

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes ofAspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of β-glucosidase. The polysaccharide-β-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free β-glucosidase. The β-glucosidase-polysaccharide complex was 18-, 36-, 40-, and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free β-glucosidase. The activation energy of polysaccharide-complexed β-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/K _m) for 4-nitrophenyl β-d-glucopyranoside between polysaccharide-free and polysaccharide-complexed β-glucosidase. We suggest that the function of these polysaccharides secreted by fungi includingA. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.     

Structural and functional integrity of chondrocytes immediately following isolation

Chondrocytes were isolated from costal cartilage in young rats after digestion with collagenase and hyaluronidase. The immediate survival of the cells was investigated with the use of different criteria for viability, namely structural integrity and metabolic activity. Structural integrity was studied by transmission and scanning electron microscopy, trypan blue exclusion and NADH oxidation. Metabolic activity was measured both as O2 consumption and as proline and sulphate incorporation, as indicators of collagen and proteoglycan synthesis. The cellular content of glutathione was also measured. The chondrocytes isolated were found to be structurally intact and metabolically active. Early after isolation the chondrocytes varied considerably in size similarly to the native tissue. A selective loss of the larger sized cells was observed during further incubation for 24 h.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

THE SMOOTH MUSCLE CELL : II. Growth of Smooth Muscle in Culture and Formation of Elastic Fibers

Smooth muscle derived from the inner media and intima of immature guinea pig aorta were grown for up to 8 wk in cell culture. The cells maintained the morphology of smooth muscle at all phases of their growth in culture. After growing to confluency, they grew in multiple overlapping layers. By 4 wk in culture, microfibrils (110 A) appeared within the spaces between the layers of cells. Basement membrane-like material also appeared adjacent to the cells. Analysis of the microfibrils showed that they have an amino acid composition similar to that of the microfibrillar protein of the intact elastic fiber. These investigations coupled with the radioautographic observations of the ability of aortic smooth muscle to synthesize and secrete extracellular proteins demonstrate that this cell is a connective tissue synthetic cell.     

Changes in the molecular packing of fibrillin microfibrils during extension indicate intrafibrillar and interfibrillar reorganization in elastic response

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.     

Raman microscopy and X-ray diffraction,a combined study of fibrillin-rich microfibrillar elasticity

Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfibrillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman microscopy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.     

How do expansins control plant growth? A model for cell wall loosening via defect migration in cellulose microfibrils

Expansins are plant cell wall-loosening proteins that promote cell growth and are essential for many critical developmental processes and stress responses. The molecular basis for expansin action is uncertain. Recently, it has been proposed that expansins loosen the wall by means of the generation of mobile conformational defects at the surface of cellulose microfibrils. The present work addresses this hypothesis by elaborating three assumptions: (1) microfibril–matrix interfaces cause steep stress gradients on the microfibril surface, (2) stress gradients drive the motion of conformational defects along the microfibril surface toward the microfibril–matrix interfaces, and (3) the approach of the defects to the microfibril–matrix interfaces facilitates the dissociation of matrix polysaccharides from cellulose microfibrils.     

The effect of the presence of globular proteins and elongated polymers on enzyme activity

We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.