Extracellular Matrices Of Connective Tissues Research Articles

Overview on Keloid Disorder: Phenotypic Spectrum, Connective Tissue Pathology, and Treatment Development

Overview on Keloid Disorder: Phenotypic Spectrum, Connective Tissue Pathology, and Treatment Development

Defective Initiation of Glycosaminoglycan Synthesis due to B3GALT6 Mutations Causes a Pleiotropic Ehlers-Danlos-Syndrome-like Connective Tissue Disorder

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs(∗)163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals' fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.

The first and second 'laws' of chemical morphology, exemplified in mammalian extracellular matrices.

Tissues are supramolecular organisations. The permanent and semi-permanent biopolymers therein function collaboratively in specifically bonded frameworks of macromolecules according to the physico-chemical laws that govern the behaviour of all molecules. In this paper two 'laws' or principles are discussed which give insights into the development and function of tissues, particularly the extracellular matrices (ECMs) of connective tissues. The first 'law' is qualitative;- The shape of a tissue is implicit in the shapes of the biopolymers from which it is constructed. The tissue biopolymers are jigsaw pieces, if they don't fit precisely, there is no picture. The second 'law' is quantitative;-The composition of a tissue is determined by the stable, specific interactions between the macromolecules of which it is constructed. These basic ideas underlie the discipline of chemical morphology. The term chemical morphology implies both the chemistry of shape and the shape of chemicals. The first meaning is well exemplified in the ECMs of connective tissues, in which the shape of an organism is defined and maintained. Specific relationships between the fibrillar (collagenous) components and the soluble polymers (proteoglycans) are set in the context of the first law. Tissue electron histochemistry (the morphology of the tissue) and knowledge of secondary and tertiary structures of the participating biopolymers (the shapes of the chemicals) together provide a model susceptible to quantitative testing. Simple calculation shows that the amount of any ligand (e.g. a proteoglycan) specifically bound at a single binding site per unit of collagen fibril length (the D period) increases linearly with the fibril diameter. Given the amount of collagen (measured as hydroxyproline) and its density, the constant of proportionality is approximately 42. Comparisons of the quantitative relationship between collagen and proteoglycans predicted from the model agree well with those obtained by biochemical analyses of different tendons from three species at all stages of development. Thus, the second 'law' appears to hold in this case.

Decorin Transfection Induces Proteomic and Phenotypic Modulation in Breast Cancer Cells 8701-BC

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decorin.

Stress transfer in collagen fibrils reinforcing connective tissues: Effects of collagen fibril slenderness and relative stiffness

Unlike engineering fibre composite materials which comprise of fibres that are uniform cylindrical in shape, collagen fibrils reinforcing the proteoglycan-rich (PG) gel in the extra-cellular matrices (ECMs) of connective tissues are taper-ended (paraboloidal in shape). In an earlier paper we have discussed how taper of a fibril leads to an axial stress up-take which differs from that of a uniform cylindrical fibre and implications for fibril fracture. The present paper focuses on the influence of fibre aspect ratio, q (slenderness), and Young's modulus (stiffness), relative to that of the gel phase, E R , on the magnitude of the axial tensile stresses generated within a fibril and wider implications on failure at tissue level. Fibre composite models were evaluated using finite element (FE) and mathematical analyses. When the applied force is low, there is elastic stress transfer between the PG gel and a fibril. FE modelling shows that the stress in a fibril increases with E R and q. At higher applied forces, there is plastic stress transfer. Mathematical modelling predicts that the stress in a fibril increases linearly with q. For small q values, fibrils may be regarded as fillers with little ability to provide tensile reinforcement. Large q values lead to high stress in a fibril. Such high stresses are beneficial provided they do not exceed the fracture stress of collagen. Modulus difference regulates the strain energy release density, u, for interfacial rupture; large E R not only leads to high stress in a fibril but also insures against interfacial rupture by raising the value of u.

Transforming growth factor-beta1 in a sterilized tissue derived from the pig small intestine submucosa.

The extracellular matrices of connective tissues contain growth factors such as transforming growth factor (TGF)-beta1. The possibility arises, therefore, that animal connective tissues that have been excised and rendered acellular in the sterilization, lyophilization, and other preparative processes for human use may still retain active growth factors that could contribute to the clinical efficacy of the product. We therefore analyzed 4M guanidine HCl extracts of a sterilized, acellular matrix, Oasis Wound Matrix, for the presence of TGF-beta1 by a sandwich enzyme-linked immunosorbent assay using the soluble type II receptor for TGF-beta to capture the growth factor, and for biological activity by testing the capacity of the extracts to inhibit 3[H]thymidine incorporation into Mv1Lu cells (mink lung epithelial cells). The enzyme-linked immunosorbent assay determined that TGF-beta1 was present at a concentration of 710.9 +/- 157.7 pg/g dry weight of tissue in Oasis and 768.1 +/- 182.1 pg/g dry weight of tissue in SIS (porcine small intestinal submucosa), the disinfected precursor of Oasis. The growth inhibition assays demonstrated that the Oasis extracts inhibited the proliferation of Mv1Lu cells in culture, consistent with the TGF-beta1 in the material having biological activity. Most of the TGF-beta1 survives the sterilization and lyophilization processes in the preparation of the Oasis Wound Matrix, and is functional in its ability to bind to its receptor and, apparently, in its capacity to inhibit growth.

Identification, characterization and expression analysis of a new fibrillar collagen gene, COL27A1.

The fibrillar collagens provide structural scaffolding and strength to the extracellular matrices of connective tissues. We identified a partial sequence of a new fibrillar collagen gene in the NCBI databases and completed the sequence with bioinformatic approaches and 5' RACE. This gene, designated COL27A1, is approximately 156 kbp long and has 61 exons located on chromosome 9q32-33. The homologous mouse gene is located on chromosome 4. The gene encodes amino- and carboxyl-terminal propeptides similar to those in the 'minor' fibrillar collagens. The triple-helical domain is, however, shorter and contains 994 amino acids with two imperfections of the Gly-Xaa-Yaa repeat pattern. There were three sites of alternative RNA splicing, only one of which led to the intact mRNA that encodes this full-length collagen proalpha chain. Phylogenetic analyses indicated that COL27A1 forms a clade with COL24A1 that is distinct from the two known lineages of fibrillar collagens. Expression analyses of the mouse col27a1 gene demonstrated high expression in cartilage, the eye and ear, but also in lung and colon. It is likely that the major protein product of COL27A1, proalpha1(XXVII), is a component of the extracellular matrices of cartilage and these other tissues. Study of this collagen should yield insights into normal chondrogenesis, and provide clues to the pathogenesis of some chondrodysplasias and disorders of other tissues in which this gene is expressed.

The structure of interfibrillar proteoglycan bridges (shape modules') in extracellular matrix of fibrous connective tissues and their stability in various chemical environments.

Collagen fibrils in extracellular matrices of connective tissues (tendon, cornea, etc.) are bridged and linked by the anionic glycosaminoglycans (AGAGs) of the small proteoglycans (decoron, etc.). It was proposed that these bridges and ties maintain the collagen fibril dispositions in relation to each other, helping to define tissue shape, and hence called shape modules. This investigation describes chemical and physicochemical conditions in which these structures are stable and what treatments cause their disruption. The effects on fixed and unfixed sections of tendon, cornea, lung and ear from rat, mouse and rabbit of pH, electrolyte concentration, EDTA, mercaptoethanol, hydrogen peroxide, free radicals, periodate, acetylation, urea, nonionic detergent and organic solvents were assessed by staining with Cupromeronic blue or Alcec blue in CEC techniques to localise AGAG bridges or their disintegration products. Ca2+ was not involved in the structures, oxidation/reduction had no effect and Triton X100, a nonionic detergent did not damage them. They were stable between pH 4.5 and 9.5. Periodate as a glycol-cleaving reagent did not affect them. High concentrations of urea (> 2.0 M) and MgCl2 (0.5 M) disrupted the tissues. The combination of Triton and urea at concentrations too low to cause damage separately was disruptive. Free radicals in periodate solutions were damaging. Organic solvents caused collapse and rearrangements of the AGAG filaments. Acetylation caused considerable disruption of shape modules. Dermochondan but not keratan sulphate AGAGs were removed by treatment with NaOH. After fixing with glutaraldehyde only free radical and NaOH treatments were severely disruptive of shape modules. The results are compatible with a previously proposed structure for the shape modules, stabilised by hydrophobic and hydrogen bonding.

Gelatinase B in chronic synovitis: immunolocalization with a monoclonal antibody.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in the remodelling of extracellular matrices of connective tissues. With the use of specific monoclonal antibodies against human gelatinase B, the producer cell types were pinpointed in histopathological sections of a number of arthritic diseases. In cases of acute joint trauma, chondromatosis, villonodular synovitis and a cyst of a bursa, high numbers of strongly immunopositive neutrophils were observed in addition to weaker staining macrophages. Activated macrophages with giant cell morphology clearly stained with the gelatinase B-specific monoclonal antibody in the case of villonodular synovitis and in an epidermoid cyst. However, in the sections from patients with rheumatoid arthritis, no immunostaining was seen. In other cases of chronic synovitis, however, within the lymphocyte nodular aggregates a strong gelatinase B expression was observed in morphologically identified dendritic cells. In conclusion, gelatinase B production in joint disease seems to be predominantly by neutrophils and cell types of the macrophage/antigen-presenting cell lineage.

Function of proteoglycans in the extracellular matrix

Proteoglycans are glycosylated proteins which have covalently attached highly anionic glycosaminoglycans. Many forms of proteoglycans are present in virtually all extracellular matrices of connective tissues. The major biological function of proteoglycans derives from the physicochemical characteristics of the glycosaminoglycan component of the molecule, which provides hydration and swelling pressure to the tissue enabling it to withstand compressional forces. This function is best illustrated by the most abundant proteoglycan in cartilage tissues, aggrecan. During the past decade, diverse species of proteoglycans have been identified in many connective tissues, on cell surfaces and in intracellular compartments. These proteoglycans have distinct biological functions apart from their hydrodynamic functions, and their involvement in many aspects of cell and tissue activities has been demonstrated. For example, decorin, which is widely distributed in many connective tissues, may have functions in regulating collagen fibril formation and in modifying the activity of transforming growth factor-beta; perlecan, the major heparan sulfate proteoglycan in the glomerular basement membrane, may play an important role as the major anionic site responsible for the charge selectivity in glomerular filtration. Specific interactions between proteoglycans (through both their glycosaminoglycan and core protein components) and macromolecules in the extracellular matrix are the key factors in the functions of proteoglycans. Exciting biological functions of proteoglycans are now gradually emerging.

Ultrastructural immunolocalization of fibronectin in epiphyseal and metaphyseal bone of young rats.

Fibronectin is a well known glycoprotein of extracellular connective tissue matrices due to a specific amino acid-sequence (RGD) suggested to act as an attachment factor in cell-cell or cell-matrix interactions. Although also present in bone, little is known about the role of fibronectin in this tissue. To obtain data for discussions on function we used ultrastructural immunolocalization techniques to quantitatively examine the distribution of fibronectin in various bone matrix compartments. The study was focused on three different stages of endochondral ossification in growing long bones of young rats. The results show large amounts of fibronectin in mature bone tissue. At a higher magnification, an obvious fibronectin association to individual fibrils of collagen type I was demonstrated. Intracellular labeling was observed in Golgi-related vesicles in some active osteoblasts of metaphyseal bone, indicating local synthesis of fibronectin. In contrast to previous suggestions based on light microscopic observations, the labeling of bone or cartilage matrices facing the surface of all cell types were low. The pattern is clearly different from that of osteopontin and bone sialoprotein, two other bone matrix proteins with the same cell-binding sequence. Our results indicate that fibronectin at these stages of development participates in matrix organization rather than being an important link between cartilage or bone matrix and adjacent cells.

Scanning electron microscopic studies of the medial smooth muscles in human major intracranial arteries.

The three-dimensional arrangement of the medial smooth muscle cells of human major intracranial arteries was studied by scanning electron microscopy after removal of extracellular connective tissue matrices with a KOH-collagenase digestion method. In the straight portion of the major arteries, the smooth muscle cells were arranged roughly circularly, whereas the arrangement was somewhat random in the vertebral, basilar, and internal carotid arteries. Groups of the longitudinal muscle cells were also found in the vertebral, basilar, and internal carotid arteries, but were absent in the anterior, middle, and posterior cerebral arteries. At a higher magnification, smooth muscle cells in these arteries formed anastomosed bundles about 5-30 micrograms in diameter. The smooth muscle cells in the dichotomous branching and the uniting portions were arranged circularly, but multidirectionally and longitudinally oriented smooth muscle cell groups were present in the facial and dorsal walls of the forked vessels. These multidirectional muscle cell groups were small in number in the portion where the major arteries gave off lateral branches at a right angle. So called "medial defects" were found at the crotch of the bifurcating region in two cases out of four. Smooth muscle cells near the defects tapered off toward the center of the defect where internal elastic lamina with oval fenestrations were exposed. However, no special arrangement of the smooth muscle cells was observed around the "medial defects" as compared with that in the ordinary bifurcating region.

Function of proteoglycans in the extracellular matrix.

Proteoglycans are glycosylated proteins which have covalently attached highly anionic glycosaminoglycans. Many forms of proteoglycans are present in virtually all extracellular matrices of connective tissues. The major biological function of proteoglycans derives from the physicochemical characteristics of the glycosaminoglycan component of the molecule, which provides hydration and swelling pressure to the tissue enabling it to withstand compressional forces. This function is best illustrated by the most abundant proteoglycan in cartilage tissues, aggrecan. During the past decade, diverse species of proteoglycans have been identified in many connective tissues, on cell surfaces and in intracellular compartments. These proteoglycans have distinct biological functions apart from their hydrodynamic functions, and their involvement in many aspects of cell and tissue activities has been demonstrated. For example, decorin, which is widely distributed in many connective tissues, may have functions in regulating collagen fibril formation and in modifying the activity of transforming growth factor-beta; perlecan, the major heparan sulfate proteoglycan in the glomerular basement membrane, may play an important role as the major anionic site responsible for the charge selectivity in glomerular filtration. Specific interactions between proteoglycans (through both their glycosaminoglycan and core protein components) and macromolecules in the extracellular matrix are the key factors in the functions of proteoglycans. Exciting biological functions of proteoglycans are now gradually emerging.

FACIT collagens: diverse molecular bridges in extracellular matrices

The collagens form a large family of proteins. Collagen fibrils, composed of staggeref arrays of fibrillar collagen molecules (types I, II, III, V and XI), provide a supporting scaffold for extracellular matrices of connective tissues. The non-fibrillar collagens are less abundant than the fibrillar collagens, but it is becoming clear that they have important functions in the matrix. Recently, a group with unique structural characteristics has been defined and named the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) group. There is evidence that these collagens may serve as molecular bridges that are important for the organization and stability of extracellular matrices.

Cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels

The three-dimensional cytoarchitecture of the smooth muscles and pericytes of rat cerebral blood vessels was studied by scanning electron microscopy after removing extracellular connective tissue matrices with the KOH-collagenase digestion method. The tunica media of major intracranial arteries such as the internal carotid, vertebral, basilar, and other cerebral arteries measuring more than 100 microns in outer diameter consisted of spindle-shaped smooth-muscle cells arranged circularly to the long axis of the vessel. Muscle cells at the branching points, however, showed a variety of shapes, sizes, and arrangements. As the vessel size decreased, smooth-muscle cells showed bi- or trifurcations at the cell poles. In the precapillary arterioles, smooth-muscle cells which had helically surrounded the endothelial tubes had bulging cell bodies with various cytoplasmic processes extending from the cell poles. Distinct specializations presumed to be sphincters were not found on the arteries or arterioles. Pericytes of the capillary had become extended along the vessel axis, having fusiform cell bodies with longitudinally oriented long cytoplasmic processes. Cells located periendothelially in the venules and veins were stellate in shape with many cytoplasmic processes which were interwoven to form complicated cellular networks around the endothelial tube.

The three-dimensional organization and ultrastructure of lymphatics in the rat intestinal mucosa as revealed by scanning electron microscopy after KOH-collagenase treatment.

The three-dimensional organization and ultrastructure of lymphatics in the rat intestinal mucosa were studied by scanning electron microscopy using the KOH-collagenase digestion method (to remove extracellular connective tissue matrices) and by conventional transmission electron microscopy. Each villus possessed three or more lymphatics in the deeper layer of the lamina propria. These initial lymphatics were 5-15 microns in diameter and ran parallel to each other in the longitudinal direction of the villus. These lymphatics were free from adhering periendothelial cells, but had numerous cytoplasmic folds and projections of the endothelium on their abluminal surface. At the villous base, the lymphatics emptied into several thicker (about 70-80 microns) lymphatics, which further connected together to form a flat and wide sinus (intra-villous lymphatic sinus). This sinus was relatively smooth in abluminal surface. From the bottom of each sinus, several lymphatics descended perpendicularly to drain into lymphatics in the submucosa. The villous lymphatics described above seemed to be partially accompanied by bundles of smooth muscle cells, which ran longitudinally in the villous core. The submucosal lymphatics were smooth in abluminal surface and possessed no periendothelial cells. These lymphatics ran in a transverse direction just beneath the muscularis mucosae to form a two-dimensional mesh-work by anastomosing with one another. This submucosal lymphatic network was often closely associated with the muscle cells of the muscularis mucosae, but was not accompanied by submucosal arteries.

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.