Non-collagenous Matrix Components Research Articles

Keratocyte-Derived Myofibroblasts: Functional Differences With Their Fibroblast Precursors.

In this study, we aim to elucidate functional differences between fibroblasts and myofibroblasts derived from a keratocyte lineage to better understand corneal scarring. Corneal fibroblasts, derived from a novel triple transgenic conditional KeraRT/tetO-Cre/mTmG mouse strain that allows isolation and tracking of keratocyte lineage, were expanded, and transformed by exposure to transforming growth factor (TGF)-β1 to myofibroblasts. The composition and organization of a fibroblast-built matrix, deposited by fibroblasts in vitro, was analyzed and compared to the composition of an in vitro matrix built by myofibroblasts. Second harmonic generation microscopy (SHG) was used to study collagen organization in deposited matrix. Different extracellular matrix proteins, expressed by fibroblasts or myofibroblasts, were analyzed and quantified. Functional assays compared latent (TGF-β) activation, in vitro wound healing, chemotaxis, and proliferation between fibroblasts and myofibroblasts. We found significant differences in cell morphology between fibroblasts and myofibroblasts. Fibroblasts expressed and deposited significantly higher quantities of fibril forming corneal collagens I and V. In contrast, myofibroblasts expressed and deposited higher quantities of fibronectin and other non-collagenous matrix components. A significant difference in the activation of latent TGF-β activation exists between fibroblasts and myofibroblasts when measured with a functional luciferase assay. Fibroblasts and myofibroblasts differ in their morphology, extracellular matrix synthesis, and deposition, activation of latent TGF-β, and chemotaxis. The differences in the expression and deposition of extracellular matrix components by fibroblasts and myofibroblasts are likely related to critical roles they play during different stages of corneal wound healing.

Role of collagen XII in skin homeostasis and repair

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis.This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFβ, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing.Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.

AB0032 Association of ADAMTS12 polymorphisms with rheumatoid arthritis in korean population

BackgroundA disintegrin and metalloproteinase (ADAM) with thrombospondin type 1 motif, 12 (ADAMTS12) is a degradative enzyme that interacts with the degradable fragments of cartilage oligomeric matrix protein, which is a...

Association of ADAMTS12 polymorphisms with rheumatoid arthritis

a disintegrin and metalloproteinase (ADAM) with thrombospondin type 1 motif 12 (ADAMTS12) is a degradative enzyme that interacts with the degradable fragments of cartilage oligomeric matrix protein, which is a prominent non-collagenous matrix component in articular cartilage. ADAMTS12 has been observed in the cartilage, synovial fluid and serum of arthritic patients, and may play an important role in the pathogenesis of arthritis. In the present study, we investigated whether genetic polymorphisms of ADAMTS12 are associated with rheumatoid arthritis (RA). To observe the association between ADAMTS12 and RA, we genotyped three single nucleotide polymorphisms (SNPs) (rs1364044, intron C/T; rs10461703, intron C/T; rs25754, missense Thr1495Ile) of ADAMTS12 using a direct sequencing method in 303 RA patients and 495 control subjects. Multiple logistic regression models were performed to analyze the genetic data. SNPStats and SNPAnalyzer Pro programs were used to estimate the odds ratios, 95% confidence intervals and p-values. Bonferroni's correction (pc) was conducted to obtain a defined result. Of the three SNPs, the genotype frequency of rs10461703 was associated with the development of RA (pc=0.0024 in the co-dominant model; pc=0.0009 in the dominant model; pc=0.0006 in the log-additive model). The allele frequency of rs10461703 also showed a significant difference between RA and controls (pc<0.0001). The C allele frequency of rs10461703 was lower in the RA group (36.6%) compared to the control group (45.7%), whereas the T allele frequency of rs10461703 in the RA group (63.4%) was higher compared to that in the control group (54.3%). The other two SNPs (rs1364044 and rs25754) were not associated with the development of RA. However, we did not find any association between the three tested SNPs and RA patients according to clinical features, including erythrocyte sedimentation rate, C-reactive protein level, rheumatoid factor (+ and -) and bone erosion (+ and -). Our results suggest that ADAMTS12 may be a susceptibility gene for RA development.

Interleukin (IL)-4, IL-13, and IL-17A differentially affect the profibrotic and proinflammatory functions of fibrocytes from asthmatic patients.

Fibrocytes contribute to the fibrotic changes most frequently observed in forms of asthma where inflammation is driven by T helper type 2 (Th2) cells. The mechanisms that regulate the profibrotic function of asthmatic fibrocytes are largely unknown. We isolated circulating fibrocytes from patients with allergen-exacerbated asthma, who showed the presence of fibrocytes, together with elevated concentrations of interleukin (IL)-4 and IL-13 and slightly increased concentrations of the Th17 cell-derived IL-17A, in induced sputum. Fibrocytes stimulated with IL-4 and IL-13 produced high levels of collagenous and non-collagenous matrix components and low levels of proinflammatory cytokines. Conversely, fibrocytes stimulated with IL-17A proliferated and released proinflammatory factors that may promote neutrophil recruitment and airway hyperresponsiveness. IL-17A also indirectly increased α-smooth muscle actin but not collagen expression in fibrocytes. Thus, fibrocytes may proliferate and express a predominant profibrotic or proinflammatory phenotype in asthmatic airways depending on the local concentrations of Th2- and Th17-derived cytokines.

The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins

Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.

The influence of swelling and matrix degradation on the microstructural integrity of tendon

Tendon is multi-level fibre composite material, responsible for the transmission of forces from muscles to the skeleton. It is composed of a hierarchical arrangement of collagenous units surrounded by a proteoglycan-rich matrix, arranged to support strain transfer, and thus contribute to the mechanical behaviour of tendon. This study examines the effect of swelling and enzymatic degradation on structural integrity at different levels of the tendon hierarchy. Biochemical and microstructural analysis are used to examine the effects of incubation on the composition and swelling of the matrix, prior to a mechanical characterisation of sample integrity. Results indicated significant swelling of tendon fibrils and interfibrillar matrix after incubation in phosphate buffered saline, leading to a reduction in ultimate tensile load, with failure initiated between fibrils and sub-fibrils. In contrast, incubation with the enzyme chondroitinase ABC resulted in a total removal of glycosaminoglycan from the samples, and a subsequent reduction in the extent of swelling. These fascicles also demonstrated an increase in failure loads, with failure predominating between fibres. The findings from this work confirm the importance of the non-collagenous matrix components in controlling strain transfer within tendon structures. It also highlights the necessity to maintain samples within a suitable and controlled environment prior to testing.

ADAMTS-12 Associates with and Degrades Cartilage Oligomeric Matrix Protein

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.

Removal of noncollagenous components affects dentin bonding

The structural integrity of fibrillar type I collagen is critical for effective dentin bonding. Since most noncollagenous matrix components in dentin are closely associated with collagen, we hypothesized that they may also contribute to dentin bonding. To test this hypothesis, bovine dentin was acid-etched, treated with chondroitinase ABC (C-ABC), endo-beta-galactosidase (Endo-beta), or trypsin. Controls were prepared in the same manner but without the enzymes. All control and experimental specimens were then bonded with One-Step. Bond strength data were analyzed by one-way ANOVA and Fisher's PLSD test (p < 0.05). When dentin was treated with C-ABC or trypsin, bond strengths significantly decreased for the rewetted groups (p < 0.05). The treatment with Endo-beta showed no effects on bond strengths (p > 0.05). When the treated dentin surfaces were observed under SEM, the C-ABC and trypsin treated groups revealed significant loss of collagen fibril architecture. The results indicate that chondroitin sulfate glycosaminoglycans and trypsin-digestible noncollagenous proteins play roles in maintaining the open dimensions of the collagen fibril scaffold, which is essential for optimal dentin bonding.

The Influence of Noncollagenous Matrix Components on the Micromechanical Environment of Tendon Fascicles

Tendon is composed of type I collagen fibers, interspersed with proteoglycan matrix and cells. Glycosaminoglycans may play a role in maintaining the structural integrity of tendon, preventing excessive shearing between collagen components. This study tests the hypothesis that tendon extension mechanisms can be altered by modifying the composition of noncollagenous matrix. Tendon explants were treated with phosphate buffered saline (PBS) or PBS + 0.5 U ml(-1) chondroitinase ABC. Structural changes were examined using TEM and biochemical analysis, while strain response was examined using confocal microscopy and gross mechanical characterization. Chondroitinase ABC removed 90% of glycosaminoglycans from the matrix. Results demonstrated significant swelling of fibrils and surrounding matrix when incubated in either solution. In response to applied strain, PBS incubated samples demonstrated significantly less sliding between adjacent fibers than nonincubated, and a 33% reduction in maximum force. By contrast, fascicles incubated in chondroitinase ABC demonstrated a similar strain response to nonincubated. Data indicate that collagen-proteoglycan binding characteristics can be influenced by incubation and this, in turn, can influence the preferred extension mechanisms adopted by fascicles. This highlights the importance of maintaining fascicles within their natural environment to prevent structural or mechanical changes prior to subsequent analysis.

Mechanisms of hepatic fibrosis.

Mechanisms of hepatic fibrosis.

Mineral characterization in calcifying tissues: atomic, molecular and macromolecular perspectives.

This paper presents a general overview of calcification and involves aspects of the chemical, physical, and biological nature of mineral crystals in invertebrate and vertebrate tissues, with selected examples of the latter. Two broad areas are described: mineral structure and composition. Mineral formation is detailed in an incidental fashion. Both classical research and recent data appropriate to mineralization studies are noted in order to convey basic principles, as well as the sense and direction of current investigations on the mineral phases of calcified tissues. In this context, novel analytical and imaging techniques from a number of different laboratories lately have helped characterize crystal size, shape, and composition; mineral association with respect to collagen; atomic lattice structure of crystal surfaces; interrelationships between non-collagenous matrix components and mineral; and stereochemical organization of putative matrix nucleation sites. Together, this work has provided a more complete understanding of the mineral-matrix atomic, molecular, and macromolecular interactions that underlie the general mechanism of calcification in biological tissues.

Immunogold study of non-collagenous matrix components in normal and exfoliative iris.

The present investigation was undertaken to determine if some of the components of exfoliation material in iris tissue were unique to exfoliation or were part of normal iris architecture. Eleven normal iris specimens and 10 exfoliative iris specimens were processed for cryoultramicrotomy and London resin white embedding. Immunogold electron microscopy was used to investigate the fine structural distribution of amyloid P component, elastin, entactin, fibronectin, gp115, and vitronectin in normal iris and their association with exfoliation material. Exfoliation material was positive for amyloid P component and possibly gp115, neither of which were present in normal iris tissue. Elastin and fibronectin were present in the normal iris stroma but were not associated with exfoliation material. The distribution of amyloid P component in the vessel lumen and wall led to the conclusion that amyloid P is a serum contaminant. The presence of gp115 in exfoliation material represents the synthesis of a component novel to the iris vascular cell synthetic repertoire.

Cellular Sources of Noncollagenous Matrix Proteins: Role of Fat-Storing Cells in Fibrogenesis

The main types of noncollagenous ECM proteins in liver are FN, laminin, PGs and elastin. Also present is a pure carbohydrate polymer, hyaluronic acid. Their concentrations increase during fibrogenesis in a specific sequence and spatial distribution. FN is among the first to appear. The accumulation of matrix proteins in the space of Disse (perisinusoidal fibrosis) is most important for some clinical consequences of fibrosis. Perisinusoidal FSC are the cell type responsible for exaggerated ECM formation at sites of tissue injury, since these cells synthesize at a high rate in vitro all the noncollagenous matrix components occurring in fibrotic liver matrix (FN, laminin, dermatan sulfate, chondroitin sulfate, hyaluronic acid) and since FSC proliferate and transform into myofibroblast-like cells with an enhanced matrix protein synthesis rate. Recent experimental data suggest strong cellular cooperation of these cells with activated liver macrophages or Kupffer cells, platelets, and regenerating hepatocytes, which leads via a paracrine mechanism involving TGF beta, TGF alpha/EGF, PDGF, and probably other cytokines to stimulated proteoglycan synthesis, proliferation, and transformation into myofibroblast-like cells. The latter cell type has the potential of auto-stimulation (by an autocrine process) involving secretion of TGF beta and TGF alpha/EGF. Based on these findings, a model of FSC activation is proposed (Fig. 10).