Collagen type XIX regulates cardiac extracellular matrix structure and ventricular function.

The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas-without gross changes in myocardial collagen content or basement membrane morphology. Col19a1N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway-a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function-potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Regeneration of collagen fibrils at the papillary dermis by reconstructing basement membrane at the dermal-epidermal junction.

The epidermal basement membrane deteriorates with aging. We previously reported that basement membrane reconstruction not only serves to maintain epidermal stem/progenitor cells in the epidermis, but also increases collagen fibrils in the papillary dermis. Here, we investigated the mechanism of the latter action. Collagen fibrils in the papillary dermis were increased in organotypic human skin culture treated with matrix metalloproteinase and heparinase inhibitors. The expression levels of COL5A1 and COL1A1 genes (encoding collagen type V α 1 chain and collagen type I α 1 chain, respectively) were increased in fibroblasts cultured with conditioned medium from a skin equivalent model cultured with the inhibitors and in keratinocytes cultured on laminin-511 E8 fragment-coated plates. We then examined cytokine expression, and found that the inhibitors increased the expression of PDGF-BB (platelet-derived growth factor consisting of two B subunits) in epidermis. Expression of COL5A1 and COL1A1 genes was increased in cultured fibroblasts stimulated with PDGF-BB. Further, the bifunctional inhibitor hydroxyethyl imidazolidinone (HEI) increased skin elasticity and the thickness of the papillary dermis in the skin equivalent. Taken together, our data suggests that reconstructing the basement membrane promotes secretion of PDGF-BB by epidermal keratinocytes, leading to increased collagen expression at the papillary dermis.

Increasing the level of cytoskeletal protein Flightless I reduces adhesion formation in a murine digital flexor tendon model.

BACKGROUND: Surgical repair of tendons is common, but function is often limited due to the formation of flexor tendon adhesions which reduce the mobility and use of the affected digit and hand. The severity of adhesion formation is dependent on numerous cellular processes many of which involve the actin cytoskeleton. Flightless I (Flii) is a highly conserved cytoskeletal protein, which has previously been identified as a potential target for improved healing of tendon injuries. Using human in vitro cell studies in conjunction with a murine model of partial laceration of the digital flexor tendon, we investigated the effect of modulating Flii levels on tenocyte function and formation of adhesions. METHODS: Human tenocyte proliferation and migration was determined using WST-1 and scratch wound assays following Flii knockdown by siRNA in vitro. Additionally, mice with normal and increased levels of Flii were subjected to a partial laceration of the digital flexor tendon in conjunction with a full tenotomy to immobilise the paw. Resulting adhesions were assessed using histology and immunohistochemistry for collagen I, III, TGF-ß1and -ß3 RESULTS: Flii knockdown significantly reduced human tenocyte proliferation and migration in vitro. Increasing the expression of Flii significantly reduced digital tendon adhesion formation in vivo which was confirmed through significantly smaller adhesion scores based on collagen fibre orientation, thickness, proximity to other fibres and crimping. Reduced adhesion formation was accompanied with significantly decreased deposition of type I collagen and increased expression of TGF-ß1 in vivo. CONCLUSIONS: These findings suggest that increasing the level of Flii in an injured tendon may be beneficial for decreasing tendon adhesion formation.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography.

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet's membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish.

Numerical Simulation of Corneal Fibril Reorientation in Response to External Loading.

PURPOSE: To simulate numerically the collagen fibril reorientation observed experimentally in the cornea. METHODS: Fibril distribution in corneal strip specimens was monitored using X-ray scattering while under gradually increasing axial loading. The data were analysed at each strain level in order to quantify the changes in the angular distribution of fibrils with strain growth. The resulting relationship between stain and fibril reorientation was adopted in a constitutive model to control the mechanical anisotropy of the tissue material. The outcome of the model was validated against the experimental measurements before using the model in simplified representations of two surgical procedures. RESULTS: The numerical model was able to reproduce the experimental measurements of specimen deformation and fibril reorientation under uniaxial loading with errors below 8.0%. With tissue removal simulated in a full eye numerical model, fibril reorientation could be predicted around the affected area, and this change both increased with larger tissue removal and reduced gradually away from that area. CONCLUSION: The presented method can successfully simulate fibril reorientation with changes in the strain regime affecting cornea tissue. Analyses based on this method showed that fibrils tend to align parallel to the tissue cut following keratoplasty operations. With the ability to simulate fibril reorientation, numerical modelling can have a greater potential in modelling the behaviour following surgery and injury to the cornea.

Role of Cyclic Adenosine Monophosphate in Myopic Scleral Remodeling in Guinea Pigs: A Microarray Analysis.

Purpose: Myopia induction accompanies increased scleral cyclic adenosine phosphate (cAMP) levels and collagen degradation in mammalian models. We compared the scleral gene expression changes following monocular form deprivation (FD) with those induced by adenylate cyclase activation with forskolin (FSK) in guinea pigs. Methods: Guinea pigs were assigned to FD, FSK-treated, and age-matched (AM) control groups. FSK was injected monocularly into the inferior palpebral subconjunctiva daily for 4 days. After scleral RNA extraction, a gene microarray scanner and software were used to evaluate the gene expression patterns, followed by pathway analysis using Gene Ontology tools. Quantitative PCR (qPCR) was used to analyze the expression of 10 candidate genes in separate sets of form-deprived, vehicle-injected, and AM animals. Results: FSK injections differentially regulated 13 collagen subtypes compared to AM and FD groups. FSK also downregulated Acta2 and Tgf-ß2 compared to the AM eyes. Collagen subtypes and Acta2 underwent larger downregulation in the FSK group than during FD. FSK differentially regulated Rarb, Rxrg, Fzd5, Ctnnd2, Dkk2, and Dkk3, which have been linked to ocular growth. Only a few genes were differentially expressed between the FD and AM groups. There was 80% agreement in the direction of gene regulation between microarray and qPCR results. No significant differences were identified between vehicle-injected and AM eyes. Conclusions: Collagen, a major scleral extracellular matrix component, is degraded during myopia. Given that FSK and FD both promote myopia through increased collagen degradation, targeting cAMP signaling pathway genes could suppress myopia development.

Mechanism of Bone Mineralization.

Mineralized "hard" tissues of the skeleton possess unique biomechanical properties to support the body weight and movement and act as a source of essential minerals required for critical body functions. For a long time, extracellular matrix (ECM) mineralization in the vertebrate skeleton was considered as a passive process. However, the explosion of genetic studies during the past decades has established that this process is essentially controlled by multiple genetic pathways. These pathways regulate the homeostasis of ionic calcium and inorganic phosphate-two mineral components required for bone mineral formation, the synthesis of mineral scaffolding ECM, and the maintainence of the levels of the inhibitory organic and inorganic molecules controlling the process of mineral crystal formation and its growth. More recently, intracellular enzyme regulators of skeletal tissue mineralization have been identified. The current review will discuss the key determinants of ECM mineralization in bone and propose a unified model explaining this process.

Articular cartilage response to a sliding load using two different-sized spherical indenters1.

BACKGROUND: Cartilage surface contact geometry influences the deformational behavior and stress distribution throughout the extracellular matrix (ECM) under load. OBJECTIVE: To test the correlation between the mechanical and cellular response of articular cartilage when loaded with two different-sized spherical indenters under dynamic reciprocating sliding motion. METHODS: Articular cartilage explants were subjected to a reciprocating sliding load using a 17.6 mm or 30.2 mm spherical ball for 2000 cycles at 10 mm/s and 4 kg axial load. Deformation of the cartilage was recorded and contact parameters were calculated according to Hertzian theory. After mechanical loading cartilage samples were collected and analyzed for ECM collagen damage, gene regulation and proteoglycan (PG) loss. RESULTS: Significantly higher ECM deformation and strain and lower dynamic effective modulus were found for explants loaded with the smaller diameter indenter whereas contact radius and stress remained unaffected. Also, the 17.6 mm indenter increased PG loss and significantly upregulated genes for ECM proteins and enzymes as compared to the 30.2 mm indenter. CONCLUSION: Sliding loads that increase ECM deformation/strain were found to induce enzyme-mediated catabolic processes in articular cartilage explants. These observations provide further understanding of how changes in cartilage contact mechanics under dynamic conditions can affect the cellular response.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvß3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvß3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3ß activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils.

Collagen XII, largest member of the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The molecule consists of three threadlike N-terminal noncollagenous NC3 domains, joined by disulfide bonds and a short interrupted collagen triple helix toward the C-terminus. Splice variants differ considerably in size and properties: "small" collagen XIIB (220 kDa subunit) is similar to collagen XIV, whereas collagen XIIA (350 kDa) has a much larger NC3 domain carrying glycosaminoglycan chains. Collagen XII binds to collagen I-containing fibrils via its collagenous domain, whereas its large noncollagenous arms interact with other matrix proteins such as tenascin-X. In dense connective tissues and bone, collagen XII is thought to regulate organization and mechanical properties of collagen fibril bundles. Accordingly, recent findings show that collagen XII mutations cause Ehlers-Danlos/myopathy overlap syndrome associated with skeletal abnormalities and muscle weakness in mice and humans.

Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER.

Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2ß1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2ß1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2ß1 integrin and α11ß1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.

[A current viewpoint on structure and evolution of collagens. II. The fibril-associated collagens with interrupted triple helices].

Fibril-associated collagens with interrupted triple helices (FACITs) form one of the subfamilies of collagen family. Being minor components of connective tissues in multicellular animals, FACITs play an important role in structurization of extracellular matrix whose peculiarities determine differences among tissues. FACITs take part in regulation of the sizes of banded collagen fibrils and are also a link between diverse components of extracellular matrix and cells in different tissues. The functional characteristics of FACIT molecules are determined by peculiarities of α-chain structure (interruptions in collagenous domains and module structure of N-terminal noncollagenous regions), trimeric molecules (trimerization domains), and supramolecular assemblies (mainly, association with banded fibrils and inability to form homopolymeric suprastructural aggregates). The evolution of FACITs is also discussed. A hypothetical model of structural changes leading to formation of FACIT subfamily is propounded.

A quantitative comparison of morphological and histological characteristics of collagen in the rabbit medial collateral ligament.

Collagen fiber is one of the critical factors in determining mechanical properties of ligaments and both the morphological and histological characteristics of collagen have been widely studied. However, there was still no consensus about whether the morphological characteristics of collagen correlated with its histological characteristics in physiological ligaments. Rabbit medial collateral ligaments (MCLs) were measured under a transmission electron microscope and a polarized light microscope plus picrosirius red-staining to obtain the distributions of collagen fibril diameters and types at different anatomical sites of rabbit MCLs, respectively. The correlation between the fibril diameter and type was determined by a correlation analysis. The collagen fibril diameters at the different anatomical sites had different distributions (unimodal or bimodal) and mean fibril diameters were found to increase significantly from the anterior part to the posterior part (P=0.0482) as well as from the proximal to the distal sections (P=0.0208). Type I collagen in the core portion of MCLs was significantly less than at the other four peripheral areas (P<0.005) but no significant variation was found in each respective portion (P>0.05). The low coefficient in the correlation analysis (r=0.3759) demonstrated collagen fibril diameters had no correlation with collagen types. This may provide a new view of collagen types in studying the mechanical behavior of ligaments.

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5.

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.

Mechanisms of collagen fibril alignment in tendon injury: from tendon regeneration to artificial tendon.

The process by which collagen fibrils are aligned following tendon injury remains unknown. Therefore, we analyzed the process of tendon regeneration by transmission electron microscopy, using a film model method. In mice, the Achilles tendon of medial head was transected. On day 3, after only the proximal end of the transected tendon was placed on film and kept in vivo, a translucent substance containing granules, called tendon gel, was secreted. On day 5, the granules assembled in a loose (L) layer, and coalesced tightly in a dense (D) layer, forming an L-D-L layered pattern. On day 10, granules showed high electron density in H layers, which developed into D-H-D layers on day 13. The distal end was placed on film to face the proximal end. On day 10, the tendon gel showed a D-H-D layer pattern. On day 11, mechanical stress from muscular constriction changed the tendon gel to aligned collagen fibrils (6 ± 2 nm in diameter). Thereafter, the diameter of the fibrils increased. Tendon gel harvested on day 5 or day 10 was pulled manually or by hanging weights (about 0.6 MPa). Aligned collagen fibrils (32 ± 7 nm in diameter) were created by traction using tendon gel harvested on day 10.

Collagen fibril disruption occurs early in primary guinea pig knee osteoarthritis.

OBJECTIVE: A major barrier inhibiting the discovery of structural modifying agents for osteoarthritis (OA) is an incomplete understanding of early disease events. Herein, we investigated the time course of collagen II cleavage and fibril disruption in the well-validated Hartley guinea pig model of spontaneous OA of the knee. METHODS: Knee joints of 46 male Hartley guinea pigs were analyzed at 3 weeks, 2, 4, 7, 10, 12, and 18 months of age for histological severity of OA, cartilage collagen fibril disruption by semi-quantitative polarized light microscopy, and expression of type II collagen degradation biomarkers, 9A4 and Coll2-1, by immunohistochemistry. In addition, serum biomarkers specific for collagen II degradation, CTX-II, C2C, and Coll2-1 were quantified. RESULTS: Collagen fibril disruption and expression of the collagenase-generated cleavage neoepitope, 9A4, were observed as early as 2 months of age, despite the appearance of histological OA at 4 months of age. Only serum Coll2-1 increased coincident with the early disruption of the collagen fibril between 3 weeks and 7 months, in contrast to serum C2C, which did not change significantly or correlate with histological severity. Inversely, CTX-II declined dramatically from 3 weeks to 4 months and remaining low thereafter, coincident with growth plate turnover. CONCLUSIONS: Collagenase cleavage and disruption of the type II collagen network are early OA disease events in this model, preceding histological evidence of proteoglycan loss. The markedly different serum profiles of collagen II-related biomarkers during the early stages of disease development suggest compartmental segregation and temporal regulation of collagen degrading enzymes.

Type VII collagen: the anchoring fibril protein at fault in dystrophic epidermolysis bullosa.

Type VII collagen is a major component of the anchoring fibrils of the dermal-epidermal adhesion on the dermal side at the lamina densa/papillary dermis interface. Dystrophic epidermolysis bullosa (DEB) emerged as a candidate for type VII collagen mutations becausing anchoring fibrils were shown to be morphologically altered, reduced in number, or completely absent in patients with different forms of DEB. Circulating autoantibodies recognize type VII collagen epitopes in epidermolysis bullosa acquisita. The suggestion that type VII collagen is required for human epidermal tumorigenesis relates to the increasing numbers of life-threatening complications associated with developing squamous cell carcinomas because of the extended life span of affected individuals with recessive DEB.

Collagen fibrillogenesis in tendon development: current models and regulation of fibril assembly.

Tendons are collagen-based fibrous tissues that connect and transmit forces from muscle to bone. These tissues, which are high in collagen type I content, have been studied extensively to understand collagen fibrillogenesis. Although the mechanisms have not been fully elucidated, our understanding has continued to progress. Here, we review two prevailing models of collagen fibrillogenesis and discuss the regulation of the process by candidate cellular and extracellular matrix molecules. Although numerous molecules have been implicated in the regulation of collagen fibrillogenesis, we focus on those that have been suggested to be particularly relevant to collagen type I fibril formation during tendon development, including members of the collagen and small leucine-rich proteoglycan families, as well as other molecules, including scleraxis, cartilage oligomeric matrix protein, and cytoskeletal proteins.

Morphometric analysis of collagen fibrils in skin of patients with spontaneous cervical artery dissection.

BACKGROUND AND AIM: The aetiopathogenesis of spontaneous cervical artery dissection (sCAD) is largely unknown. Electron microscopic (EM) examination of skin biopsies of patients with sCAD revealed very subtle pathological changes of dermal connective tissue in about half of these patients leading to the hypothesis of an underlying connective tissue disorder. However, connective tissue abnormalities did not allow clear discrimination between patients and controls in our hands. Therefore, we sought to establish an objective basis for the assessment of connective tissue abnormalities in patients with sCAD using standardised morphometric assessment of collagen fibrils. METHODS: In this study a blinded examination was performed of collagen in skin biopsies and it sought parameters which are able to discriminate between patients with sCAD and controls. Various morphometric parameters were compared between patients with sCAD (n = 20) and control subjects (n = 18). RESULTS: Previously described "flower-like" collagen fibrils in skin biopsies were extremely rare in patients and controls and did not discriminate between the groups. However, they were abundant in the skin biopsy of a patient with Ehlers-Danlos syndrome type III (EDSIII) used as a reference. Morphometric parameters such as collagen fibril diameter, fibril density and relative fibril area did not discriminate between patients and controls on an individual basis, but the mean diameter of collagen fibrils in the skin was lower in patients with sCAD compared with controls while fibril density was higher resulting in nearly equal fibril areas per unit of area (relative fibril area) comparing both groups as well as individuals. CONCLUSIONS: Blinded pathological and morphometric analysis of collagen fibres in skin biopsies was, in our hands, unable to discriminate reliably between patients with sCAD and controls on an individual basis but did show differences in collagen fibril morphometry on a group basis. Furthermore, systematic and blinded pathological studies of skin biopsies in patients with sCAD and controls are needed.