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.

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.

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 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.

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.

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.

Articular cartilage collagen: an irreplaceable framework?

Adult articular cartilage by dry weight is two-thirds collagen. The collagen has a unique molecular phenotype. The nascent type II collagen fibril is a heteropolymer, with collagen IX molecules covalently linked to the surface and collagen XI forming the filamentous template of the fibril as a whole. The functions of collagens IX and XI in the heteropolymer are far from clear but, evidently, they are critically important since mutations in COLIX and COLXI genes can result in chondrodysplasia syndromes. Here we review what is known of the collagen assembly and present new evidence that collagen type III becomes covalently added to the polymeric fabric of adult human articular cartilage, perhaps as part of a matrix repair or remodelling process.

Collagens and cartilage matrix homeostasis.

At least 27 types of collagen, the products of forty genes, are expressed in the tissues of higher vertebrates. Cartilage has a distinctive collagen phenotype. Two-thirds of the dry weight of adult articular cartilage is collagen. Proteolysis of this collagen framework is integral to the process of cartilage destruction and joint failure in osteoarthritis. Molecular studies are revealing the mechanisms of assembly of cartilage collagen fibrils. The nascent Type II collagen fibril is a heteropolymer, with collagen IX molecules covalently linked to the surface and collagen XI forming a filamentous template at the core, which regulates fibril diameter through its retained N-propeptide domains. This structure presents a challenge to understanding how fibril growth and collagen network maturation are brought about. Proteolytic remodeling, other than that mediated by collagenases, would appear to be involved, but the proteases and molecular mechanisms are still undefined. Valuable insights and predictions on the function of the individual collagen types in cartilage continue to come from the study of skeletal dysplasia syndromes caused by mutations in genes for collagens and associated matrix proteins.

Expression of FACIT collagens XII and XIV during bleomycin-induced pulmonary fibrosis in mice.

Collagens XII and XIV are members of a subfamily of fibril-associated collagens with interrupted triple-helices (FACITs) that facilitate the interactions of adjacent collagen fibrils. Using immunohistochemistry and in situ hybridization, we analyzed the spatial and temporal expression pattern of collagens XII and XIV during bleomycin-induced pulmonary fibrosis. C57Bl mice were treated with bleomycin (1 U, i.p., every other day for 8 days) or saline (control), and lung tissue samples were analyzed 2-12 weeks later. Collagen I protein expression was increased in the lung 2 weeks post bleomycin treatment and persisted for at least 12 weeks. In contrast, collagen XII and XIV expression was low until 4 weeks after bleomycin treatment. Whereas collagen XII expression was greatest between 4 weeks and 8 weeks, expression of collagen XIV persisted from 4 to 12 weeks, which suggests that these two proteins may play distinct roles in the fibrotic process. The mRNA for lysyl oxidase (LOX), an enzyme for cross-linking of collagens, had a delayed increase in the lung after bleomycin administration. It reached a maximum after 8 weeks, and persisted throughout the 12 weeks of the study. These data support the hypothesis that fibrosis is a multistep process that involves both collagen accumulation and changes in the molecules that modulate the biomechanical properties of fibrils.

Collagens XII and XIV (FACITs) in capsular opacification and in cultured lens epithelial cells.

PURPOSE: We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). METHODS: Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. RESULTS: In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. CONCLUSIONS: LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.