Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

Collagen V localizes to pericellular sites during tendon collagen fibrillogenesis

During tendon development collagen fibrillogenesis occurs in extracellular micro-domains defined by the tenocytes. This permits cellular regulation of the extracellular steps involved in the tissue-specific matrix assembly required for function. The hypothesis tested here is that collagen V associates with the tenocyte surface where it functions in regulation of collagen assembly and cell-directed fibril deposition. The in vitro and in vivo data demonstrate that collagen V is a quantitatively minor component of the tendon. It is preferentially localized on the tenocyte surface as distinct foci in tendons and in cell culture. In vitro data indicate that this interaction with the tenocyte is not HSPG GAG-dependent. Collagen V is present as the mature, processed form, is absent from the media, and is a significant part of the detergent-insoluble cell layer, presumably as part of a membrane-associated complex. In contrast, procollagen I is not efficiently processed and is found predominantly in the culture media. Our data suggest that the regulatory role of collagen V requires collagen V to occupy a different cellular niche from the structural collagen I. In monolayer cultures, the conversion to the tissue form of collagen V and its deposition with the cell layer suggest efficient engagement of procollagen V with pericellular receptors and processing enzymes. The secretion of collagen I into the media and inefficient processing of procollagen I suggest reduced accessibility to these pericellular molecules due to disengagement from the cell surface. This all points to differential spatial localization of collagen V as a mechanism to optimize its regulatory roles during the cell-surface directed steps in tendon collagen fibril assembly.     

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.     

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.     

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon.

The small dermatan sulphate proteoglycan of bovine tendon demonstrated a unique ability to inhibit fibrillogenesis of both type I and type II collagen from bovine tendon and cartilage respectively in an assay performed in vitro. None of the other proteoglycan populations from cartilage, tendon or aorta, even those similar in size and chemical structure, had this effect. Alkali treatment of the small proteoglycan of tendon eliminated its ability to inhibit fibrillogenesis, whereas chondroitinase digestion did not. This indicates that its interaction with collagen depends on the core protein. Fibrillogenesis of pepsin-digested collagens was affected similarly, indicating that interaction with the collagen telopeptides is not involved. The results suggest that interactions between collagen and proteoglycans may be quite specific both for the type of proteoglycan and its tissue of origin.     

Development of collagen fibril organization and collagen crimp patterns during tendon healing

Normal tendon comprises coaxially aligned bundles of crimped collagen fibres each of which possesses a fibrillar substructure. In acute traumatic injury this level of organization is disrupted and the mechanical function of the tendon impaired. During repair, a degree of recovery of the fibrillar structure takes place. In this tudy we have assessed the re-establishment of tendon organization after injury on the basis of the collagen fibril diameter distribution and the collagen crimp parameters. Crimp became undetectable following injury but one month later was present throughout the tissue. At this time the periodicity was greatly reduced by comparison with that of the normal tendon and normal values were not re-established within 14 months following injury. Collagen fibril diameters remained abnormally small over this same period of time. In particular, fibrils of diameters in excess of 100 nm, commonly found in normal and contralateral tendons, were totally absent from the observed distributions in the healing tendons. Such large diameter fibrils often account for as much as 50% of the total mass of collagen present in the uninjured tissue. Thus the mechanical properties of the healing tendon may remain significantly different from those of normal tendon for a minimum time of 14 months after injury.     

The crystalline structure of collagen fibrils in tendon.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with $\text{b = 75.5}\text{A}\text{?}$, β = 93 °, $\text{a =}\text{b}\text{sin}\text{β}$, a = 90 °, $\text{c = n × 668}\text{A}\text{?}$, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.     

Role of aldehydes in collagen fibrillogenesis in vitro

The process of in vitro fibrillogenesis of type I collagen from bovine tendon has been studied with particular attention to the events occurring in the lag phase. From the conditions under which those events can be reversed or stabilized and from the effects of reduction on the length of the lag phase, we conclude that one event is the reaction of aldehydes on the molecules. However, we have been unable to detect the products of these reactions or any physical changes in the solution, so the change most probably involves the inter- or intramolecular reaction of a very small fraction of the molecules. In particular, the viscosity appears unchanged in solutions in which nucleation-promoting structures have been stabilized, and so measurable numbers of polymers are not formed in this process.     

Hemopexin: structure,function, and regulation

Hemopexin (HPX) is the plasma protein with the highest binding affinity to heme among known proteins. It is mainly expressed in liver, and belongs to acute phase reactants, the synthesis of which is induced after inflammation. Heme is potentially highly toxic because of its ability to intercalate into lipid membrane and to produce hydroxyl radicals. The binding strength between heme and HPX, and the presence of a specific heme-HPX receptor able to catabolize the complex and to induce intracellular antioxidant activities, suggest that hemopexin is the major vehicle for the transportation of heme in the plasma, thus preventing heme-mediated oxidative stress and heme-bound iron loss. In this review, we discuss the experimental data that support this view and show that the most important physiological role of HPX is to act as an antioxidant after blood heme overload, rather than to participate in iron metabolism. Particular attention is also put on the structure of the protein and on its regulation during the acute phase reaction.     

The role of decorin in collagen fibrillogenesis and skin homeostasis

Decorin, a prototype member of the growing family of the small leucine-rich proteoglycans (SLRP's), plays significant roles in tissue development and assembly, as well as playing both direct and indirect signaling roles. This review will concentrate on decorin's function in collagen fibrillogenesis as determined through the study of mice with a disrupted decorin gene. The fragile skin and abnormal tendon phenotypes initially observed were found to be due to fundamental alterations in collagen fibers, highlighting the crucial role of proteoglycans in general and SLRP's in particular in collagen fibrillogenesis. The altered fibril formation within tissues in turn leads to observable and quantifiable changes at the organismal level. Research into certain fibrotic processes with concomitant upregulation or reduction of decorin makes interesting comparisons with the collagen malformations seen in Dcn ^–/– mice. Overall, decorin is shown to be a vital player in maintaining skin and tendon integrity at the molecular level, among other functions. Published in 2003.     

Influence of the telopeptides on type I collagen fibrillogenesis

Preparations have been made of acid-soluble collagens whose telopeptides have suffered different levels of proteolytic attack. The collagens with more intact telopeptides form fibrils more rapidly than those with degraded telopeptides. In addition, we have shown that a high molecular weight aggregate rich in the carboxyterminal CNBr peptide, α1CB6, can be found in cyanogen bromide digests of fibrils formed from intact collagen. A similar aggregate is found in CNBr digests of native tendons. The aggregate formed in fibrils assembled in vitro can be stabilized by reduction, and its generation is strongly dependent on the presence of intact telopeptides. The latter point is the most objective evidence that to reproduce the characteristics of native fibrils in vitro, the collagen telopeptides must be preserved from proteolysis.     

The role of small leucine-rich proteoglycans in collagen fibrillogenesis

Small leucine-rich proteoglycans/proteins (SLRPs) are associated with collagen fibril formation, and therefore important for the proper formation of extracellular matrices. SLRPs are differentially expressed in tissues and during pathological conditions, contributing to the development of connective tissue properties. The binding of SLRPs to collagens have recently been characterized, and may give some clues to the significance of these interactions. In this mini review, we summarize published work in this field, and propose several mechanisms for how SLRPs can control collagen matrix structure and function. SLRPs appear to influence collagen cross-linking patterns. We also propose that the SLRP-collagen interactions can assist in the process of juxtaposing the collagen monomers by steric hindrance or by directly connecting two collagen monomers during the fibril growth.     

Type I collagen alpha-1 chain C-telopeptide: solution structure determined by 600-MHz proton NMR spectroscopy and implications for its role in collagen fibrillogenesis

The solution conformation of the alpha-1 chain C-telopeptide has been studied by circular dichroism (CD) and 600-MHz 1H NMR spectroscopy in 60% CD3OH/40% H2O solution. The C-telopeptide contains 27 amino acids which form the C-terminal end of the alpha-1 collagen polypeptide chain. By the combined application of various two-dimensional, phase-sensitive NMR techniques (COSY, RELAY, NOESY, ROESY), a nearly complete assignment of all proton resonances was achieved. Furthermore, the backbone conformation could be established, on the basis of coupling constant and NOE data. The spectroscopic evidence indicates that large sections of the peptide exist in a nonrandom, extended conformation and that there are two segments of higher mobility around the two Gly-Gly units in positions 2,3 and 20,21. Despite these hingelike, flexible sections no measurable fold-back of any of the extended parts was evident. On the basis of this structure, a model is proposed for the simultaneous interaction of the C-telopeptide with two adjacent collagen triple helices within the growing collagen fibril.     

Type I collagen fibrillogenesis: initiation via reversible linear and lateral growth steps

Type I collagen fibrillogenesis in vitro has been studied by laser light scattering, and the results indicate that initiation of aggregation involves at least two steps. Step I of aggregation involves no change in the intensity of scattered light at an angle of 90° and is accompanied by a decrease in the diffusion coefficient. Step II is characterized by an increased intensity of scattered light and decreased diffusion coefficients. Theoretical calculations using the Stokes-Einstein equation for the translational diffusion coefficient and the Perrin equation for the frictional coefficient of a prolate ellipsoid indicate that the step I aggregates are 4D staggered linear dimers and trimers 570 and 845 nm long, whereas step II aggregates are greater than 950 nm in length. These dimensions are similar to those previously reported based on physicochemical measurements and electron microscopy. It is proposed that the rate and extent of fibrillogenesis in vitro is controlled by the concentration of the linear aggregates and that the effects of temperature and collagen concentration on fibrillogenesis previously observed are qualitatively explained in terms of their effects on the concentration of these aggregates.     

Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues

Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.