Collagen cross-linking. Synthesis of collagen cross-links in vitro with highly purified lysyl oxidase.

In this paper, the synthesis of collagen cross-links in vitro was investigated in a defined system consisting of highly purified chick cartilage lysyl oxidase and chick bone collagen fibrils. Cross-link synthesis in vitro was quite similar to the biosynthesis of collagen cross-links in vivo. Enzyme-dependent synthesis of cross-link intermediates and cross-linked collagen derived from lathyritic collagen occurred. The concentration of the two principal reducible cross-links, N6:6'-dehydro-5,5'-dihydroxylysinonorleucine and N6:6'-dehydro-5-hydroxylysinonorleucine, increased to a peak value of approximately two cross-links per molecule and then decreased. Synthesis of histidinohydroxymerodesmosine and a second polyfunctional cross-link of unknown structure began after synthesis of bifunctional cross-links was largely completed and proceeded linearly afterwards. Inhibition of lysyl oxidase after the bulk of bifunctional cross-link synthesis had occurred did not alter the rate of decrease in reducible cross-link concentration but did inhibit further histidinohydroxymerodesmosine synthesis. These results indicate that lysyl oxidase and collagen fibrils are the only macromolecules required for cross-link biosynthesis in vivo. It is likely that the decrease in reducible cross-links observed during fibril maturation results from spontaneous reactions within the collagen fibril rather than additional enzymatic reactions.     

Role of matricellular proteins in cardiac tissue remodeling after myocardial infarction

After onset of myocardial infarction (MI), the left ventricle (LV) undergoes a continuum of molecular, cellular, and extracellular responses that result in LV wall thinning, dilatation, and dysfunction. These dynamic changes in LV shape, size, and function are termed cardiac remodeling. If the cardiac healing after MI does not proceed properly, it could lead to cardiac rupture or maladaptive cardiac remodeling, such as further LV dilatation and dysfunction, and ultimately death. Although the precise molecular mechanisms in this cardiac healing process have not been fully elucidated, this process is strictly coordinated by the interaction of cells with their surrounding extracellular matrix (ECM) proteins. The components of ECM include basic structural proteins such as collagen, elastin and specialized proteins such as fibronectin, proteoglycans and matricellular proteins. Matricellular proteins are a class of non-structural and secreted proteins that probably exert regulatory functions through direct binding to cell surface receptors, other matrix proteins, and soluble extracellular factors such as growth factors and cytokines. This small group of proteins, which includes osteopontin, thrombospondin-1/2, tenascin, periostin, and secreted protein, acidic and rich in cysteine, shows a low level of expression in normal adult tissue, but is markedly upregulated during wound healing and tissue remodeling, including MI. In this review, we focus on the regulatory functions of matricellular proteins during cardiac tissue healing and remodeling after MI.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.     

Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.     

Conformation and sequence evidence for two-fold symmetry in left-handed beta-helix fold

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion.A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls.Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.     

Collagen I, laminin, and tenascin: ultrastructure and correlation with avian neural crest formation.

We have investigated the distribution of type I collagen, tenascin, and laminin in younger chick embryos than have previously been studied in detail. The initial appearance of type I collagen, but not tenascin and laminin, is exactly correlated with the beginning of neural crest migration, suggesting a role for collagen I in the migration. Light microscopy of whole mounts of 2-day-old chick embryos reveals that type I collagen is expressed in a rostral to caudal gradient; it localizes to the notochord sheath before accumulating around the neural tube and somites. Collagen I and tenascin also associate with central somite cells. Surprisingly, no extracellular matrix can be detected among the early sclerotomal cells, which suggests that little or no cell migration is involved in this epithelial-mesenchymal transformation. Electron microscopy using peroxidase antiperoxidase reveals that tenascin is present in nonstriated, 10 nm wide fibrils and in interstitial bodies, both of which have previously been reported to contain fibronectin. However, collagen I only occurs in the 10 nm fibrils and larger striated fibrils. This is the first ultrastructural study to assign tenascin to fibrils and interstitial bodies and to describe its appearance and disappearance from embryonic basement membranes. The discussion emphasizes the possible importance of type I collagen in neural crest cell migration and compares the ultrastructural associations of the ECM molecules present at this early embryonic stage.     

Extracellular matrix regulation of fibroblast function: redefining our perspective on skin aging

The dermal extracellular matrix (ECM) comprises the bulk of skin and confers strength and resiliency. In young skin, fibroblasts produce and adhere to the dermal ECM, which is composed primarily of type I collagen fibrils. Adherence allows fibroblasts to spread and exert mechanical force on the surrounding ECM. In this state, fibroblasts display a “youthful” phenotype characterized by maintenance of the composition and structural organization of the dermal ECM. During aging, fibroblast-ECM interactions become disrupted due to fragmentation of collagen fibrils. This disruption causes loss of fibroblast spreading and mechanical force, which inextricably lead to an “aged” phenotype; fibroblasts synthesize less ECM proteins and more matrix-degrading metalloproteinases. This imbalance of ECM homeostasis further drives collagen fibril fragmentation in a self-perpetuating cycle. This article summarizes age-related changes in the dermal ECM and the mechanisms by which these changes alter the interplay between fibroblasts and their extracellular matrix microenvironment that drive the aging process in human skin.     

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.     

Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates.

Collagen fibrils are the principal source of mechanical strength of connective tissues such as tendon, skin, cornea, cartilage and bone. The ability of these tissues to withstand tensile forces is directly attributable to the length and diameter of the fibrils, and to interactions between individual fibrils. Although electron microscopy studies have provided information on fibril diameters, little is known about the length of fibrils in tissue and how fibrils interact with each other. The question of fibril length has been difficult to address because fibril ends are rarely observed in cross-sections of tissue. The paucity of fibril ends, or tips, has led to controversy about how long individual fibrils might be and how the fibrils grow in length and diameter. This review describes recent discoveries that are relevant to these questions. We now know that vertebrate collagen fibrils are synthesised as short (1-3 microm) early fibrils that fuse end-to-end in young tissues to generate very long fibrils. The diameter of the final fibril is determined by the diameter of the collagen early fibrils. During a late stage of tissue assembly fibril tips fuse to fibril shafts to generate branched networks. Of direct relevance to fibril fusion is the fact that collagen fibrils can be unipolar or bipolar, depending on the orientation of collagen molecules in the fibril. Fusion relies on: (1) specific molecular interactions at the carboxyl terminal ends of unipolar collagen fibrils; and (2) the insulator function of small proteoglycans to shield the surfaces of fibrils from inappropriate fusion reactions. The fusion of tips to shafts to produce branched networks of collagen fibrils is an elegant mechanism to increase the mechanical strength of tissues and provides an explanation for the paucity of fibril tips in older tissue.     

Time-dependent increase in the stability of collagen fibrils formed in vitro. I. Effects of pH and salt concentration on the dissolution of the fibrils.

The time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37 degrees C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.     

ColBuilder: A server to build collagen fibril models

Type I collagen is the main structural component of many tissues in the human body. It provides excellent mechanical properties to connective tissue and acts as a protein interaction hub. There is thus a wide interest in understanding the properties and diverse functions of type I collagen at the molecular level. A precondition is an atomistic collagen I structure as it occurs in native tissue. To this end, we built full-atom models of cross-linked collagen fibrils by integrating the low-resolution structure of collagen fibril available from x-ray fiber diffraction with high-resolution structures of short collagen-like peptides from x-ray crystallography and mass spectrometry data. We created a Web resource of collagen models for 20 different species with a large variety of cross-link types and localization within the fibril to facilitate structure-based analyses and simulations of type I collagen in health and disease. To easily enable simulations, we provide parameters of the modeled cross-links for an Amber force field. The repository of collagen models is available at https://colbuilder.h-its.org.     

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

Fibronectin matrix polymerization increases tensile strength of model tissue

The composition and organization of the extracellular matrix (ECM) contribute to the mechanical properties of tissues. The polymerization of fibronectin into the ECM increases actin organization and regulates the composition of the ECM. In this study, we examined the ability of cell-dependent fibronectin matrix polymerization to affect the tensile properties of an established tissue model. Our data indicate that fibronectin polymerization increases the ultimate strength and toughness, but not the stiffness, of collagen biogels. A fragment of fibronectin that stimulates mechanical tension generation by cells, but is not incorporated into ECM fibrils, did not increase the tensile properties, suggesting that changes in actin organization in the absence of fibronectin fibril formation are not sufficient to increase tensile strength. The actin cytoskeleton was needed to initiate the fibronectin-induced increases in the mechanical properties. However, once fibronectin-treated collagen biogels were fully contracted, the actin cytoskeleton no longer contributed to the tensile strength. These data indicate that fibronectin polymerization plays a significant role in determining the mechanical strength of collagen biogels and suggest a novel mechanism by which fibronectin can be used to enhance the mechanical performance of artificial tissue constructs.     

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.     

Towards a fibrous composite with dynamically controlled stiffness: lessons from echinoderms

Sea urchins and sea cucumbers, like other echinoderms, control the tensile properties of their connective tissues by regulating stress transfer between collagen fibrils. The collagen fibrils are spindle-shaped and up to 1 mm long with a constant aspect ratio of approx. 2000. They are organized into a tissue by an elastomeric network of fibrillin microfibrils. Interactions between the fibrils are regulated by soluble macromolecules that are secreted by local, neurally controlled, effector cells. We are characterizing the non-linear viscoelastic properties of sea cucumber dermis under different conditions, as well as the structures, molecules and molecular interactions that determine its properties. In addition, we are developing reagents that will bind covalently to fibril surfaces and reversibly form cross-links with other reagents, resulting in a chemically controlled stress-transfer capacity. The information being developed will lead to the design and construction of a synthetic analogue composed of fibres in an elastomeric matrix that contains photo- or electro-sensitive reagents that reversibly form interfibrillar cross-links.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.