Aggrecan Molecules Research Articles

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilageduring movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as duringthe development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthriticdiseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

Cartilage polymers: From viscoelastic solutions to weak gels*

AbstractThe dynamic properties of the polymeric components of cartilage are important for understanding the many biological functions of this biological tissue. Knowledge of the rheology of these materials is also crucial for the development of therapeutic treatments for diseases, such as arthritis, in which cartilage loses its functional physiological properties. In the present work complementary noninvasive techniques, rheology and dynamic light scattering (DLS) are used to probe the structural and dynamic properties of solutions of the principal proteoglycan components of cartilage extracellular matrix at different levels of structural hierarchy (chondroitin sulfate, aggrecan, aggrecan‐hyaluronic acid complex). It is shown that the bottlebrush shaped aggrecan molecules form large, loosely organized polyelectrolyte domains comprised of molecules organized into diffuse branched polymer network structures permeated by a diffuse cloud of counter‐ions. The rheological behaviors of these polymer solutions vary from viscoelastic solutions, consistent with the formation of dynamic polymer clusters in solution, to “weak gel” materials exhibiting approximate power law shear stress relaxation or creep over a wide range of timescales.

Rheological Properties of Cartilage Glycosaminoglycans and Proteoglycans

Glycosaminoglycans (GAGs) are molecules that govern the load-bearing and frictional properties of cartilage and the lubricating properties of synovial fluid of joints. Most GAGs in the body form proteoglycans (PGs), and the dominant PG in cartilage is the bottlebrush-shaped aggrecan, comprised of a protein core decorated by GAG side chains consisting mainly of chondroitin sulfate (CS) and keratan sulfate. Additionally, hyaluronic acid (HA) induces aggrecan to self-assemble into complexes having up to 100 aggrecan molecules attached to each HA chain “backbone”. Here, we report the rheological properties of CS, HA, aggrecan, and aggrecan–HA complexes in water and salt solutions. We find that CS solutions are viscous liquids, while HA solutions exhibit viscoelasticity and shear thinning. Specifically, in the case of HA, the storage modulus G′ exceeds the loss modulus G″ at high frequencies (ω) while the reverse is true at low ω. Thus, the rheologies of CS and HA exhibit somewhat distinct viscoelastic properties. Aggrecan, on the other hand, shows a rheology consistent with an incipient “weak gel” in which G′ and G″ cross at a specific ω and follow a power-law scaling over a wide ω range. Moreover, aggrecan samples do not attain an observable plateau in the viscosity under steady shear and low shear rates. Finally, the complexation of aggrecan and HA over a period of 48 h results in a slow evolution (“aging”) in measured rheological properties, with G′ at low ω progressively increases in time, while G″ remains essentially unchanged. This suggests that aggrecan and HA gradually form a macroscopic network in which molecular complexes act as physical cross-links.

Decorin Regulates the Aggrecan Network Integrity and Biomechanical Functions of Cartilage Extracellular Matrix.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

Expression of aggrecan components in perineuronal nets in the mouse cerebral cortex

Specific regions of the cerebral cortex are highly plastic in an organism’s lifetime. It is thought that perineuronal nets (PNNs) regulate plasticity, but labeling for Wisteria floribunda agglutinin (WFA), which is widely used to detect PNNs, is observed throughout the cortex. The aggrecan molecule—a PNN component—may regulate plasticity, and may also be involved in determining region-specific vulnerability to stress. To clarify cortical region-specific plasticity and vulnerability, we qualitatively analyzed aggrecan-positive and glycosylated aggrecan-positive PNNs in the mature mouse cerebral cortex. Our findings revealed the selective expression of both aggrecan-positive and glycosylated aggrecan-positive PNNs in the cortex. WFA-positive PNNs expressed aggrecan in a region-specific manner in the cortex. Furthermore, we observed variable distributions of PNNs containing WFA- and aggrecan-positive molecules. Together, our findings suggest that PNN components and their function differ depending on the cortical region, and that aggrecan molecules may be involved in determining region-specific plasticity and vulnerability in the cortex.

Biomimetic molecules lower catabolic expression and prevent chondroitin sulfate degradation in an osteoarthritic ex vivo model.

Aggrecan, the major proteoglycan in cartilage, serves to protect cartilage tissue from damage and degradation during the progression of osteoarthritis (OA). In cartilage extracellular matrix (ECM) aggrecan exists in an aggregate composed of several aggrecan molecules that bind to a single filament of hyaluronan. Each molecule of aggrecan is composed of a protein core and glycosaminoglycan sides chains, the latter of which provides cartilage with the ability to retain water and resist compressive loads. During the progression of OA, loss of aggrecan is considered to occur first, after which other cartilage matrix components become extremely susceptible to degradation. Proteolytic cleavage of the protein core of aggrecan by enzymes such as aggrecanases, prevent its binding to HA and lower cartilage mechanical strength. Here we present the use of HA-binding or collagen type II-binding molecules that functionally mimic aggrecan but lack known cleavage sites, protecting the molecule from proteolytic degradation. These molecules synthesized with chondroitin sulfate backbones conjugated to hyaluronan- or collagen type II- binding peptides, are capable of diffusing through a cartilage explant and adhering to the ECM of this tissue. The objective of this study was to test the functional efficacy of these molecules in an ex vivo osteoarthritic model to discern the optimal molecule for further studies. Different variations of chondroitin sulfate conjugated to the binding peptides were diffused through aggrecan depleted explants and assessed for their ability to enhance compressive stiffness, prevent CS degradation, and modulate catabolic (MMP-13 and ADAMTS-5) and anabolic (aggrecan and collagen type II) gene expression. A pilot in vivo study assessed the ability to retain the molecule within the joint space of an osteoarthritic guinea pig model. The results indicate chondroitin sulfate conjugated to hyaluronan-binding peptides is able to significantly restore equilibrium modulus and prevent CS degradation. All molecules demonstrated the ability to lower catabolic gene expression in aggrecan depleted explants. In order to enhance biosynthesis and regeneration, the molecules need to be coupled with an external stimulant such as a growth factor. The chondroitin sulfate molecule synthesized with HA-binding peptides demonstrated adherence to cartilage tissue and retention up to 6 hours in an ambulatory joint. Further studies will monitor the in vivo residence time and ability of the molecules to act as a disease-modifying agent.

Interaction of Cartilage Biopolymers

SummaryAggrecan is a charged biopolymer whose complexes with hyaluronic acid provide the compressive resistance of cartilage. In cartilage extracellular matrix aggrecan‐hyaluronic acid complexes are enmeshed within a network of collagen fibrils. Osmotic pressure measurements and small angle neutron scattering (SANS) measurements are reported on solutions of aggrecan and aggrecan‐hyaluronic acid complexes. These techniques probe the length scales relevant to the biological functions of cartilage. Osmotic pressure measurements indicate that dissolved aggrecan monomers form microgel‐like assemblies. The osmotic pressure of aggrecan‐hyaluronic acid complexes decreases with decreasing the ratio of aggrecan to hyaluronic acid. SANS reveals that there is no significant interpenetration between the neighboring aggrecan molecules. Both osmotic pressure measurements and SANS indicate weak interactions between the aggrecan bottlebrushes and collagen fibers.

The role of aggrecan in normal and osteoarthritic cartilage.

Aggrecan is a large proteoglycan bearing numerous chondroitin sulfate and keratan sulfate chains that endow articular cartilage with its ability to withstand compressive loads. It is present in the extracellular matrix in the form of proteoglycan aggregates, in which many aggrecan molecules interact with hyaluronan and a link protein stabilizes each interaction. Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events. Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains, whereas those due to degradation cause cleavage of all components of the aggregate. These latter changes can be viewed as being detrimental to cartilage function and are enhanced in osteoarthritic cartilage, resulting in aggrecan depletion and predisposing to cartilage erosion. Matrix metalloproteinases and aggrecanases play a major role in aggrecan degradation and their production is upregulated by mediators associated with joint inflammation and overloading. The presence of increased levels of aggrecan fragments in synovial fluid has been used as a marker of ongoing cartilage destruction in osteoarthritis. During the early stages of osteoarthritis it may be possible to retard the destructive process by enhancing the production of aggrecan and inhibiting its degradation. Aggrecan production also plays a central role in cartilage repair techniques involving stem cell or chondrocyte implantation into lesions. Thus aggrecan participates in both the demise and survival of articular cartilage.

Molecular Therapy for Disk Degeneration and Pain

The nucleus pulposus of the intervertebral disk contains high amounts of the proteoglycan aggrecan, which confers the disk with a remarkable ability to resist compression. Other molecules such as collagens and noncollagenous proteins in the extracellular matrix are also essential for function. During disk degeneration, aggrecan and other molecules are lost due to proteolysis. This can result in loss of disk height, which can ultimately lead to pain. Biological therapy of intervertebral disk degeneration aims at preventing or restoring primarily aggrecan content and other molecules using therapeutic molecules. The purpose of the article is to review recent advances in biological repair of degenerate disks and pain.

Enhanced Hyaline Cartilage Matrix Synthesis in Collagen Sponge Scaffolds by Using siRNA to Stabilize Chondrocytes Phenotype Cultured with Bone Morphogenetic Protein-2 Under Hypoxia

Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

Mechanical strain stimulates hedgehog signalling in adult articular chondrocytes and reduces primary cilia length

Mechanical strain stimulates hedgehog signalling in adult articular chondrocytes and reduces primary cilia length

Hierarchical Organization of Cartilage Proteoglycans

The hierarchical organization of cartilage proteoglycans is investigated on different length and time scales using osmotic pressure measurements, small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), static and dynamic light scattering and neutron spin echo techniques. Osmotic pressure measurements reveal association of aggrecan bottlebrushes into microgel-like assemblies. SAXS, SANS and light scattering results indicate weak interpenetration between neighboring aggrecan molecules. As opposed to DNA and many synthetic polyelectrolytes, which display great sensitivity to ion valence, aggrecan exhibits exceptional insensitivity to calcium ions in the physiological ion concentration range and beyond. This property allows aggrecan to play a role of ion reservoir that can mediate calcium metabolism in cartilage and bone.

Probing Aggrecan Interactions by Atomic Force Microscopy

Aggrecan, the major extracellular matrix proteoglycan in cartilage, is a highly charged bottlebrush shaped macromolecule. It consists of negatively charged glycosaminoglycan (GAG) chains attached to a protein backbone. The bottlebrush structure enables aggrecan to maintain an extended conformation responsible for the high osmotic pressure sustaining compressive loads in cartilage. Alterations in aggrecan bottlebrush structure with age and disease lead to bone deformities, dwarfism, arthritis, and other pathological conditions. In solution, aggrecan bottlebrushes show distinct osmotic pressure versus concentration regimes. They self-assemble and form large clusters. Using Atomic Force Microscopy aggrecan molecules adsorbed on controlled mica surfaces were imaged. On positively charged APS mica, the average extension and height of the bottlebrush side chains were practically unaffected by the presence of calcium ions. With increasing aggrecan concentration transition takes place from dispersed, non-interacting bottlebrushes to clusters of conforming chains. At higher concentrations aggrecan molecules form a continuous monolayer. These surface observations are consistent with aggrecan properties in solution. On negatively charged mica, aggrecan shows interesting network patterns at higher concentrations. Understanding aggrecan adsorption onto charged surfaces provides insight into its interactions with bone and implants in the biological milieu.

Single-Molecule Force Spectroscopy of Cartilage Aggrecan Self-Adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca 2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x β = 0.31 ± 0.08 nm. Whereas the x β -value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

Probing Interactions between Aggrecan and Mica Surface by the Atomic Force Microscopy.

Aggrecan is a bottlebrush shaped macromolecule found in the extracellular matrix of cartilage. The negatively charged glycosaminoglycan (GAG) chains attached to its protein backbone give aggrecan molecules a high charge density, which is essential for exerting high osmotic swelling pressure and resisting compression under external load. In solution aggrecan assemblies are insensitive to the presence of calcium ions, and show distinct osmotic pressure versus concentration regimes. The aim of this study is to investigate the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well-controlled mica surfaces. The conformation of the aggrecan were visualized using Atomic Force Microscopy. On positively charged APS mica the GAG chains of the aggrecan molecules are distinguishable, and their average dimensions are practically unaffected by the presence of salt ions. With increasing aggrecan concentration they form clusters, and at higher concentrations they form a continuous monolayer of conforming molecules. On negatively charged mica, the extent of aggrecan adsorption varies with salt composition. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu.

A validated model of GAG deposition, cell distribution, and growth of tissue engineered cartilage cultured in a rotating bioreactor

In this work a new phenomenological model of growth of cartilage tissue cultured in a rotating bioreactor is developed. It represents an advancement of a previously derived model of deposition of glycosaminoglycan (GAG) in engineered cartilage by (i) introduction of physiological mechanisms of proteoglycan accumulation in the extracellular matrix (ECM) as well as by correlating (ii) local cell densities and (iii) tissue growth to the ECM composition. In particular, previously established predictions and correlations of local oxygen concentrations and GAG synthesis rates are extended to distinguish cell secreted proteoglycan monomers free to diffuse in cell surroundings and outside from the engineered construct, from large aggrecan molecules, which are constrained within the ECM and practically immovable. The model includes kinetics of aggregation, that is, transformation of mobile GAG species into immobile aggregates as well as maintenance of the normal ECM composition after the physiological GAG concentration is reached by incorporation of a product inhibition term. The model also includes mechanisms of the temporal evolution of cell density distributions and tissue growth under in vitro conditions. After a short initial proliferation phase the total cell number in the construct remains constant, but the local cell distribution is leveled out by GAG accumulation and repulsion due to negative molecular charges. Furthermore, strong repulsive forces result in expansion of the local tissue elements observed macroscopically as tissue growth (i.e., construct enlargement). The model is validated by comparison with experimental data of (i) GAG distribution and leakage, (ii) spatial-temporal distributions of cells, and (iii) tissue growth reported in previous works. Validation of the model predictive capability--against a selection of measured data that were not used to construct the model--suggests that the model successfully describes the interplay of several simultaneous processes carried out during in vitro cartilage tissue regeneration and indicates that this approach could also be attractive for application in other tissue engineering systems.

Structure and Interactions of Aggrecans: Statistical Thermodynamic Approach

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration on the behavior is explored in detail. The repulsive interactions between two cylindrical-shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity, the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH, the number of charges increases, causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolyte layers in general and cartilage in particular.

Hyaluronan Binding to Link Module of TSG-6 and to G1 Domain of Aggrecan Is Differently Regulated by pH

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.

The influence of sub-micron inhibitory clusters on growth cone substratum attachments and CD44 expression

Proteoglycan expression patterns in the central nervous system guide neuronal pathfinding during development, but also disrupt regeneration after injuries. To deepen our understanding of the molecular level effects of proteoglycan spatial arrangements on neuronal pathfinding, we designed micropatterning stamps for the precise placement of near single molecule chondroitin sulfate proteoglycan (CSPG) clusters into regularly spaced arrays. Actin ultrastructural analysis in dorsal root ganglion neurons grown on laminin-coated substrata patterned with aggrecan cluster arrays revealed filopodial and lamellapodial edge contact avoidance of individual clusters, while growth cone lamellapodia and central domains were able to span multiple clusters over a range of cluster densities. Total internal reflection fluorescence microscopy interrogation of growth cone substratum morphology further revealed persistence of integrin mediated substratum adhesion and local out-of-plane membrane bending over clusters on the height scale of aggrecan glycosaminoglycan side chains. Direct imaging of cell adhesion molecule CD44 expression in growth cones revealed an aggrecan dose dependent upregulation in CD44 molecules. Evidence of CD44 clustering coinciding with underlying aggrecan molecules implies CSPG–CD44 interactions. The results reveal the limited local repulsive effect of CSPGs on neuronal structures and provide evidence that CD44 upregulation in neurons is affected by local CSPG expression.

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile

BackgroundRheumatoid arthritis (RA) is a chronic auto-immune disease with extensive articular cartilage destruction. Aggrecan depletion, mediated by aggrecanases is one of the first signs of early cartilage erosion. We investigated, whether measurement of aggrecan and fragments thereof in serum, could be used as biomarkers for joint-disease in RA patients and furthermore characterized the fragments found in the circulation.MethodsThe study consisted of 38 patients, 12 males (62.2 ± 16.0 years) and 26 females (59.8 ± 20.7 years) diagnosed with RA: 41.5 ± 27.5 mm/h erythrocyte sedimentation rate (ESR), 38.4 ± 34.7 mg/ml C-reactive protein (CRP) and 4.8 ± 1.7 disease activity score (DAS) and 108 healthy age-matched controls. Aggrecan levels were measured using two immunoassays, i.e. the 374ARGSVI-G2 sandwich ELISA measuring aggrecanase-mediated aggrecan degradation and the G1/G2 sandwich assay, detecting aggrecan molecules containing G1 and/or G2 (total aggrecan) We further characterized serum samples by western blots, by using monoclonal antibodies F-78, binding to G1 and G2, or by BC-3, detecting the aggrecanase-generated N-terminal 374ARGSVI neo-epitope.ResultsTotal aggrecan levels in RA patients were significantly decreased from 824.8 ± 31 ng/ml in healthy controls to 570.5 ± 30 ng/ml (31% decrease, P < 0.0001), as measured by the G1/G2 ELISA. Western blot analysis with F-78 showed one strong band at 10 kDa, and weaker bands at 25 and 45 kDa in both healthy controls and RA patients. In contrast, staining for aggrecanase-activity revealed only one strong band in RA patients of 45 kDa.ConclusionThis is the first study, which characterizes different aggrecan fragments in human serum. The data strongly suggests that total aggrecan levels, i.e. aggrecan molecules containing G1 and/or G2 are lower in RA patients, and that RA patients have at least one specific subpopulation of aggrecan fragments, namely aggrecanse generated 374ARGSVI fragments. Further clinical studies are needed to investigate the potential of G1/G2 as a structure-related biochemical marker in destructive joint-diseases.