Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

Dysfunctional Extracellular Matrix Remodeling Supports Perianal Fistulizing Crohn's Disease by a Mechanoregulated Activation of the Epithelial-to-Mesenchymal Transition.

BACKGROUND AND AIMS: Perianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation. METHODS: Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed. RESULTS: A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli. CONCLUSIONS: By mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.

The Link module of human TSG-6 (Link_TSG6) promotes wound healing, suppresses inflammation and improves glandular function in mouse models of Dry Eye Disease.

PURPOSE: To investigate the potential of the Link_TSG6 polypeptide comprising the Link module of human TSG-6 (TNF-stimulated gene/protein-6) as a novel treatment for dry eye disease (DED). METHODS: We analyzed the therapeutic effects of topical application of Link_TSG6 in two murine models of DED, the NOD.B10.H2b mouse model and the desiccating stress model. The effects of Link_TSG6 on the ocular surface and DED were compared with those of full-length TSG-6 (FL_TSG6) and of 0.05% cyclosporine (Restasis®). Additionally, the direct effect of Link_TSG6 on wound healing of the corneal epithelium was evaluated in a mouse model of corneal epithelial debridement. RESULTS: Topical Link_TSG6 administration dose-dependently reduced corneal epithelial defects in DED mice while increasing tear production and conjunctival goblet cell density. At the highest dose, no corneal lesions remained in ∼50% of eyes treated. Also, Link_TSG6 significantly suppressed the levels of inflammatory cytokines at the ocular surface and inhibited the infiltration of T cells in the lacrimal glands and draining lymph nodes. Link_TSG6 was more effective in decreasing corneal epithelial defects than an equimolar concentration of FL_TSG6. Link_TSG6 was significantly more potent than Restasis® at ameliorating clinical signs and reducing inflammation. Link_TSG6 markedly and rapidly facilitated epithelial healing in mice with corneal epithelial debridement wounds. CONCLUSION: Link_TSG6 holds promise as a novel therapeutic agent for DED through its effects on the promotion of corneal epithelial healing and tear secretion, the preservation of conjunctival goblet cells and the suppression of inflammation.

Isolation and Purification of Versican and Analysis of Versican Proteolysis.

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. Versican is crucial for several developmental processes in the embryo ranging from cardiac development to digit separation, and there is an increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and an N-terminal versican fragment named versikine that are provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

Association of plasma trace element levels with neovascular age-related macular degeneration.

Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 µg/L (standard deviation [SD] 0.71) in nAMD and 1.15 µg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 µg/L (SD 0.22) in nAMD and 0.46 µg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 µg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 µg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.

TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis.

Little is known about the impact of viral infections on lung matrix despite its important contribution to mechanical stability and structural support. The composition of matrix also indirectly controls inflammation by influencing cell adhesion, migration, survival, proliferation and differentiation. Hyaluronan is a significant component of the lung extracellular matrix and production and degradation must be carefully balanced. We have discovered an imbalance in hyaluronan production following resolution of a severe lung influenza virus infection, driven by hyaluronan synthase 2 from epithelial cells, endothelial cells and fibroblasts. Furthermore hyaluronan is complexed with inter-α-inhibitor heavy chains due to elevated TNF-stimulated gene 6 expression and sequesters CD44-expressing macrophages. We show that intranasal administration of exogenous hyaluronidase is sufficient to release inter-α-inhibitor heavy chains, reduce lung hyaluronan content and restore lung function. Hyaluronidase is already used to facilitate dispersion of co-injected materials in the clinic. It is therefore feasible that fibrotic changes following severe lung infection and inflammation could be overcome by targeting abnormal matrix production.

TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.

Tumor necrosis factor- (TNF) stimulated gene-6 (TSG-6) is an inflammation-associated secreted protein that has been implicated as having important and diverse tissue protective and anti-inflammatory properties, e.g. mediating many of the immunomodulatory and beneficial activities of mesenchymal stem/stromal cells. TSG-6 is constitutively expressed in some tissues, which are either highly metabolically active or subject to challenges from the environment, perhaps providing protection in these contexts. The diversity of its functions are dependent on the binding of TSG-6 to numerous ligands, including matrix molecules such as glycosaminoglycans, as well as immune regulators and growth factors that themselves interact with these linear polysaccharides. It is becoming apparent that TSG-6 can directly affect matrix structure and modulate the way extracellular signalling molecules interact with matrix. In this review, we focus mainly on the literature for TSG-6 over the last 10 years, summarizing its expression, structure, ligand-binding properties, biological functions and highlighting TSG-6's potential as a therapeutic for a broad range of disease indications.

Oocyte-specific ablation of N- and O-glycans alters cumulus cell signalling and extracellular matrix composition.

Cumulus-oocyte complex (COC) expansion is essential for ovulation and fertilisation and is linked to oocyte quality. Hyaluronan (HA), the major matrix constituent, is cross-linked via inter-α-inhibitor heavy chains (HCs), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene 6 (TSG-6). All except HCs are secreted by cumulus cells in response to oocyte-secreted factors, which signal via SMAD pathways. The double mutant (DM) mouse generates oocytes lacking complex N- and O-glycans due to oocyte-specific deletion of core 1 ß1,3-galactosyltransferase (C1galt1) and N-acetylglucosaminyltransferase I (Mgat1) and has modified cumulus expansion. We compared COCs before expansion (48 h-post-pregnant mare serum gonadotrophin (PMSG)) and at late-stage expansion (9 h-post-human chorionic gonadotrophin (hCG); control n=3 mice, DM n=3 per group). Using histochemistry the levels of HA, HCs, PTX3, TSG-6 and phosphorylated-SMAD1/5/8 and -SMAD2 (12-25 COCs per group) were assessed. DM COCs did not differ from Controls in cumulus size or cell density at 9 h-post-hCG; however, HA and HC levels and phosphorylated-SMAD1/5/8 were reduced. Furthermore, no correlations were found between the levels of matrix molecules and cumulus area in DM or Control samples. These data suggest that HA and HCs can support cumulus expansion provided that they are present above minimum threshold levels. We propose that oocyte-specific ablation of C1galt1 and Mgat1 may affect bone morphogenetic protein 15 synthesis or bioactivity, thereby reducing SMAD1/5/8 phosphorylation and HA production.

A Novel Choroidal Endothelial Cell Line Has a Decreased Affinity for the Age-Related Macular Degeneration-Associated Complement Factor H Variant 402H.

Purpose: Choroidal endothelial cells play a central role in the pathogenesis of age-related macular degeneration (AMD). Protocols for isolating primary choroidal endothelial cells have been described but require access to human donor eyes, which is a limiting factor. Therefore, a conditionally immortalized choroidal endothelial cell (ciChEnC) line has been established. Methods: Choroidal endothelial cells were selected by magnetic-activated cell sorting and conditionally immortalized using temperature-sensitive simian virus 40 large T antigen and human telomerase. The cell line obtained was characterized based on expression of endothelial marker proteins and endothelial cell-specific responses to various stimuli. Binding of AMD-associated and non-AMD variants of complement factor H in the context of a recombinant CCP6-8 (complement control protein domains 6-8) construct was determined using ELISA. Results: ciChEnCs maintained morphology and von Willebrand factor and vascular endothelial cadherin expression for up to 27 passages. The cells internalized acetylated low-density lipoprotein, formed tubes on Matrigel, and increased intercellular adhesion molecule-1 expression in response to tumor necrosis factor-α. Cells grew into dense monolayers with barrier function and showed characteristics of choriocapillary cells, such as expression of plasmalemma vesicle-associated protein, human leukocyte antigen ABC, carbonic anhydrase IV, and membrane indentations reflecting fenestrations. ciChEnCs synthesized glycosaminoglycans chondroitin sulfate and the complement factor H ligand heparan sulfate. Interestingly, binding of the AMD-associated 402H variant of factor H to ciChEnC was significantly decreased compared to the 402Y variant. Conclusions: A novel ciChEnC cell line with choriocapillary characteristics has been established and should greatly facilitate investigation of the pathogenesis of AMD in the context of the choriocapillary microenvironment.

Complement factor H in host defense and immune evasion.

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Is Constitutively Expressed in Adult Central Nervous System (CNS) and Associated with Astrocyte-mediated Glial Scar Formation following Spinal Cord Injury.

Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) binds to hyaluronan and can reorganize/stabilize its structure, also enhancing the binding of this glycosaminoglycan to its cell surface receptor, CD44. TSG-6 is rapidly up-regulated in response to inflammatory cytokines protecting tissues from the damaging effects of inflammation. Despite TSG-6 treatment having been shown to improve outcomes in an experimental model of traumatic brain injury, TSG-6 expression has not been extensively studied in the central nervous system (CNS). We hereby analyzed the expression profile of TSG-6 in the developing CNS and following injury. We show that TSG-6 is expressed in the rat CNS by GFAP(+) and CD44(+) astrocytes, solely in the mature brain and spinal cord, and is not present during the development of the CNS. TSG-6(-/-) mice present a reduced number of GFAP(+) astrocytes when compared with the littermate TSG-6(+/-) mice. TSG-6 expression is drastically up-regulated after injury, and the TSG-6 protein is present within the glial scar, potentially coordinating and stabilizing the formation of this hyaluronan-rich matrix. This study shows that TSG-6 is expressed in the CNS, suggesting a role for TSG-6 in astrocyte activation and tissue repair. We hypothesize that within this context TSG-6 could participate in the formation of the glial scar and confer anti-inflammatory properties. Further studies are required to elucidate the therapeutic potential of targeting TSG-6 after CNS injury to promote its protective effects while reducing the inhibitory properties of the glial scar in axon regeneration.

G1 Domain of Versican Regulates Hyaluronan Organization and the Phenotype of Cultured Human Dermal Fibroblasts.

Variants of versican have wide-ranging effects on cell and tissue phenotype, impacting proliferation, adhesion, pericellular matrix composition, and elastogenesis. The G1 domain of versican, which contains two Link modules that bind to hyaluronan (HA), may be central to these effects. Recombinant human G1 (rhG1) with an N-terminal 8 amino acid histidine (His) tag, produced in Nicotiana benthamiana, was applied to cultures of dermal fibroblasts, and effects on proliferation and pericellular HA organization determined. rhG1 located to individual strands of cell surface HA which aggregated into structures resembling HA cables. On both individual and aggregated strands, the spacing of attached rhG1 was similar (~120 nm), suggesting interaction between rhG1 molecules. Endogenous V0/V1, present on HA between attached rhG1, did not prevent cable formation, while treatment with V0/V1 alone, which also bound to HA, did not induce cables. A single treatment with rhG1 suppressed cell proliferation for an extended period. Treating cells for 4 weeks with rhG1 resulted in condensed layers of elongated, differentiated α actin-positive fibroblasts, with rhG1 localized to cell surfaces, and a compact extracellular matrix including both collagen and elastin. These results demonstrate that the G1 domain of versican can regulate the organization of pericellular HA and affect phenotype.

Age and Smoking Related Changes in Metal Ion Levels in Human Lens: Implications for Cataract Formation.

Age-related cataract formation is the primary cause of blindness worldwide and although treatable by surgical removal of the lens the majority of sufferers have neither the finances nor access to the medical facilities required. Therefore, a better understanding of the pathogenesis of cataract may identify new therapeutic targets to prevent or slow its progression. Cataract incidence is strongly correlated with age and cigarette smoking, factors that are often associated with accumulation of metal ions in other tissues. Therefore this study evaluated the age-related changes in 14 metal ions in 32 post mortem human lenses without known cataract from donors of 11 to 82 years of age by inductively coupled plasma mass spectrometry; smoking-related changes in 10 smokers verses 14 non-smokers were also analysed. A significant age-related increase in selenium and decrease in copper ions was observed for the first time in the lens tissue, where cadmium ion levels were also increased as has been seen previously. Aluminium and vanadium ions were found to be increased in smokers compared to non-smokers (an analysis that has only been carried out before in lenses with cataract). These changes in metal ions, i.e. that occur as a consequence of normal ageing and of smoking, could contribute to cataract formation via induction of oxidative stress pathways, modulation of extracellular matrix structure/function and cellular toxicity. Thus, this study has identified novel changes in metal ions in human lens that could potentially drive the pathology of cataract formation.

Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization.

The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely onin vitrostudies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HAin vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposedin vivofunctions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte traffickingin vivo.

Nuclear Magnetic Resonance Insight into the Multiple Glycosaminoglycan Binding Modes of the Link Module from Human TSG-6.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that is essential for stabilizing and remodeling the extracellular matrix (ECM) during ovulation and inflammatory disease processes such as arthritis. The Link module, one of the domains of TSG-6, is responsible for binding hyaluronan and other glycosaminoglycans found in the ECM. In this study, we used a well-defined chondroitin sulfate (CS) hexasaccharide (ΔC444S) to determine the structure of the Link module, in solution, in its chondroitin sulfate-bound state. A variety of nuclear magnetic resonance techniques were employed, including chemical shift perturbation, residual dipolar couplings (RDCs), nuclear Overhauser effects, spin relaxation measurements, and paramagnetic relaxation enhancements from a spin-labeled analogue of ΔC444S. The binding site for ΔC444S on the Link module overlapped with that of HA. Surprisingly, ΔC444S binding induced dimerization of the Link module (as confirmed by analytical ultracentrifugation), and a second weak binding site that partially overlapped with a previously identified heparin site was detected. A dimer model was generated using chemical shift perturbations and RDCs as restraints in the docking program HADDOCK. We postulate that the molecular cross-linking enhanced by the multiple binding modes of the Link module might be critical for remodeling the ECM during inflammation/ovulation and might contribute to other functions of TSG-6.

Isolation and purification of versican and analysis of versican proteolysis.

Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. It is crucial for several developmental processes in the embryo and there is increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and a versican fragment provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.

Hyaluronan and hyaluronan-binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor-stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.

TSG-6 inhibits neutrophil migration via direct interaction with the chemokine CXCL8.

TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to proinflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanisms underlying this activity remain unknown. In this study, we have shown that TSG-6 inhibits chemokine-stimulated transendothelial migration of neutrophils via a direct interaction (KD, ∼ 25 nM) between TSG-6 and the glycosaminoglycan binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Furthermore, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it, to our knowledge, the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or following transplantation.