TNF-stimulated gene 6 promotes formation of hyaluronan-inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the periventricular white matter, which results in arrested maturation of OPCs and myelination failure. HA is a major component of the extracellular matrix of the brain, which regulates inflammation through CD44 and TLR2/4 receptors. Here, we show two mechanism-based strategies that effectively enhanced myelination and neurological recovery in preterm rabbit model of IVH. First, degrading HA by hyaluronidase treatment reduced CD44 and TLR4 expression, proinflammatory cytokines, and microglial infiltration, as well as promoted oligodendrocyte maturation and myelination. Second, intraventricular injection of HA oligosaccharide reduced inflammation and enhanced myelination, conceivably by depleting HC-HA levels.

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.

Pathological Hyaluronan Matrices in Cystic Fibrosis Airways and Secretions.

Hyaluronan (HA) has been used in treatment of cystic fibrosis (CF) via a nebulizer and has demonstrated success in clinical outcomes. HA is an important glycosaminoglycan that is cross-linked by heavy chains (HCs) from inter-α-inhibitor during inflammation. HC cross-linked HA (HC-HA) becomes significantly more adhesive for leukocytes than non-cross-linked HA, which can enhance inflammation. Our studies tested the hypothesis that HC-HA is present in CF airways and that altered ratios of HC-HA to its degradation into relatively lower molecular weight HA contribute to the pathophysiology of chronic inflammation in CF. We evaluated the distribution, levels, and size of HC-HA within CF, healthy, and diseased control lung, bronchus, and sputum tissues by histological and biochemical approaches. HC-HA was significantly elevated in CF, with deposits around the pulmonary vasculature, airway submucosa, and in the stroma of the submucosal glands. The increased infiltration of leukocyte populations correlated with the distribution of HC-HA matrices in the airways. Elevated lung tissue HC-HA correlated with decreased HA levels in CF mucus and sputum compared with controls, suggesting that aberrant degradation and cross-linking of HA in lung tissue is a unique feature of CF. The accumulation and degradation of proinflammatory HC-HA in CF lung tissue suggests that aberrant HA catabolism and cross-linking may contribute to chronic inflammation in airway tissues and affect mucus viscosity in CF airways.

Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.

Hyaluronan and Its Heavy Chain Modification in Asthma Severity and Experimental Asthma Exacerbation.

Hyaluronan (HA) is a large (>1500 kDa) polysaccharide of the extracellular matrix that has been linked to severity and inflammation in asthma. During inflammation, HA becomes covalently modified with heavy chains (HC-HA) from inter-α-inhibitor (IαI), which functions to increase its avidity for leukocytes. Our murine model of allergic pulmonary inflammation suggested that HC-HA may contribute to inflammation, adversely effecting lower airway remodeling and asthma severity. Our objective was to characterize the levels of HA and HC-HA in asthmatic subjects and to correlate these levels with asthma severity. We determined the levels and distribution of HA and HC-HA (i) from asthmatic and control lung tissue, (ii) in bronchoalveolar lavage fluid obtained from non-severe and severe asthmatics and controls, and (iii) in serum and urine from atopic asthmatics after an experimental asthma exacerbation. HC-HA distribution was observed (i) in the thickened basement membrane of asthmatic lower airways, (ii) around smooth muscle cells of the asthmatic submucosa, and (iii) around reserve cells of the asthmatic epithelium. Patients with severe asthma had increased HA levels in bronchoalveolar lavage fluid that correlated with pulmonary function and nitric oxide levels, whereas HC-HA was only observed in a patient with non-severe asthma. After an experimental asthma exacerbation, serum HA was increased within 4 h after challenge and remained elevated through 5 days after challenge. Urine HA and HC-HA were not significantly different. These data implicate HA and HC-HA in the pathogenesis of asthma severity that may occur in part due to repetitive asthma exacerbations over the course of the disease.

Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.

A balanced turnover of dermal fibroblasts is crucial for structural integrity and normal function of the skin. During recovery from environmental injury (such as UV exposure and physical wounding), apoptosis is an important mechanism regulating fibroblast turnover. We are interested in the role that hyaluronan (HA), an extracellular matrix molecule synthesized by HA synthase enzymes (Has), plays in regulating apoptosis in fibroblasts. We previously reported that Has1 and Has3 double knock-out (Has1/3 null) mice show accelerated wound closure and increased numbers of fibroblasts in the dermis. In the present study, we report that HA levels and Has2 mRNA expression are higher in cultured Has1/3 null primary skin fibroblasts than in wild type (WT) cells. Apoptosis induced by two different environmental stressors, UV exposure and serum starvation (SS), was reduced in the Has1/3 null cells. Hyaluronidase, added to cultures to remove extracellular HA, surprisingly had no effect upon apoptotic susceptibility to UVB or SS. However, cells treated with 4-methylumbelliferone to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. When fibroblasts were transfected with Has2-specific siRNA that lowered Has2 mRNA and HA levels by 90%, both Has1/3 null and WT cells became significantly more sensitive to apoptosis. The exogenous addition of high molecular weight HA failed to reverse this effect. We conclude that Has1/3 null skin fibroblasts (which have higher levels of Has2 gene expression) are resistant to stress-induced apoptosis.

Irreversible heavy chain transfer to chondroitin.

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.

Modification of hyaluronan by heavy chains of inter-α-inhibitor in idiopathic pulmonary arterial hypertension.

We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA "cable" structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.

TSG-6 protein is crucial for the development of pulmonary hyaluronan deposition, eosinophilia, and airway hyperresponsiveness in a murine model of asthma.

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6(-/-) mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6(-/-) mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6(-/-) mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.

Tumor necrosis factor-stimulated gene-6 (TSG-6) amplifies hyaluronan synthesis by airway smooth muscle cells.

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Irreversible heavy chain transfer to hyaluronan oligosaccharides by tumor necrosis factor-stimulated gene-6.

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

Correlation of hyaluronan deposition with infiltration of eosinophils and lymphocytes in a cockroach-induced murine model of asthma.

Asthma is a chronic inflammatory disease that exhibits airway remodeling with changes in the extracellular matrix (ECM). The role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in many biological processes in diseases. This study investigates the processes involved in HA synthesis, deposition and localization during the propagation of cockroach-induced asthma. Mice were sensitized and challenged with cockroach antigen, and sacrificed at various time points during an 8-week challenge protocol. Analysis of bronchoalveolar lavage (BAL) fluid revealed an increase in total nucleated cells as early as 6h, which peaked at 6 days. Histopathologic analysis of the lung tissue revealed an influx of inflammatory cells at the peribronchial and perivascular regions starting at 12 h, which peaked at 6 days and persisted to 8 weeks. Eosinophils predominated in the early time points while lymphocytes predominated during the late time points. Quantitative polymerase chain reaction (PCR) data showed that hyaluronan synthase 1 (HAS1) mRNA peaked within 6 h and then declined. HAS2 mRNA also peaked within 6 h but remained elevated throughout the 8-week exposure course. HA levels in lung tissue and BAL increased at 12 h and peaked by 6 and 8 days, respectively. Inflammatory cells and new collagen formation localized in areas of HA deposition. Taken together, these data support a role for HA in the pathogenesis in asthma.

TSG6 hyaluronan matrix remodeling dampens the inflammatory response during colitis.

In response to tissue injury, changes in the extracellular matrix (ECM) can directly affect the inflammatory response and contribute to disease progression or resolution. During inflammation, the glycosaminoglycan hyaluronan (HA) becomes modified by tumor necrosis factor stimulated gene-6 (TSG6). TSG6 covalently transfers heavy chain (HC) proteins from inter-α-trypsin inhibitor (IαI) to HA in a transesterification reaction and is to date is the only known HC-transferase. By modifying the HA matrix, TSG6 generates HC:HA complexes that are implicated in mediating both protective and pathological responses. Inflammatory bowel disease (IBD) is a lifelong chronic disorder with well-described remodeling of the ECM and increased mononuclear leukocyte influx into the intestinal mucosa. Deposition of HC:HA matrices is an early event in inflamed gut tissue that precedes and promotes leukocyte infiltration. However, the mechanisms by which TSG6 contributes to intestinal inflammation are not well understood. The aim of our study was to understand how the TSG6 and its enzymatic activity contributes to the inflammatory response in colitis. Our findings indicate that inflamed tissues of IBD patients show an elevated level of TSG6 and increased HC deposition and that levels of HA strongly associate with TSG6 levels in patient colon tissue specimens. Additionally, we observed that mice lacking TSG6 are more vulnerable to acute colitis and exhibit an aggravated macrophage-associated mucosal immune response characterized by elevated pro-inflammatory cytokines and chemokines and diminished anti-inflammatory mediators including IL-10. Surprisingly, along with significantly increased levels of inflammation in the absence of TSG6, tissue HA levels in mice were found to be significantly reduced and disorganized, absent of typical "HA-cable" structures. Inhibition of TSG6 HC-transferase activity leads to a loss of cell surface HA and leukocyte adhesion, indicating that the enzymatic functions of TSG6 are a major contributor to stability of the HA ECM during inflammation. Finally, using biochemically generated HC:HA matrices derived by TSG6, we show that HC:HA complexes can attenuate the inflammatory response of activated monocytes. In conclusion, our data suggests that TSG6 exerts a tissue-protective, anti-inflammatory effect via the generation of HC:HA complexes that become dysregulated in IBD.

Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis.

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFß signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

Quantification of hyaluronan (HA) using a simplified fluorophore-assisted carbohydrate electrophoresis (FACE) procedure.

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.

Midgut Laterality Is Driven by Hyaluronan on the Right.

For many years, biologists have focused on the role of Pitx2, expressed on the left side of developing embryos, in governing organ laterality. Here, we identify a different pathway during left-right asymmetry initiated by the right side of the embryo. Surprisingly, this conserved mechanism is orchestrated by the extracellular glycosaminoglycan, hyaluronan (HA) and is independent of Pitx2 on the left. Whereas HA is normally synthesized bilaterally as a simple polysaccharide, we show that covalent modification of HA by the enzyme Tsg6 on the right triggers distinct cell behavior necessary to drive the conserved midgut rotation and to pattern gut vasculature. HA disruption in chicken and Tsg6-/- mice results in failure to initiate midgut rotation and perturbs vascular development predisposing to midgut volvulus. Our study leads us to revise the current symmetry-breaking paradigm in vertebrates and demonstrates how enzymatic modification of HA matrices can execute the blueprint of organ laterality.

Chronic asthma and Mesenchymal stem cells: Hyaluronan and airway remodeling.

BACKGROUND: Previous studies have demonstrated that ovalbumin sensitization promotes chronic asthma phenotype in murine asthma model. Human mesenchymal stem cells (hMSCs) are multipotent cells in vitro that have been shown to decrease inflammation and can reverse airway remodeling when infused into an in vivo chronic asthma model. However, the mechanism by which hMSCs reverse remodeling is still unclear. In this study, we hypothesized that hMSCs influence remodeling by decreasing extracellular matrix (ECM) deposition, more specifically by decreasing collagen I, collagen III, and hyaluronan synthesis. METHODS: Chronic asthma phenotype was produced in an in vitro model with 3 T3 murine airway fibroblast cells by stimulating with GM-CSF. Collagen I and collagen III gene expression was investigated using RT-PCR and Taqman techniques. Hyaluronan was evaluated using FACE and Western Blots. The chronic asthma phenotype was produced in vivo in murine model using sensitization with ovalbumin with and without hMSC infusion therapy. ECM deposition (specifically trichrome staining, soluble and insoluble collagen deposition, and hyaluronan production) was evaluated. Image quantification was used to monitor trichrome staining changes. RESULTS: GM-CSF which induced collagen I and collagen III production was down-regulated with hMSC using co-culture. In the in vivo model, Ovalbumin induced enhanced ECM deposition, soluble and insoluble collagen production, and lung elastance. hMSC infusions decreased ECM deposition as evidenced by decreases in soluble and insoluble collagen production. CONCLUSION: hMSCs participate in improved outcomes of remodeling by reversing excess collagen deposition and changing hyaluronan levels.

Dysregulation of hyaluronan homeostasis during aortic valve disease.

Aortic valve disease (AVD) is one of the leading causes of cardiovascular mortality. Abnormal expression of hyaluronan (HA) and its synthesizing/degrading enzymes have been observed during latent AVD however, the mechanism of impaired HA homeostasis prior to and after the onset of AVD remains unexplored. Transforming growth factor beta (TGFß) pathway defects and biomechanical dysfunction are hallmarks of AVD, however their association with altered HA regulation is understudied. Expression of HA homeostatic markers was evaluated in diseased human aortic valves and TGFß1-cultured porcine aortic valve tissues using histology, immunohistochemistry and Western blotting. Further, porcine valve interstitial cell cultures were stretched (using Flexcell) and simultaneously treated with exogenous TGFß1±inhibitors for activated Smad2/3 (SB431542) and ERK1/2 (U0126) pathways, and differential HA regulation was assessed using qRT-PCR. Pathological heavy chain HA together with abnormal regional expression of the enzymes HAS2, HYAL1, KIAA1199, TSG6 and IαI was demonstrated in calcified valve tissues identifying the collapse of HA homeostatic machinery during human AVD. Heightened TSG6 activity likely preceded the end-stage of disease, with the existence of a transitional, pre-calcific phase characterized by HA dysregulation. TGFß1 elicited a fibrotic remodeling response in porcine aortic valves similar to human disease pathology, with increased collagen and HYAL to HAS ratio, and site-specific abnormalities in the expression of CD44 and RHAMM receptors. Further in these porcine valves, expression of HAS2 and HYAL1 was found to be differentially regulated by the Smad2/3 and ERK1/2 pathways, and CD44 expression was highly responsive to biomechanical strain. Leveraging the regulatory pathways that control both HA maintenance in normal valves and early postnatal dysregulation of HA homeostasis during disease may identify new mechanistic insight into AVD pathogenesis.

The Rise and Fall of Hyaluronan in Respiratory Diseases.

In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases.