Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

Hyaluronan in articular cartilage: Analysis of hip osteoarthritis and osteonecrosis of femoral head.

Hyaluronan (HA) plays crucial roles in the maintenance of high-quality cartilage extracellular matrix. Several studies have reported the HA in synovial fluid in patients with osteoarthritis (OA), but few have described the changes of HA in articular cartilage of OA or idiopathic osteonecrosis of the femoral head (ONFH). KIAA1199 was recently reported to have strong hyaluronidase activity. The aim of this study was to clarify the HA metabolism in OA and ONFH, particularly the involvement of KIAA1199. Immunohistochemical analysis of KIAA1199 and HA deposition was performed for human OA (n = 10), ONFH (n = 10), and control cartilage (n = 7). The concentration and molecular weight (MW) of HA were determined by competitive HA ELISA and Chromatography, respectively. Regarding HA metabolism-related molecules, HAS1, HAS2, HAS3, HYAL1, HYAL2, and KIAA1199 gene expression was assessed by reverse transcriptase polymerase chain reaction. Histological analysis showed the overexpression of KIAA1199 in OA cartilage, which was accompanied by decreased hyaluronic acid binding protein (HABP) staining compared with ONFH and control. Little KIAA1199 expression was observed in cartilage at the collapsed area of ONFH, which was accompanied by a slight decrease in HABP staining. The messenger RNA (​​​​​mRNA) expression of HAS2 and KIAA1199 was upregulated in OA cartilage, while the mRNA expression of genes related to HA catabolism in ONFH cartilage showed mostly a downward trend. The MW of HA in OA cartilage increased while that in ONFH cartilage decreased. HA metabolism in ONFH is suggested to be generally indolent, and is activated in OA including high expression of KIAA1199. Interestingly, MW of HA in OA cartilage was not reduced.

Possible Repositioning of an Oral Anti-Osteoporotic Drug, Ipriflavone, for Treatment of Inflammatory Arthritis via Inhibitory Activity of KIAA1199, a Novel Potent Hyaluronidase.

KIAA1199 has a strong hyaluronidase activity in inflammatory arthritis. This study aimed to identify a drug that could reduce KIAA1199 activity and clarify its effects on inflammatory arthritis. Rat chondrosarcoma (RCS) cells were strongly stained with Alcian blue (AB). Its stainability was reduced in RCS cells, which were over-expressed with the KIAA1199 gene (RCS-KIAA). We screened the drugs that restore the AB stainability in RCS-KIAA. The effects of the drug were evaluated by particle exclusion assay, HA ELISA, RT-PCR, and Western blotting. We further evaluated the HA accumulation and the MMP1 and three expressions in fibroblast-like synoviocytes (FLS). In vivo, the effects of the drug on symptoms and serum concentration of HA in a collagen-induced arthritis mouse were evaluated. Ipriflavone was identified to restore AB stainability at 23%. Extracellular matrix formation was significantly increased in a dose-dependent manner (p = 0.006). Ipriflavone increased the HA accumulation and suppressed the MMP1 and MMP3 expression on TNF-α stimulated FLS. In vivo, Ipriflavone significantly improved the symptoms and reduced the serum concentrations of HA. Conclusions: We identified Ipriflavone, which has inhibitory effects on KIAA1199 activity. Ipriflavone may be a therapeutic candidate based on its reduction of KIAA1199 activity in inflammatory arthritis.

A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate.

Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification. This study aims to establish a method for quantifying 3-O-sulfated components of heparan sulfate, focusing on non-AT-binding units. We previously examined the reaction products of human 3OST isoforms and identified five 3-O-sulfated components, including three non-AT-binding disaccharides and two AT-binding tetrasaccharides, as digestion products of heparin lyases. In this study, we prepared these five components as a standard saccharide for HPLC analysis. Together with eight non-3-O-sulfated disaccharides, a standard mixture of 13 units was prepared. Using reverse-phase ion-pair HPLC with a postcolumn fluorescent labeling system, the separation conditions were optimized to quantify the 13 units. Finally, we analyzed the compositional changes of 3-O-sulfated units in heparan sulfate from P19 cells before and after neuronal differentiation. We successfully detected the 3-O-sulfated units specifically expressed in the differentiated neurons. This is the first report that shows the quantification of three non-AT-binding 3-O-sulfated units and establishes a new approach to explore the physiological functions of 3-O-sulfate.

Forced expression of KIAA1199, a novel hyaluronidase, inhibits tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of a novel hyaluronidase, KIAA1199, on ECM formation as well as antitumor effects on chondrosarcoma. To clarify the roles of KIAA1199 in chondrosarcoma, mouse KIAA1199 was stably transfected to Swarm rat chondrosarcoma (RCS) cells (histologically grade 1). We investigated the effects of KIAA1199 on RCS cells in vitro and an autografted model in vivo. HA binding protein (HABP) stainability and ECM formation in KIAA1199-RCS was markedly suppressed compared with that of control cells. No significant changes in messenger RNA expression of Has1, Has2, Has3, Hyal1, or Hyal2 were observed. KIAA1199 expression did not affect proliferation or apoptosis but inhibited migration and invasion of RCS cells. In contrast, the expression of KIAA1199 significantly inhibited the growth of grafted tumors and suppressed the stainability of alcian blue in tumor tissues. Although there was no direct inhibitory effect on proliferation in vitro, induction of KIAA1199 showed the antitumor effects in grafted tumor growth in vivo possibly due to changes in the tumor microenvironment such as inhibition of ECM formation. Forced expression of KIAA1199 exhibits antitumor effects on low-grade chondrosarcoma, which has chemo- and radio-therapy resistant features. Together, KIAA1199 could be a novel promising therapeutic tool for low-grade chondrosarcoma, mediated by the degradation of HA.

Roles of hyaluronan in cardiovascular and nervous system disorders.

Hyaluronan is a widely occurring extracellular matrix molecule, which is not only a supporting structural component, but also an active regulator of cellular functions. The chemophysical and biological properties of hyaluronan are greatly affected by its molecular size and several hyaluronan-binding proteins, making hyaluronan a fascinating molecule with great functional diversity. This review summarizes our current understanding of the roles of hyaluronan in cardiovascular and nervous system disorders, such as atherosclerosis, myocardial infarction, and stroke, with the aim to provide a foundation for future research and clinical trials.

Emerging evidence for the essential role of hyaluronan in cutaneous biology.

Hyaluronan (HA), a linear non-sulfated glycosaminoglycan, participates in a variety of biological processes in the skin, such as cell-matrix interactions and activation of chemokines/cytokines, enzymes, and growth factors. In these activation events, HA acts as a damage-associated molecular pattern (DAMP). This review discusses the progress in functional research on HA, and its associated factors, in several aspects of cutaneous biology; e.g., immunity and wound repair.

Inter-α-inhibitor Ameliorates Endothelial Inflammation in Sepsis.

PURPOSE: Vascular endothelial cells demonstrate severe injury in sepsis, and a reduction in endothelial inflammation would be beneficial. Inter-α-Inhibitor (IαI) is a family of abundant plasma proteins with anti-inflammatory properties and has been investigated in human and animal sepsis with encouraging results. We hypothesized that IαI may protect endothelia from sepsis-related inflammation. METHODS: IαI-deficient or sufficient mice were treated with endotoxin or underwent complement-induced lung injury. VCAM-1 and ICAM-1 expression was measured in blood and lung as marker of endothelial activation. Human endothelia were exposed to activated complement C5a with or without IαI. Blood from human sepsis patients was examined for VCAM-1 and ICAM-1 and levels were correlated with blood levels of IαI. RESULTS: IαI-deficient mice showed increased endothelial activation in endotoxin/sepsis- and complement-induced lung injury models. In vitro, levels of endothelial pro-inflammatory cytokines and cell growth factors induced by activated complement C5a were significantly decreased in the presence of IαI. This effect was associated with decreased ERK and NFκB activation. IαI levels were inversely associated with VCAM-1 and ICAM-1 levels in a human sepsis cohort. CONCLUSIONS: IαI ameliorates endothelial inflammation and may be beneficial as a treatment of sepsis.

Inter-α-inhibitor deficiency in the mouse is associated with alterations in anxiety-like behavior, exploration and social approach.

In recent years, several genome-wide association studies have identified candidate regions for genetic susceptibility in major mood disorders. Most notable are regions in a locus in chromosome 3p21, encompassing the genes NEK4-ITIH1-ITIH3-ITIH4. Three of these genes represent heavy chains of the composite protein inter-α-inhibitor (IαI). In order to further establish associations of these genes with mood disorders, we evaluated behavioral phenotypes in mice deficient in either Ambp/bikunin, which is necessary for functional ITIH1 and ITIH3 complexes, or in Itih4, the gene encoding the heavy chain Itih4. We found that loss of Itih4 had no effect on the behaviors tested, but loss of Ambp/bikunin led to increased anxiety-like behavior in the light/dark and open field tests and reduced exploratory activity in the elevated plus maze, light/dark preference and open field tests. Ambp/bikunin knockout mice also exhibited a sex-dependent exaggeration of acoustic startle responses, alterations in social approach during a three-chamber choice test, and an elevated fear conditioning response. These results provide experimental support for the role of ITIH1/ITIH3 in the development of mood disorders.

A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.

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.

Versican A-subdomain is required for its adequate function in dermal development.

Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor ß (TGFß) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFß storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFß signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFß signaling.

The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

The hyaluronan (HA)-rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride-gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions.

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4-methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0 mM. Cell proliferation was significantly reduced by MU at 24 h. Cell motility and invasion were suppressed in a dose-dependent manner by MU. No significant changes in mRNA expression of Has1-3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell-clustering structure. HABP stainability was markedly decreased in the MU-treated group. These results suggest that MU exhibits antitumor effects on low-grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off-label medication for chondrosarcomas. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1573-1580, 2018.

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Conditional knockdown of hyaluronidase 2 in articular cartilage stimulates osteoarthritic progression in a mice model.

The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal -/- ) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2 -/- mice. Hyal2 -/- mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2 -/- mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2 -/- mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.

Serum-Derived Hyaluronan-Associated Protein Is a Novel Biomarker for Inflammatory Bowel Diseases.

BACKGROUND/AIMS: We evaluated the role of serum-derived hyaluronan-associated protein (SHAP) in inflammatory bowel disease (IBD) pathogenesis and its potential as a novel IBD biomarker. METHODS: We studied the SHAP expression in a mouse model of colitis and in human intestinal samples of IBD and compared serum concentrations with normal controls. RESULTS: SHAP was expressed in the connective tissue derived from inflamed regions of the intestine. In mice, serum levels of SHAP-hyaluronic acid (SHAP-HA) were positively correlated with the histological damage of the colon (r = 0.566, p < 0.001). Serum concentration of SHAP-HA complex was significantly higher in patients with active ulcerative colitis than in those in remission, and this value was positively correlated with the erythrocyte sedimentation rate, serum level of tumor necrosis factor (TNF)-α, and endoscopic damage (r = 0.568, p < 0.001; r = 0.521, p < 0.001, and r = 0.641, p < 0.001). In patients with Crohn's disease, the serum SHAP-HA level correlated only with TNF-α (r = 0.630, p = 0.002). CONCLUSION: SHAP is a novel IBD biomarker that is related to disease activity in certain types of colitis, and it may affect disease pathogenesis. Future studies are needed to evaluate the therapeutic potential of this complex.

Antitumor effects of 4-methylumbelliferone, a hyaluronan synthesis inhibitor, on malignant peripheral nerve sheath tumor.

Hyaluronan (HA) has been shown to play important roles in the growth, invasion and metastasis of malignant tumors. Our previous study showing that high HA expression in malignant peripheral nerve sheath tumors (MPNST) is predictive of poor patient prognosis, prompted us to speculate that inhibition of HA synthesis in MPNST might suppress the tumorigenicity. The aim of our study was to investigate the antitumor effects of 4-methylumbelliferone (MU), an HA synthesis inhibitor, on human MPNST cells and tissues. The effects of MU on HA accumulation and tumorigenicity in MPNST cells were analyzed in the presence or absence of MU in an in vitro as well as in vivo xenograft model using human MPNST cell lines, sNF96.2 (primary recurrent) and sNF02.2 (metastatic). MU significantly inhibited cell proliferation, migration and invasion in both MPNST cell lines. HA binding protein (HABP) staining, particle exclusion assay and quantification of HA revealed that MU significantly decreased HA accumulation in the cytoplasms and pericellular matrices in both MPNST cell lines. The expression levels of HA synthase2 (HAS2) and HA synthase3 (HAS3) mRNA were downregulated after treatment with MU. MU induced apoptosis of sNF96.2 cells, but not sNF02.2 cells. MU administration significantly inhibited the tumor growth of sNF96.2 cells in the mouse xenograft model. To the best of our knowledge, our study demonstrates for the first time the antitumor effects of MU on human MPNST mediated by inhibition of HA synthesis. Our results suggest that MU may be a promising agent with novel antitumor mechanisms for MPNST.