High Hyaluronan Research Articles

Fragmented hyaluronan has no alarmin function assessed in arthritis synovial fibroblast and chondrocyte cultures.

Hyaluronan (HA) is a large polymer and an important component of the extracellular matrix. During homeostasis, high molecular mass HA is the predominant form, but upon inflammation, degradation products of HA accumulate. These HA fragments (HA-fs) have been reported to possess pro-inflammatory activities and thus act as alarmins, notifying immune cells of danger via TLR4 and CD44. HA is found in large quantities in synovial joint fluid. In order to reveal a potential role of HA-fs in arthritis pathogenesis, the in vitro effects of HA of various molecular masses (from 1680 kDa to oligosaccharide HA) on synovial fibroblasts and chondrocytes from rheumatoid arthritis patients, and on peripheral blood mononuclear cells from healthy donors, were investigated. TLR4 and CD44 surface expression was confirmed by immunocytochemistry, and cell activation was determined based on cytokine and chemokine production. While the cell types investigated expressed TLR4 and CD44, no increased release of IL-1ß, IL-6, IL-8, IL-10, IL-12 or TNF-α was detected after HA stimulation. Similarly, HA did not enhance activation after priming cells with low doses of LPS or by forming complexes with LPS. Hence, this study does not support the common view of HA-fs being pro-inflammatory mediators and it is not likely that HA-fs generated during arthritis contribute to disease pathogenesis.

Modulation of hyaluronan polymer size regulates proliferation of perimysial fibroblasts in thyroid eye disease

In active thyroid eye disease (TED), the extraocular muscles feature excessive hyaluronan (HA) accumulation and increased fibroblast proliferation. To investigate the effects of HA on proliferation, we cultured perimysial fibroblasts from extraocular muscles of active TED patients, and adopted IGF-1 and PH20 as modulators for HA concentration and HA polymer size. Based on the results, IGF-1 increased HA concentration, promoted high molecular weight HA (HMW-HA) proportion and stimulated fibroblast proliferation. Hyaluronidase PH20 decreased HA concentration, but caused HMW-HA accumulation and exaggerating proliferation as well. Combined treatment with both reagents resulted in retention of low molecular weight HA (LMW-HA), and suppressed fibroblast proliferation. Pearson correlation demonstrated no significance between HA concentration and proliferation. Mitogenic investigation unveiled the stimulatory effects of HMW-HA via membrane depolarization and inhibitive effects of LMW-HA via membrane hyperpolarization. Our findings offer insights into the essential role of HA molecular weight during TED pathogenesis.

Hyaluronan Isolation from Mouse Mammary Gland.

The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis ( Calabro et al., 2000 ).

Local injection of high-molecular hyaluronan promotes wound healing in old rats by increasing angiogenesis.

Impaired angiogenesis contributes to delayed wound healing in aging. Hyaluronan (HA) has a close relationship with angiogenesis and wound healing. However, HA content decreases with age. In this study, we used high molecular weight HA (HMW-HA) (1650 kDa), and investigated its effects on wound healing in old rats by local injection. We found that HMW-HA significantly increases proliferation, migration and tube formation in endothelial cells, and protects endothelial cells against apoptosis. Local injection of HMW-HA promotes wound healing by increasing angiogenesis in old rats. HMW-HA increases the phosphorylation of Src, ERK and AKT, leading to increased angiogenesis, suggesting that local injection of HMW-HA promotes wound healing in elderly patients.

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia.

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E2, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKCε, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E2 is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.SIGNIFICANCE STATEMENT We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

A Trickster in Disguise: Hyaluronan’s Ambivalent Roles in the Matrix

Hyaluronan (HA) is a simple but diverse glycosaminoglycan. It plays a major role in aging, cellular senescence, cancer, and tissue homeostasis. In which way HA affects the surrounding tissues greatly depends on the molecular weight of HA. Whereas high molecular weight HA is associated with homeostasis and protective effects, HA fragments tend to be linked to the pathologic state. Furthermore, the interaction of HA with its binding partners, the hyaladherins, such as CD44, is essential for sustaining tissue integrity and is likewise related to cancer. The naked mole rat, a rodent species, possesses a special form of very high molecular weight (vHMW) HA, which is associated with the extraordinary cancer resistance and longevity of those animals. This review addresses HA and its diverse facets: from HA synthesis to degradation, from oligomeric HA to vHMW-HA and from its beneficial properties to the involvement in pathologies. We further discuss the functions of HA in the naked mole rat and compare them to human conditions. Though intensively researched, this simple polymer bears some secrets that may hold the key for a better understanding of cellular processes and the development of diseases, such as cancer.

Absence of differences among low, middle, and high molecular weight hyaluronan in activating murine immune cells in vitro

Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW.

Hyaluronan in the experimental injury of the cartilage: biochemical action and protective effects.

Our knowledge of extracellular matrix (ECM) structure and function has increased enormously over the last decade or so. There is evidence demonstrating that ECM provides signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation. ECM presents many domains that become active after proteolytic cleavage. These active ECM fragments are called matrikines which play different roles; in particular, they may act as potent inflammatory mediators during cartilage injury. A major component of the ECM that undergoes dynamic regulation during cartilage damage and inflammation is the non-sulphated glycosaminoglycan (GAG) hyaluronan (HA). In this contest, HA is the most studied because of its different activity due to the different polymerization state. In vivo evidences have shown that low molecular weight HA exerts pro-inflammatory action, while high molecular weight HA possesses anti-inflammatory properties. Therefore, the beneficial HA effects on arthritis are not only limited to its viscosity and lubricant action on the joints, but it is especially due to a specific and effective anti-inflammatory activity. Several in vitro experimental investigations demonstrated that HA treatment may regulate different biochemical pathways involved during the cartilage damage. Emerging reports are suggesting that the ability to recognize receptors both for the HA degraded fragments, whether for the high-polymerized native HA involve interaction with integrins, toll-like receptors (TLRs), and the cluster determinant (CD44). The activation of these receptors induced by small HA fragments, via the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) mediation, directly or other different pathways, produces the transcription of a large number of damaging intermediates that lead to cartilage erosion. This review briefly summarizes a number of findings of the recent studies focused on the protective effects of HA, at the different polymerization states, on experimental arthritis in vitro both in animal and human cultured chondrocytes.

Thermo-and pH-sensitive hydrogel membranes composed of poly(N-isopropylacrylamide)-hyaluronan for biomedical applications: Influence of hyaluronan incorporation on the membrane properties

Interpenetrating hydrogel membranes consisting of pH-sensitive hyaluronan (HA) and thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAM) were synthesized using redox polymerization, followed by N,N-methylenebisacrylamide (BIS) and epichlorohydrin (EPI) were added as chemical crosslinkers. The interaction between membrane compositions has been characterized by FTIR spectroscopy and discussed intensively. The result indicates that HA incorporation in membranes increase the gel fraction, swelling uptake, and the flexibility/elasticity of crosslinked membranes, however it reduced oppositely the mechanical elongation of membranes. PNIPAAm-HA hydrogels responded to both temperature and pH changes and the stimuli-responsiveness was reversible. However, in vitro bioevaluation results revealed that the released ampicillin during the burst release time was sharply influenced and increased with increasing HA contents in membranes; afterwards it became sustainable. Whereas, high HA contents in hydrogels unexpectedly impacted negatively on the cells viability, owing to the viscosity of cell culture media changed. A big resistance was observed against microbial growth of Staphylococcus aureus, Salmonella typhi, and Candida albicans in case of pure PNIPAAm hydrogel membranes without HA or ampicillin. However, HA incorporation or the loaded ampicillin in membranes showed unexpected easily microbial growth. The fast release performance with dual pH-thermo-sensitive hydrogels were suggested as promising materials for quick drug carrier in the biomedical field.

The Effect of in vivo High Molecular Weight Hyaluronan Treatment on Airway Reactivity in a Neonatal Mouse Model of Continuous Positive Airway Pressure (CPAP)

Although continuous positive airway pressure (CPAP) is a commonly used mode of respiratory support in the care of pre‐term infants, its long‐term effects on lung development and airway reactivity are largely unknown. Previously, we developed a neonatal mouse model of CPAP and showed that it caused a long‐term increase in airway reactivity to methacholine challenge. In the current study we tested the hypotheses that hyaluronan (HA), a major component of lung extracellular matrix, plays a role in the airway hyper‐reactivity (AHR) following neonatal CPAP and whether it can be prevented by exogenous administration of high molecular weight HA. Neonatal mice received CPAP (6cm H2O) for 3 hours per day for the first 7 postnatal days. Control animals experienced the same conditions as experimental animals only with no CPAP. On days 3 and 7, a subset of mice in each group received sub‐cutaneous injections of 1% high molecular weight HA (>1000kD, 20μl/g). Two weeks after CPAP ended (postnatal (P) age 21 days), mice were sacrificed and lungs prepared for measurements of airway reactivity to increasing doses of methacholine (0.25μM‐8μM) using the precision cut lung slice method. Airway reactivity to methacholine was increased in P21 mice that received CPAP during the first postnatal week compared to untreated control mice. Gel electrophoresis analysis of whole lung homogenates revealed CPAP also increased total lung HA levels. Immunohistochemical staining of HA with an antibody selective for the hyaluronan binding protein (HABP) showed increased HA expression around the airways within or in the vicinity of airway smooth muscle. However, mice that also received high molecular weight HA during CPAP displayed decreased reactivity to methacholine challenge when compared to airways from animals that received CPAP alone. High molecular weight HA administration also prevented the increased total HA expression caused by CPAP. We conclude that lung HA plays a role in the long‐term increase in AHR following neonatal CPAP, although exogenous administration of high molecular weight HA appears to be protective against such adverse effects of CPAP. These data may have important implications for the way CPAP may contribute to the pathophysiology of wheezing disorders commonly seen in preterm infants.Support or Funding InformationNHLBI PO1HL107147

Mesenchymal Stromal Cell Growth and Glycocalyx Morphology are Regulated by Media Glucose Levels and Hypoxia

Physiological conditions such as hypoxia and/or chemical environments such as glucose levels in the media function as key regulators of cell behavior. Mesenchymal stromal cells (MSCs) are unique cells which have played an important role in many regenerative medicine technologies. They are characterized by a multi‐lineage potential, ability to self‐renew and immunomodulatory properties. The microenvironment of stem cells may play a crucial role in maintaining their stemness and regenerative potential, and the cellular glycocalyx may be an important cell based regulator within this microenvironment. Hyaluronan (HA), a non‐sulfonated linear glycosaminoglycan found in the glycocalyx of MSCs and other cell types has been implicated in regulation of various physiological processes including inflammation, tissue regeneration, cell proliferation and migration.Here we investigated the effect of hypoxic environments (1% O2 or 5% O2) as well as different glucose concentrations in minimal media on MSC identity, viability and potency as well as production of HA levels in the glycocalyx.MSCs were derived from bone marrow and maintained in different oxygen conditions (1% O2, 5% O2 or room air (~21% O2)). Additionally, bone marrow derived MSCs were also grown in alpha minimal essential media (αMEM) containing either low (1 mg/mL) or high (4.5 mg/mL) levels of glucose. Cell viability was measured microscopically using Trypan blue dye exclusion as well by flow cytometry with annexin V and propidium iodide staining. MSCs were characterized for identity through flow cytometry and potency through the induction of IDO by IFN‐gamma. HA levels were detected with biotin‐conjugated HA binding protein (HABP) followed by FITC‐conjugated streptavidin.Significantly higher numbers of MSCs were derived from bone marrow in 5% O2 (21067 ±1579 cells/cm2) when compared to 1% (14000 ± 5327 cells/cm2) or 21% O2 (10721 ± 3782 cells/cm2). Additionally, cells grown in 5% O2 had significantly lower doubling times of 42.5 ± 3.8 h in passage 1 in comparison to 52.8 ± 4.9 h for cells grown in 21% O2. However, no significant differences in cell morphology, cell identity and cell viability were observed between cells grown in 1%, 5% or 21% O2.Cells grown in high glucose media resulted in significantly enhanced cell yields (39333 ± 6839 cells/cm2) in comparison to those cultured in low glucose media (13111 ± 1736 cells/cm2). MSCs also displayed large intricate networks of high molecular weight (HMW) HA in the glycocalyx as shown by HABP binding and fluorescence microscopy. In comparison, MSCs grown in low glucose media displayed disperse networks of HA. Additionally, there was a 3‐fold increase observed in levels of HA when MSCs were cultured in high glucose media versus cells grown in low glucose media. MSCs in high glucose also displayed enhanced levels of CD44, one of the receptors of HA. Finally, Hyaluronidase treated cells grown in high glucose media displayed a lack of network formation of high molecular HA in the glycocalyx and a 2‐fold decrease in cell yield.These results suggest that the physiological and chemical environment of MSCs can play an important role in their growth characteristics. The balance between HMW and LMW HA as observed through the presence or absence of intricate networks of HA as a part of the cellular glycocalyx could be a significant factor regulating MSC growth and potency.

Modulating Three-Dimensional Microenvironment with Hyaluronan of Different Molecular Weights Alters Breast Cancer Cell Invasion Behavior.

Hyaluronan (HA), a polymer with various molecular weights (MW) found in tumor microenvironments, is associated with malignant progression of breast cancer. Reducing the amount of high-MW HA in the microenvironment by hyaluronidase is a promising approach for breast cancer treatment. However, whether the generation of HA fragments negatively affects breast cancer cells remains to be determined. Furthermore, HA forms three-dimensional (3D) networks by cross-linking with other extracellular molecules to function. Therefore, a model mimicking the cross-linked HA network is required to determine the effect of HA fragments on breast cancer cells. To clarify the differential roles of low (HA35) versus high (HA117) MW HA on cancer cell phenotype, a 3D culture system was set up by covalently cross-linking HA with alginate and investigating the behavior of 4T-1 and SKBR3 breast cancer cells alongside a two-dimensional (2D) control. The results show the invasion and migration abilities of 4T-1 and SKBR3 cells are significantly enhanced by the presence of HA35 but inhibited by HA117 in both 2D monolayers and 3D spheroids. The differential effects of HA35 and HA117 on cancer cell epithelial-mesenchymal transition (EMT) phenotype were further confirmed in terms of differential regulation of E-cadherin and vimentin as important EMT markers at both the cellular and mRNA levels. Additional experiments show the CD44-Twist signaling pathway might be involved in the differential effects of HA35 and HA117. These results have important implications with respect to understanding the role of HA in breast cancer development and for the design of therapeutic approaches based on the eradication of HA with hyaluronidase.

Hyaluronan mediates the adhesion of porcine peripheral blood mononuclear cells to poly (I:C)-treated intestinal cells and modulates their cytokine production

Hyaluronan (HA), a major component of the extracellular matrix (ECM), has been increasingly recognized as a regulator of inflammation. Its role is complex since it has pro- and anti-inflammatory actions by modulating the expression of inflammatory genes, the recruitment of inflammatory cells and the production of inflammatory cytokines, but also by attenuating the course of inflammation and providing protection against tissue damage. Certain viruses and other inflammatory stimuli induce organization of HA into cable-like structures, which may be responsible for leukocyte recruitment and, on the other hand, low molecular weight fragments of HA have been shown to activate various inflammatory responses.The aim of the present study was to analyze the effects of a simulated infection with the viral mimetic Poly (I:C) on HA deposition on different porcine intestinal cells (primary colonic muscular smooth muscle cells (SMC), and epithelial IPEC-J2 and IPI-2I cell lines) and on the recruitment of peripheral blood mononuclear cells (PBMC) to intestinal cell layers. We show that Poly (I:C) treatment induces the formation of an HA-based pericellular matrix coat in muscular SMC and in intestinal epithelial cells (IECs) and that, on differentiated IPEC-J2 cells, HA accumulates in the basolateral membrane. Porcine PBMCs bind to Poly (I:C)-treated cells and this binding is dependent on HA, since the increase in adhesion is abolished by hyaluronidase treatment of the cell layers. A second goal was to study the effect of different molecular weight HA forms on the production of pro-inflammatory cytokines and chemokines (TNF-α, IL-1β and IL-8) by porcine PBMCs. Low molecular weight HA fragments (100–150kDa), in contrast to high molecular weight HA (2500kDa), stimulate the release of these pro-inflammatory mediators by porcine PBMCs. Our results suggest that HA is involved in the inflammatory response against pathogenic insults to the porcine gut.

HYAL-2–WWOX–SMAD4 Signaling in Cell Death and Anticancer Response

Hyaluronidase HYAL-2 is a membrane-anchored protein and also localizes, in part, in the lysosome. Recent study from animal models revealed that both HYAL-1 and HYAL-2 are essential for the metabolism of hyaluronan (HA). Hyal-2 deficiency is associated with chronic thrombotic microangiopathy with hemolytic anemia in mice due to over accumulation of high molecular size HA. HYAL-2 is essential for platelet generation. Membrane HYAL-2 degrades HA bound by co-receptor CD44. Also, in a non-canonical signal pathway, HYAL-2 serves as a receptor for transforming growth factor beta (TGF-β) to signal with downstream tumor suppressors WWOX and SMAD4 to control gene transcription. When SMAD4 responsive element is overly driven by the HYAL-2–WWOX–SMAD4 signaling complex, cell death occurs. When rats are subjected to traumatic brain injury, over accumulation of a HYAL-2–WWOX complex occurs in the nucleus to cause neuronal death. HA induces the signaling of HYAL-2–WWOX–SMAD4 and relocation of the signaling complex to the nucleus. If the signaling complex is overexpressed, bubbling cell death occurs in WWOX-expressing cells. In addition, a small synthetic peptide Zfra (zinc finger-like protein that regulates apoptosis) binds membrane HYAL-2 of non-T/non-B spleen HYAL-2+ CD3− CD19− Z lymphocytes and activates the cells to generate memory anticancer response against many types of cancer cells in vivo. Whether the HYAL-2–WWOX–SMAD4 signaling complex is involved is discussed. In this review and opinion article, we have updated the current knowledge of HA, HYAL-2 and WWOX, HYAL-2–WWOX–SMAD4 signaling, bubbling cell death, and Z cell activation for memory anticancer response.

Absorption of Orally Administered Hyaluronan.

Hyaluronan (HA) has been utilized as a supplement. However, the absorption of orally administrated HA remains controversial. The degradation and absorption of HA in the intestine were investigated in this study. HA excretion into the feces, degradation in the intestinal tract, absorption through the large intestine, and translocation to the blood and skin were examined. HA administered orally was not detected in rat feces. HA was degraded by cecal content, but not by artificial gastric juice and intestinal juice. Oligosaccharide HA passed through excised large intestine sacs. Furthermore, disaccharides, tetrasaccharides, and polysaccharides HA were distributed to the skin of rats following oral administration of high molecular weight HA (300 kDa). The results of the study suggest that orally administered HA is degraded to oligosaccharides by intestinal bacteria, and oligosaccharide HA is absorbed in the large intestine and is subsequently distributed throughout the tissues, including the skin.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

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.

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.

Rapid evolution of hyaluronan synthase to improve hyaluronan production and molecular mass in Bacillus subtilis.

To improve the production and molecular mass of the glycosaminoglycan hyaluronan (HA) in Bacillus subtilis by engineering hyaluronan synthase (HAS) from Streptococcus zooepidemicus. By mutating regions within HAS intracellular domains, five positive variants exhibiting higher HA production (from 1.22 to 2.24gl-1) and molecular mass values (from 1.20 to 1.36×106Da) were constructed and characterized. Overexpression of the V5 variant and the genes tuaD and glmU increased HA production and molecular mass to 2.8gl-1 and 2.4×106Da, respectively. This study provides a novel strategy for improving HA production and its molecular mass.

Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks

Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.