Low Molecular Weight Hyaluronan Research Articles

Sexual dimorphism in the nociceptive effects of hyaluronan.

Intradermal administration of low-molecular-weight hyaluronan (LMWH) in the hind paw induced dose-dependent (0.1, 1, or 10 µg) mechanical hyperalgesia of similar magnitude in male and female rats. However, the duration of LMWH hyperalgesia was greater in females. This sexual dimorphism was eliminated by bilateral ovariectomy and by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to the G-protein-coupled estrogen receptor (GPR30) mRNA in females, indicating estrogen dependence. To assess the receptors at which LMWH acts to induce hyperalgesia, LMWH was administered to groups of male and female rats that had been pretreated with ODN antisense (or mismatch) to the mRNA for 1 of 3 hyaluronan receptors, cluster of differentiation 44 (CD44), toll-like receptor 4, or receptor for hyaluronan-mediated motility (RHAMM). Although LMWH-induced hyperalgesia was attenuated in both male and female rats pretreated with ODN antisense for CD44 and toll-like receptor 4 mRNA, RHAMM antisense pretreatment only attenuated LMWH-induced hyperalgesia in males. Oligodeoxynucleotide antisense for RHAMM, however, attenuated LMWH-induced hyperalgesia in female rats treated with ODN antisense to GPR30, as well as in ovariectomized females. Low-molecular-weight hyaluronan-induced hyperalgesia was significantly attenuated by pretreatment with high-molecular-weight hyaluronan (HMWH) in male, but not in female rats. After gonadectomy or treatment with ODN antisense to GPR30 expression in females, HMWH produced similar attenuation of LMWH-induced hyperalgesia to that seen in males. These experiments identify nociceptors at which LMWH acts to produce mechanical hyperalgesia, establishes estrogen dependence in the role of RHAMM in female rats, and establishes estrogen dependence in the inhibition of LMWH-induced hyperalgesia by HMWH.

Effects of Hyaluronan Molecular Weight on the Lubrication of Cartilage-Emulating Boundary Layers.

Osteoarthritic joints contain lower-molecular-weight (MW) hyaluronan (hyaluronic acid, HA) than healthy joints. To understand the relevance of this HA size effect for joint lubrication, the friction and surface structure of cartilage-emulating surfaces with HA of different MWs were studied using a surface force balance (SFB) and atomic force microscopy (AFM). Gelatin (gel)-covered mica surfaces were coated with high-MW HA (HHA), medium-MW HA (MHA), or low-MW HA (LHA), and lipids of hydrogenated soy l-α-phosphatidylcholine (HSPC) in the form of small unilamellar vesicles, using a layer-by-layer assembly method. SFB results indicate that the gel-HHA-HSPC boundary layer provides very efficient lubrication, attributed to hydration lubrication at the phosphocholine headgroups exposed by the HA-attached lipids, with friction coefficients (COF) as low as 10–3–10–4 at contact stresses at least up to P = 120 atm. However, for the gel-MHA-HSPC and gel-LHA-HSPC surfaces, the friction, initially low, increases sharply at much lower pressures (up to 30–60 atm at most). This higher friction with the shorter chains may be due to their weaker total adhesion energy to the gelatin, where the attraction between the negatively charged HA and the weakly positively charged gelatin is attributed largely to counterion-release entropy. Thus, the complexes of LHA and MHA with the lubricating HSPC lipids are more easily removed by shear during sliding, especially at high stresses, than the HHA-HSPC complex, which is strongly adhered to gelatin. This is ultimately the reason for lower-pressure lubrication breakdown with the shorter polysaccharides. Our results provide molecular-level insight into why the decrease in HA molecular weight in osteoarthritic joints may be associated with higher friction at the articular cartilage surface, and may have relevance for treatments of osteoarthritis involving intra-articular HA injections.

Hyaluronan goes to great length.

Hyaluronan is a major non-protein component of extracellular matrix that affects biomechanical properties of tissues and interacts with cell receptors. Hyaluronan is a linear glycosaminoglycan composed of repeating disaccharides of (β, 1–4)-glucuronic acid (GlcUA) and (β, 1-3)-N-acetyl glucosamine (GlcNAc). The length of hyaluronan can range from an oligomer to an extremely long form up to millions of daltons. The concept that emerged in the field is that high (HMW-HA) and low (LMW-HA) molecular weight hyaluronans have different biological properties and trigger different signaling cascades within the cells. LMW-HA is associated with inflammation, tissue injury and metastasis, while HMW-HA improves tissue homeostasis and has anti-inflammatory and antimetastatic properties. HMW-HA is used in the clinic to treat arthritis, and as a filler in surgery and in the form of rinses to treat local inflammation. However, HMW-HA products used in the clinic come in a range of sizes between 0.5-6 mDa that are used interchangeably. Remarkably, the tissues of a long-lived and cancer-resistant rodent, the naked mole rat, contain abundant HA of very high molecular weight. While human fibroblasts secrete HA up to 2 MDa, naked mole rat fibroblasts produce HA of 6-12 MDa. Does this very high HMW-HA (vHMW-HA) differ functionally from HMW-HA? We found that vHMW-HA has superior cytoprotective properties compared to HMW-HA, and interacts differently with the CD44 receptor leading to distinct transcriptional changes (Takasugi et al. (2020), Nat Commun). These results indicate that vHMW-HA has greater therapeutic benefits than the standard HMW-HA.

An in vitro study to assess the effect of hyaluronan-based gels on muscle-derived cells: Highlighting a new perspective in regenerative medicine.

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.

Hyaluronic acid coating of gold nanoparticles for intraocular drug delivery: Evaluation of the surface properties and effect on their distribution

Due to the unique anatomical structure of the eye, ocular drug delivery is a promising delivery route for the treatment of several ocular diseases, such as the ocular neovascularization that contributes to diabetic retinopathy. This disease is triggered by inflammation, retinal ischemia, and/or deposits of advanced-glycation end-products (AGEs), as well as increased levels of vascular endothelial growth factor (VEGF), interleukins, or reactive oxygen species (ROS). Gold has unique antioxidant and antiangiogenic properties and can inhibit angiogenic molecules. Furthermore, gold nanoparticles (GNPs) are not only biocompatible, they are easy to synthesize, they absorb and scatter visible light, and they can be made with precise control over size and shape. GNPs are an excellent candidate for ocular drug delivery because they can be conjugated to an extraordinarily diverse array of different biomolecules, and surface functionalization can improve the mobility of GNPs across the physiological barriers of the eye, such as the vitreous humour or the inner limiting membrane. For this purpose, we employed low molecular weight hyaluronan (HA) to increase the mobility of the nanoparticles as well as target them to HA receptors that are expressed in different cells of the eye. In this study, the combination of gold and HA enhanced the stability of the whole carrier and promoted their distribution across ocular tissues and barriers to reach the retina. Moreover, analysis in vitro, ex vivo, and in ovo revealed the protective and antiangiogenic effect of GNPs as inhibitors of AGEs-mediated- retinal pigment epithelial cell death and neovascularization. We demonstrated that conjugation with HA enhances GNP stability and distribution due to a specific CD44 receptor interaction. The capacity of HA-GNPs to distribute through the vitreous humour and their avidity for the deeper retinal layers ex vivo, suggest that HA-GNPs are a promising delivery system for the treatment of ocular neovascularization and related disorders.

Hyaluronan promotes the regeneration of vascular smooth muscle with potent contractile function in rapidly biodegradable vascular grafts

The regeneration of smooth muscle with physiological functions has been a key challenge in vascular tissue engineering. Hyaluronan (HA), as a major component of the extracellular matrix, plays a vital role in regulating tissue injury and repair. In this study, a biomimetic vascular graft was prepared by co-electrospinning of synthetic degradable polymers and native ECM components including collagen type-I as well as low and high molecular weight HA (LMW HA and HMW HA). Upon implantation in the rat abdominal aorta, the grafts exhibited sustained HA release that effectively enhanced the regeneration of vascular smooth muscle. Besides, LMW HA loaded vascular grafts demonstrated rapid endothelialization compared to the other groups. More importantly, HA-loaded poly(L-lactide-co-caprolactone) grafts demonstrated an optimal vascular media layer accompanied by well-organized elastin fibers after long-term implantation (6 months), and they maintained potent physiological function up to 1/3 that of the native artery. In contrast, inadequate smooth muscle regeneration was observed in poly(ε-caprolactone) grafts due to slow degradation restricting the regeneration. The mechanism was further investigated and explained by the HA-induced migration of smooth muscle cell (SMC) via CD44-mediated signaling. Besides, low molecular weight HA can promote the migration of vascular progenitor cells that further differentiate into SMCs. These results highlight the importance of HA in the regeneration of functional vascular smooth muscle, and provide a new insight into the fabrication of tissue engineering vascular grafts (TEVGs) via combining rapidly degradable polymers and bioactive ECM components that hold great translational potential.

Effect of hyaluronate and splinting alone <em>versus</em> combined treatment (splinting and hyaluronate) on thumb carpometacarpal osteoarthritis

Thumb carpometacarpal osteoarthritis can lead to global hand dysfunctions and its symptoms are pain and inability. The purpose of this study is to determine the effectiveness of hyaluronate in relieving these symptoms, and to compare it to orthosis and combined treatment (orthosis and hyaluronate). We enrolled 39 patients, evaluated at the baseline by using numeric rating scale (NRS) for pain, Disability of the Arm, Shoulder and Hand (DASH) and Dreiser Scale for disability degree, and Digital Hydraulic Pinch Gauge for grip strength. Eligible participants were randomly assigned to one of the three treatments: injection of hyaluronate (group I), combined treatment (hyaluronate and orthosis, Group II) and orthosis (hard-resting splint, Group III). Patients of Group I and Group II were injected by low molecular weight Hyaluronate once a week for three consecutive weeks. Injections were performed by means of the so-called blind technique. The data analysis indicated a significant decrease (P<0.01) of pain at week 4, further manifested at week 26 by all groups treated. The same occurred for functional symptoms, and grip strength. This improvement appears more evident in group I that received HA.

Proteoglycan-4 and hyaluronan composition in synovial fluid and serum from clinical equine subjects: relationship to cartilage boundary lubrication and viscosity of synovial fluid

ABSTRACT Purpose: In experimental models of equine joint-injury and osteoarthritis synovial fluid (SF) composition (proteoglycan-4, hyaluronan) can vary, along with changes to SF mechanical function (lubrication, viscosity). The study hypotheses were a) clinical equine joint-injury and disease results in altered SF composition and diminished mechanical function, and b) serum composition (proteoglycan-4 or hyaluronan) changes concurrently. The objectives were to characterize composition (proteoglycan-4, hyaluronan), and function of SF and serum from normal horses compared to clinical groups: osteoarthritis, acute-joint-injury, and osteochondrosis. Materials and Methods: Equine samples of SF (from various joints) and blood were collected at the point-of-care. Proteoglycan-4 concentrations were measured by amplified-luminescence-proximity-assay and enzyme-linked-immunosorbent-assay in SF and serum, respectively. Molecular-weight of hyaluronan was characterized by agarose-gel-electrophoresis, and concentrations were measured by enzyme-linked-immunosorbent-assay kit. Biomechanical function of SF was characterized by an in vitro cartilage-on-cartilage friction test, and viscosity test. Results: SF proteoglycan-4 concentration increased in acute-joint-injury (1185 ± 276 versus normal 205 ± 106 µg/mL, µ± SEM, p < 0.01), with increased percentage of lower molecular-weight hyaluronan in acute-joint-injury and osteochondrosis. SF and serum proteoglycan-4 concentrations were correlated in normal horses (r2 = 0.85, p < 0.05), but not in clinical groups. Cartilage-lubricating ability was unchanged, although steady-shear viscosity of acute-joint-injury SF decreased from normal. Conclusion: Composition of SF from cases of equine acute-joint-injury changed; both proteoglycan-4 concentration and hyaluronan molecular-weight were altered, with decreased SF viscosity, but no associated changes to serum. Serum proteoglycan-4 and hyaluronan concentrations alone may not be useful biomarkers for equine joint-injury or disease.

CD44 Can Compensate for IgSF11 Deficiency by Associating with the Scaffold Protein PSD-95 during Osteoclast Differentiation.

Differentiation of osteoclasts, which are specialized multinucleated macrophages capable of bone resorption, is driven primarily by receptor activator of NF-κB ligand (RANKL). Additional signaling from cell surface receptors, such as cell adhesion molecules (CAMs), is also required for osteoclast maturation. Previously, we have demonstrated that immunoglobulin superfamily 11 (IgSF11), a member of the immunoglobulin-CAM (IgCAM) family, plays an important role in osteoclast differentiation through association with the scaffold protein postsynaptic density protein 95 (PSD-95). Here, we demonstrate that the osteoclast-expressed CAM CD44 can compensate for IgSF11 deficiency when cell–cell interaction conditions are suboptimal by associating with PSD-95. Impaired osteoclast differentiation in IgSF11-deficient (IgSF11−/−) cultures was rescued by antibody-mediated stimulation of CD44 or by treatment with low-molecular-weight hyaluronan (LMW-HA), a CD44 ligand. Biochemical analysis revealed that PSD-95, which is required for osteoclast differentiation, associates with CD44 in osteoclasts regardless of the presence or absence of IgSF11. RNAi-mediated knockdown of PSD-95 abrogated the effects of either CD44 stimulation or LMW-HA treatment on osteoclast differentiation, suggesting that CD44, similar to IgSF11, is functionally associated with PSD-95 during osteoclast differentiation. Taken together, these results reveal that CD44 can compensate for IgSF11 deficiency in osteoclasts through association with PSD-95.

Characterization of Chitosan/Hyaluronan Complex Coacervates Assembled by Varying Polymers Weight Ratio and Chitosan Physical-Chemical Composition

Herein, we synthetized and characterized polysaccharide-based complex coacervates starting from two water-soluble biopolymers, i.e., hydrochloride chitosans and sodium hyaluronan. We used chitosans encompassing a range of molecular weights from 30,000 to 400,000 and showing different fraction of acetylated units (i.e., FA = 0.16, 0.46, and 0.63). This set of chitosans was mixed with a low molecular weight hyaluronan to promote electrostatic interactions. Resulting colloids were analyzed in terms of size, polydispersity and surface charge by Dynamic Light Scattering. The weight ratio between the two polyelectrolytes was studied as additional parameter influencing the liquid-liquid phase separation. Main results include the following: the polymers weight ratio was fundamental in dictating the colloids surface charge, whereas chitosan physical-chemical features influenced the dimension and homogeneity of colloids. This contribution presents additional understanding of the complex coacervation between these two oppositely charged polysaccharides, with the potential translation of present system in food and biomedical sectors.

Low Molecular Weight Hyaluronan Induces an Inflammatory Response in Ovarian Stromal Cells and Impairs Gamete Development In Vitro.

The ovarian stroma, the microenvironment in which female gametes grow and mature, becomes inflamed and fibrotic with age. Hyaluronan is a major component of the ovarian extracellular matrix (ECM), and in other aging tissues, accumulation of low molecular weight (LMW) hyaluronan fragments can drive inflammation. Thus, we hypothesized that LMW hyaluronan fragments contribute to female reproductive aging by stimulating an inflammatory response in the ovarian stroma and impairing gamete quality. To test this hypothesis, isolated mouse ovarian stromal cells or secondary stage ovarian follicles were treated with physiologically relevant (10 or 100 μg/mL) concentrations of 200 kDa LMW hyaluronan. In ovarian stromal cells, acute LMW hyaluronan exposure, at both doses, resulted in the secretion of a predominantly type 2 (Th2) inflammatory cytokine profile as revealed by a cytokine antibody array of conditioned media. Additional qPCR analyses of ovarian stromal cells demonstrated a notable up-regulation of the eotaxin receptor Ccr3 and activation of genes involved in eosinophil recruitment through the IL5-CCR3 signaling pathway. These findings were consistent with an age-dependent increase in ovarian stromal expression of Ccl11, a major CCR3 ligand. When ovarian follicles were cultured in 10 or 100 μg/mL LMW hyaluronan for 12 days, gametes with compromised morphology and impaired meiotic competence were produced. In the 100 μg/mL condition, LMW hyaluronan induced premature meiotic resumption, ultimately leading to in vitro aging of the resulting eggs. Further, follicles cultured in this LMW hyaluronan concentration produced significantly less estradiol, suggesting compromised granulosa cell function. Taken together, these data demonstrate that bioactive LMW hyaluronan fragments may contribute to reproductive aging by driving an inflammatory stromal milieu, potentially through eosinophils, and by directly compromising gamete quality through impaired granulosa cell function.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105U/mL. Finally, LHyal activity of 2.12 × 106U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.

A 3D "In Vitro" Model to Study Hyaluronan Effect in Nasal Epithelial Cell Line Exposed to Double-Stranded RNA Poly(I:C).

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an “in vitro” model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G’ and viscous G’’), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D “in vitro” model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

Corrigendum to "Effect of High, Medium, and Low Molecular Weight Hyaluronan on Inflammation and Oxidative Stress in an In Vitro Model of Human Nasal Epithelial Cells".

[This corrects the article DOI: 10.1155/2016/8727289.].

A simple method for the production of low molecular weight hyaluronan by in situ degradation in fermentation broth

AbstractFermentation of hyaluronan (HA) by Streptococcus zooepidemicus was carried out in a 10-L fermentor. When the medium pH was controlled at 7.0 and the temperature was maintained at 38°C for 12 h followed by 35°C for 8 h, the yield of HA was 4.83 g/L with a molecular weight of 1,890 kDa. After the cells were removed by centrifugation from the fermentation broth, HA was slowly degraded to low molecular weight HA by hyaluronidase at a suitable temperature without a decrease in HA concentration. If the time and temperature for enzymatic degradation were controlled, the desired low molecular weight HA could be obtained by in situ degradation in the fermentation broth. The method does not require the addition of exogenous hyaluronidase, and is a simple way to produce low molecular weight HA.

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-κΒ) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-κB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-κB signaling axis.

High-molecular-weight hyaluronan produced by activated pancreatic stellate cells promotes pancreatic cancer cell migration via paracrine signaling

BackgroundThe polysaccharide hyaluronan (HA) is abundant in pancreatic cancer (PC) tissue and promotes pancreatic cancer cell (PCC) motility in vitro. However, it is controversial as to whether high-molecular-weight HA (HMW-HA) or low-molecular weight HA(LMW-HA) is present in the pancreatic cancer stroma and whether PCC or pancreatic stellate cell (PSC) in PC tissue produces HA. We thereby aim to characterize the molecular weight and source of HA in PC tissue that promotes cancer cell motility. MethodsWe analyzed the expression of hyaluronan synthase 2 (HAS2) and the hydrolyzing enzyme hyaluronidase 1 (HYAL1) in PCC lines and pancreatic stellate cells (PSCs) using real-time PCR. HA production in the supernatant of PCC lines and PSCs and in PC tissues was quantitatively and qualitatively examined. Finally, we knocked down HYAL1 expression in one of the PCC line PANC-1 cells and analyzed the impact on cell migration. ResultsHAS2 was abundantly expressed in activated PSCs (aPSCs) but less so in quiescent PSCs (qPSCs) and PCC lines. The baseline expression of HYAL1 did not differ among the cell types. The concentration of HMW-HA was higher in the supernatant of aPSCs than in that of PCC lines. Treatment with exogenous HMW-HA promoted PANC-1 cell motility. Knockdown of HYAL1 decreased HMW-HA-promoted PANC-1 cell migration, which was accompanied by a decrease in intracellular HA levels. ConclusionaPSCs are an important source of stromal HMW-HA, which promotes PCC migration in an HYAL1-dependent manner in PC.

Safety of Hyaluronan 35 in Healthy Human Subjects: A Pilot Study.

Background. Hyaluronan (HA) is a naturally occurring glycosaminoglycan polymer produced in all vertebrates, and usually present at the high molecular weight (>106 Da). Low molecular weight HA has signaling properties, and fragments ~35 kDa size (HA35) have biological activity in eliciting epithelial β-defensins and tight junction proteins, notably ZO1, important components of innate host defense arsenal of the gut barrier in preclinical models. Safety, tolerability, impact on metabolism, gut permeability, and microbiome composition in healthy human subjects were all evaluated prospectively. Methods. Pharmaceutical grade HA35 (140 mg in water once daily for seven days), was administered orally to 20 healthy subjects (30.7 ± 5.6 years). Demographical, clinical, biochemical laboratory tests, metabolic function and stool microbiome composition were measured on Day 0, 8 and 28. Results. HA35 was tolerated well in all subjects with no serious adverse events in any subjects. No statistical differences in any of the measurements were seen among the study group over the course of the trial. In aggregate there were no changes in demographical, clinical, biochemical laboratory tests, and metabolic function or microbiome composition during the 28-day study. Conclusion. Oral HA35 administration (140 mg/day) is a safe treatment in healthy individuals and does not affect metabolic, inflammatory or microbiome parameters.

Colorectal cancer-associated ~6kDa hyaluronan serves as a novel biomarker for cancer progression and metastasis.

Low molecular weight hyaluronan (LMW-HA) is believed to accumulate in tumors and to exert protumor effects. This study aimed to identify colorectal cancer (CRC)-associated LMW-HA, precisely determine its MW, and elucidate its role in predicting tumor progression. The MW distribution of HA extracted from CRC and paired noncancerous tissues was evaluated. We found that the level of HA with a MW below 30kDa was markedly elevated in CRC tissues, and we defined HA with a MW of ~6kDa as CRC-associated LMW-HA. In line with this finding, ~6kDa HA was significantly accumulated in cancer tissues relative to total HA, and this LMW-HA played a critical role in tumor metastasis. Moreover, serum ~6kDa HA levels in CRC patients were significantly increased and positively correlated with the levels in matched cancer tissues. Elevated serum ~6kDa HA levels could be used to discriminate patients with or without CRC and was associated with early relapse, advanced tumor-node-metastasis stage, lymphovascular invasion, and lymph node (LN) metastasis. Notably, serum ~6kDa HA levels were significantly reduced after tumor resection. Our study suggests that ~6kDa HA may serve as a new biomarker for estimating tumor progression, predicting LN metastasis, and monitoring tumor recurrence.