HA Fragments Research Articles

Hyaluronan‐protein interactions: Lilliput revisited

AbstractHyaluronan (HA) is a huge linear polysaccharide composed entirely of a simple repeating disaccharide that has been preserved unchanged since the evolution of vertebrates ~520 million years ago. It is present in all mammalian tissues, being synthesised within the cell membrane and extruded into the extracellular space where it dictates tissue elasticity, hydration and permeability. HA also directs cell behaviour via engagement with cell surface receptors. These properties allow it to mediate diverse functions in a wide range of physiological and pathological processes, including development, mammalian reproduction and inflammation. There is growing evidence that the way in which the HA biopolymer is differentially organised, through its interaction with a repertoire of HA‐binding proteins (HABPs), is key to the diversity of its biological functions. This review summarises current knowledge of HA‐protein interactions and how their diversity may lead to the formation of HA/protein complexes with distinct molecular architectures that in turn underpin different physical properties and receptor‐mediated effects. Potentially controversial areas such the pro‐inflammatory effects of low molecular weight HA fragments and how short HA‐binding peptides modulate HA function are considered from a molecular perspective.

Abstract 4272: Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms

Abstract Hyaluronan (HA) is the main constituent of the brain extracellular matrix and is an essential component of the glioblastoma (GBM) tumor microenvironment. The biosynthesis and catabolism of hyaluronan has multiple roles in tissue architecture and cell signaling. Dysregulation of these mechanisms is crucial in pathological processes such as cancer, inflammation or tissue remodeling. The role of HA is believed to be strongly dependent on its size (molecular weight), location, and cell receptor density and activity. HA is produced intracellularly by HA synthases (HAS1-3) and can also be catabolized endogenously by hyaluronidases (HYAL1-3).The fabrication of controlled microenvironments provided by three dimensional (3D) models helps to elucidate the role of HA signaling in GBM tumors. We exploit here the advantages of these models to understand the role of hyaluronan in the local behavior of GBM after radiation therapy and selected targeted inhibitors. We have established engineered brain tumor biomaterials based on functionalized hydrogels to monitor the response of patient-derived xenograft cell populations with different molecular signatures in combination with microfluidic devices. We have analyzed shifts in metabolism, hyaluronan secretion, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient derived GBM cells. We reveal that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype dependent manner. We also show that HA fragments can enhance tumor metabolism and growth through ligand dependent and independent mechanisms. We provide bioengineered platforms and strategies to predict and stratify tumors in order to achieve more efficient combinatorial treatments targeting endogenous hyaluronan production. This work emphasizes the potential of these preclinical models to predict and accelerate cancer treatments, more relevant in malignancies with limited treatment options. Citation Format: Edward R. Neves, Joseph Mueller, Achal Anand, Kimberly Selting, Hui Xu, Roddel Remy, Brendan Harley, Sara Pedron-Haba. Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4272.

The Effect of Mechanical Stress on Hyaluronan Fragments' Inflammatory Cascade: Clinical Implications.

It is a common experience, reported by patients who have undergone manual therapy that uses deep friction, to perceive soreness in treatment areas; however, it is still not clear what causes it and if it is therapeutically useful or a simple side effect. The purpose of this narrative review is to determine whether manual and physical therapies can catalyze an inflammatory process driven by HA fragments. The literature supports the hypothesis that mechanical stress can depolymerize into small pieces at low molecular weight and have a high inflammatory capacity. Many of these pieces are then further degraded into small oligosaccharides. Recently, it has been demonstrated that oligosaccharides are able to stop this inflammatory process. These data support the hypothesis that manual therapy that uses deep friction could metabolize self-aggregated HA chains responsible for increasing loose connective tissue viscosity, catalyzing a local HA fragment cascade that will generate soreness but, at the same time, facilitate the reconstitution of the physiological loose connective tissue properties. This information can help to explain the meaning of the inflammatory process as well as the requirement for it for the long-lasting resolution of these alterations.

Hyaluronic Acid Oligosaccharide Derivatives Alleviate Lipopolysaccharide-Induced Inflammation in ATDC5 Cells by Multiple Mechanisms.

High molecular weight hyaluronic acids (HMW-HAs) have been used for the palliative treatment of osteoarthritis (OA) for decades, but the pharmacological activity of HA fragments has not been fully explored due to the limited availability of structurally defined HA fragments. In this study, we synthesized a series glycosides of oligosaccharides of HA (o-HAs), hereinafter collectively referred to as o-HA derivatives. Their effects on OA progression were examined in a chondrocyte inflammatory model established by the lipopolysaccharide (LPS)-challenged ATDC5 cells. Cell Counting Kit-8 (CCK-8) assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that o-HA derivatives (≤100 μg/mL) exhibited no cytotoxicity and pro-inflammatory effects. We found that the o-HA and o-HA derivatives alleviated LPS-induced inflammation, apoptosis, autophagy and proliferation-inhibition of ATDC5 cells, similar to the activities of HMW-HAs. Moreover, Western blot analysis showed that different HA derivatives selectively reversed the effects of LPS on the expression of extracellular matrix (ECM)-related proteins (MMP13, COL2A1 and Aggrecan) in ATDC5 cells. Our study suggested that o-HA derivatives may alleviate LPS-induced chondrocyte injury by reducing the inflammatory response, maintaining cell proliferation, inhibiting apoptosis and autophagy, and decreasing ECM degradation, supporting a potential oligosaccharides-mediated therapy for OA.

Improving Current Knowledge on Seroprevalence and Genetic Characterization of Swine Influenza Virus in Croatian Pig Farms: A Retrospective Study.

In a total of 1536 blood serum samples analysed by ELISA, antibodies for IAV nucleoprotein (NP) were detected in 30.3%. Results from HI show that the most common subtype of swIAV in the Croatian pig population was H1N1 (44.6%), followed by H3N2 (42.7%) and H1N2 (26.3%). Antibodies to at least one subtype were detected in 62.19% of blood serum samples. Detection of swIAV antigen was performed by IHC and detected in 8 of 28 lung samples collected post-mortem. The matrix (M) gene was detected in nine of one hundred and forty-two lung tissue samples and in seven of twenty-nine nasopharyngeal swabs. Phylogenetic analysis of amplified HA and NA gene fragments in Croatian isolates suggests the presence of swIAV H1avN1av.

A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection.

Influenza pandemics pose public health threats annually for lacking vaccine that provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens that induce cross-protective antibody responses with epitopes that activate cross-protective T cell responses might be an attractive strategy for developing a universal vaccine. In this study, we constructed a recombinant protein named NMHC that consists of influenza viral conserved epitopes and a superantigen fragment. NMHC promoted the maturation of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2, and Th17 subtypes. Mice vaccinated with NMHC produced high levels of immunoglobulins that cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtype) and group 2 (H3 subtype) influenza virus strains. Furthermore, purified anti-NMHC antibodies bound to multiple HAs with high affinities. NMHC vaccination effectively protected mice from infection and lung damage when exposed to two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T cell responses that cleared the virus from infected tissues and prevented virus spread. In conclusion, this study provides proof of concept that NMHC vaccination triggers B and T cell immune responses against multiple influenza virus infections. Therefore, NMHC might be a candidate universal broad-spectrum vaccine for the prevention and treatment of multiple influenza viruses.

Dynamic role of LMW-hyaluronan fragments and Toll-like receptors 2,4 in progression of bleomycin induced lung parenchymal injury to fibrosis

BackgroundPulmonary fibrosis (PF) is a progressive and lethal lung disease of elderly whose incidence has been increasing following the Covid-19 pandemic caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). PF immunopathogenesis involves progressive alveolar epithelial cell damage, release of damage-associated molecular patterns (DAMPs), and extracellular matrix (ECM) injury. We assessed the dynamic role of LMW-hyaluronan (LMW-HA) as DAMP in initiation of host immune TLR-2,4 responses and as determinant in progression of ECM injury to fibrosis. Male Wistar rats were divided into Group I (saline control, n = 24) and Group II (intratracheal bleomycin, 7 U/kg/animal, n = 24). Animals were euthanized on 0, 7, 14, and 28 days. The time course of release of LMW-HA, TLR-2,4 mRNA and protein levels, and NF-κB-p65 levels after bleomycin injury were correlated with the development of parenchymal inflammation, remodelling, and fibrosis.ResultsAcute lung injury caused by bleomycin significantly increases the pro-inflammatory LMW-HA levels and elevates TLR-2,4 levels on day 7. Subsequently, TLR-2 upregulation, TLR-4 downregulation, and NF-κB signalling follow on days 14 and 28. This results in progressive tissue inflammation, alveolar and interstitial macrophage accumulation, and fibrosis.ConclusionsLMW-HA significantly increases in PF caused by non-infectious and infectious (Covid-19) etiologies. The accumulating HA fragments function as endogenous DAMPs and trigger inflammatory responses, through differential TLR2 and TLR4 signalling, thus promoting inflammation and macrophage influx. LMW-HA are reflective of the state of ongoing tissue inflammation and may be considered as a natural biosensor for fibrotic lung diseases and as potential therapeutic targets.

Hyaluronan oligosaccharides modulate inflammatory response, NIS and thyreoglobulin expression in human thyrocytes

Autoimmune thyroid diseases, such as Hashimoto's thyroiditis, are characterized by lymphocytic infiltration and altered function of the thyroid. During inflammation, it has been reported a decreased expression in Tg and NIS, accompanied by an increase in HA production that accumulates in the gland.HA fragments produced in different pathological states can modulate gene expression in a variety of cell types and may prime inflammatory response by interacting with the TLR-2, TLR-4 and CD44 that, in turn, induce NF-kB activation finally responsible of inflammatory mediator transcription, such as IL-1β, TNF-α and IL-6.The aim of this study was to investigate the potential inflammatory effect and the biochemical pathways activated by 6-mer HA oligosaccharides in cultured human thyrocytes. 6-mer HA treatment induced up-regulation of TLR-2, TLR-4, CD44 mRNA and related protein levels, increased HA production and NF-kB activation, that in turn increased IL-1β and IL-6 concentrations. Instead, we found evidence of an opposite effect on thyroid specific-gene Tg and NIS, that were decreased after 6-mer HA addition. Thyrocytes exposition to specific blocking antibodies for TLR-2, TLR-4 and CD44 abolished up-regulation of NF-κB activation and the consequent pro-inflammatory cytokine production, while restored Tg and NIS levels. A further goal of this study was demonstrate that also other LMW HA have pro inflammatory proprieties.These data suggest that HA fragments, through the involvement of TLR-2, TLR-4 and CD44 signaling cascade, contribute to prime the inflammatory response in thyrocytes and, by reducing the expression of thyroid-specific genes, could promote the loss of function of gland such as in Hashimoto's thyroiditis.

MiR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes

BackgroundSmall HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism.Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes. MethodsStarting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes. ResultsTreatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified. ConclusionsWe concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation. General significanceThese results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.

Microscopic visualization of cell – Cold sprayed bio-coating interfaces: An intermediate layer formed during the culturing mediates the behaviors of the cells

Cold spraying has been proved it is a promising method for biomaterials surface modification and additive manufacturing. Fundamental understanding of the cell-biomaterial interaction is essential for design and fabrication of biomaterials for their biomedical applications. Here we report the evolutional characteristics of the interfaces between osteoblast cells and cold sprayed hydroxyapatite-titanium composite coating. The in situ observation of the interfaces was carried out by focused ion beam-scanning electron microscopy, and influence of the surface characteristics of the coatings on the behaviors of protein adsorption and cell proliferation was examined. Apparent cellular sensing, adhesion, migration, and mineral dissolution after 3 h incubation of the cells were visualized. The predominant existence of fragment particles in the coatings, one of the exceptional features of cold sprayed coating, plays vital role in regulating the cell association and cellular uptake. The presence of Ti and HA fragments at the cell-coating interface had no significant effect on cell viability and cell proliferation. After 5 days culturing, in vitro cell fusion and mineralization with the formation of an intermediate layer were evidenced on the coating, suggesting rapid regeneration and accelerated establishment of bone-implant integration. These critical phenomena occurred at different periods of culturing would facilitate understanding the influence of specific surface features of biomaterials on cell responses, and shed light on the development of manufacturing techniques for biomaterials.

226 Skin controls gut immune function through innate immune ECM cross talk

Inflammatory bowel diseases (IBD) are associated with several skin inflammatory diseases but the mechanism responsible for communication between organs is unknown. Local immune activation occurs in part by the action of induced hyaluronidase in the extracellular matrix (ECM) and subsequent recognition of soluble HA fragments. We hypothesized that such HA fragments may also act to enable organ crosstalk between skin and gut. To test this, the intestine was examined in mice expressing hyaluronidase in the skin (K14/Hyal1 mice).

Hyaluronan primes the oxidative burst in human neutrophils.

Hyaluronan (HA) is a glycosaminoglycan that in its natural, high molecular mass (HMM) form, promotes tissue repair and homeostasis. With inflammation, HA metabolism and HMM HA fragmentation to low molecular mass (LMM) forms is greatly enhanced. Considerable evidence suggests that LMM HA may act as a damage-associated molecular pattern to initiate innate immune responses. However, the responsiveness of myeloid cells to LMM HA is controversial and largely unknown for neutrophils. Peripheral blood cells from healthy donors were incubated ex vivo with pharmaceutical grade HA of different molecular mass (HMM, LMM, and HA fragments<10kDa). Key innate immune functions were assessed, namely production of cytokines and reactive oxygen species release (ROS), granule mobilization, and apoptosis. None of the tested sizes of HA altered cytokine production by PBMC and neutrophils. Also, HA had no effect on neutrophil granule mobilization and apoptosis. In contrast, HA primed neutrophils for rapid and robust release of ROS in response to a secondary stimulus (N-formyl-methionyl-leucyl phenylalanine). Priming occurred within 20min of exposure to HA and was similar for all tested molecular mass. The observed effect was independent of granule mobilization and associated with the activation of intracellular signaling pathways involving Src family kinases, glycogen synthase kinase-3, and the proline-rich Akt substrate of 40kDa. Our findings provide new evidence that HA, irrespective of molecular mass, is a specific priming agent of the neutrophil oxidative burst, which is a critical, early component of an innate immune response.

Hyaluronan Fragmentation During Inflammatory Pathologies: A Signal that Empowers Tissue Damage.

The mechanisms that modulate the response to tissue injury are not fully understood. Abnormalities in the repair response are associated with a variety of chronic disease states characterized by inflammation, followed subsequently by excessive ECM deposition. As cell-matrix interactions are able to regulate cellular homeostasis, modification of ECM integrity appears to be an unspecific factor in promoting the onset and progression of inflammatory diseases. Evidence is emerging to show that endogenous ECM molecules supply signals to damage tissues and cells in order to promote further ECM degradation and inflammation progression. Several investigations have been confirmed that HA fragments of different molecular sizes exhibit different biological effects and responses. In fact, the increased deposition of HA into the ECM is a strong hallmark of inflammation processes. In the context of inflammatory pathologies, highly polymerized HA is broken down into small components, which are able to exacerbate the inflammatory response by inducing the release of various detrimental mediators such as reactive oxygen species, cytokines, chemokines and destructive enzymes and by facilitating the recruitment of leukocytes. However, strategies involving the modulation of the HA fragment with specific receptors on cell surface could represent different promising effects for therapeutic scope. This review will focus on the inflammation action of small HA fragments in recent years obtained by in vivo reports.

Controlled conversion of sodium hyaluronate into low-molecular-weight polymers without additives using high-temperature water and fast-heating-rates

In this work, molecular weight reduction of the polysaccharide biopolymer, sodium hyaluronate (NaHA), was obtained without added chemicals or catalysts using high temperature water (180 to 260) °C, fast-heating-rates (417 to 750) °C∙s−1 and short reaction times (< 18 s) in a flow reactor. Results show that hydrothermal treatment of NaHA with fast-heating-rates convert the substrate into low molecular weight oligomers with minimal byproduct formation. A kinetic model assuming random depolymerization provided data correlation and allowed estimation of molecular weight reductions. Mechanism of NaHA decomposition in high temperature water with fast-heating rates seems to be related to initial disentanglement of NaHA random coils through convective (turbulent) mixing and shear that minimizes heterogenous decomposition. Evidence for the mechanism includes lack of significant furan byproducts (fast-heating), clear product solutions and model applicability. Fast-heating with high temperature water allows selective conversion of high molecular weight NaHA into low molecular weight HA fragments without chemical modification, catalysts or additives.

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.

Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells

High levels of hyaluronan (ΗΑ), a major extracellular matrix (ECM) glycosaminoglycan, have been correlated with poor clinical outcome in several malignancies, including breast cancer. The high and low molecular weight HΑ forms exert diverse biological functions. Depending on their molecular size, ΗΑ forms either promote or attenuate signaling cascades that regulate cancer progression. In order to evaluate the effects of different ΗΑ forms on breast cancer cells' behavior, ΗΑ fragments of defined molecular size were synthesized. Breast cancer cells of different estrogen receptor (ER) status – the low metastatic, ERα-positive MCF-7 epithelial cells and the highly aggressive, ERβ-positive MDA-MB-231 mesenchymal cells – were evaluated following treatment with HA fragments. Scanning electron microscopy revealed that HA fragments critically affect the morphology of breast cancer cells in a molecular-size dependent mode. Moreover, the ΗΑ fragments affect cell functional properties, the expression of major ECM mediators and epithelial-to-mesenchymal transition (ΕΜΤ) markers. Notably, treatment with 200 kDa ΗΑ increased the expression levels of the epithelial marker Ε-cadherin and reduced the expression levels of HA synthase 2 and mesenchymal markers, like fibronectin and snail2/slug. These novel data suggest that the effects of HA in breast cancer cells depend on the molecular size and the ER status. An in-depth understanding on the mechanistic basis of these effects may contribute on the development of novel therapeutic strategies for the pharmacological targeting of aggressive breast cancer.

INT-HA induces M2-like macrophage differentiation of human monocytes via TLR4-miR-935 pathway.

As a major component of the microenvironment of solid tumors, tumor-associated macrophages (TAMs) facilitate tumor progression. Intermediate-sized hyaluronan (INT-HA) fragments have an immunological function in cell differentiation; however, their role in promoting the polarization of non-activated macrophages to an M2-like TAM phenotype has not been characterized, and the underlying mechanisms remain unclear. Here, we used a miRNA microarray to find that some miRNAs (especially miR-935) were differentially regulated in INT-HA-induced M2-like macrophages. According to RT-qPCR and Western blot, there was an association between miR-935 and C/EBPβ, that control the polarization of macrophages. Moreover, we found that INT-HA induced an M2-like phenotype via the TLR4 receptor. In our study, there was a negative correlation between plasma HA and miR-935 in monocytes from the peripheral blood of patients with solid tumors. There was also a negative correlation between miR-935 and M2-like macrophage markers in monocytes. These findings suggest that HA fragments interact with TLR4 and educate macrophage polarization to an M2-like phenotype via miR-935. Therefore, this study provides new insight into the role of miR-935 in INT-HA-induced M2-like polarization, and suggests a potential therapeutic target for antitumor treatment.

Chondroitin sulphate inhibits NF-κB activity induced by interaction of pathogenic and damage associated molecules

To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200μg/ml). We measured IL-1β release, intracellular IL-1β, proIL-1β, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P=0.02) in the OA cohort (n=40). The priming dose of LPS used for these experiments (10ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1β that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.

Is molecular size a discriminating factor in hyaluronan interaction with human cells?

Nowadays there is a great interest in investigating the effect of particular hyaluronan fragments in the biomedical field and in cosmeceutical applications. Literature has reported that very low molecular weight HA (Mw<5kDa) has an inflammatory effect, whilst HA ranging from 15 to 250 has shown controversial effects. This work aims to give better elucidation on the correlation between the different sized HA fragments and their biological functions. In this respect, a simple and effective degradation strategy is used to obtain several HA fragments. Also, an hydrodynamic and structural characterization was performed in order to obtain samples suitable to evaluate cellular response. In particular an in vitro scratch test in time lapse experiments was used to study the effect of HA fragments, ranging from 1800 to 6kDa on wound dermal reparation based on human keratinocytes. All high and low Mw HA used in this study allowed for faster wound closure compared to the un-treated cells, except for 6kDa that, on the contrary, prevented repair.In addition, TGF-β 1, TNFα and IL-6, representative biomarkers of the inflammation phase occurring in wound healing process, were quantified by RT-PCR. A general up-regulation trend of these biomarkers was found with the HA molecular weight reduction. LHA6kDa was the only treatment that induced a major inflammatory response (over 30 fold increase respect to control) confirming the recent literature outcomes. IL-6 protein level evaluated through ELISA assay corroborated the previous results. Furthermore, activation of key HA receptors, such as CD44, RHAMM, TLR4, with respect to hyaluronan size, was evaluated, at transcriptional level showing selective recognition by HA 1800, 1400, 500 for CD44, whilst the lower Mw fragments activated TLR-4 moderately at 50 and 15kDa. An increase to “alarm” level was found for 6kDa fragments. Immunofluorescence staining confirmed this data.The present research work demonstrated that the diverse pharma grade hyaluronan fragments could modulate cellular processes differently. From 1800kDa down to 50kDa, CD44 was the recognized receptor and pro-inflammatory biomarkers were only slightly up-regulated during wound healing in the presence of HA. Finally our outcomes showed that the lower the fragment size the higher the concern for inflammatory cytokines up-regulation; repair process impairment was highlighted only for 6kDa chains.

739 Hyaluronan controls adipogenesis following skin injury

Skin immune defense and repair is a coordinated response to a variety of danger signals and involves the complex interactions of several different cell types. Dermal adipogenesis is a previously unknown but critical event in this process that participates in antimicrobial defense and inflammation. We have previously shown that high-molecular-weight hyaluronan (HMW-HA) undergoes breakdown into small fragments after injury where it can then activate endothelial cells, macrophages and DCs via TLR4. In this study we investigated if HA may also influence adipogenesis and thus regulate the wound repair process. Maturation of 3T3L1 cells into adipocytes was accompanied by increased HMW-HA seen by Gel Electrophoresis and expression of HAS2 mRNA measured by qPCR (p =0.006). Conversely, digestion of HMW-HA by addition of hyaluronidase inhibited differentiation into mature adipocytes as seen by decreased mRNA for zfp521(p=0.02), zfp423(p=0.01) and Adiponectin (p =0.004), as well as decreased accumulation of lipid droplets and enhanced cell proliferation. The inhibition of early adipogenesis was not due to HA fragments since the addition of HA fragments of 6.4kDa, 50kDa, 150kDa and 1000kD had no effect. In vivo, tissue specific expression of hyaluronidase 1 (HYAL1) in mice prior to injury resulted in less mature dermal fat tissue as seen by immunohistochemistry and lower expression of mRNA for C/EBPα (p=0.002) but not Adiponeqtin(p=0.13) or PPARγ(p=0.27). In normal mice, HA immunostaining was lost 3 days following injury coincident with an increase in expression of HAS2 mRNA (p=0.0008) and mRNA for the hyaluronidase KIAA1199 (p=0.01). The loss of HMW-HA in vivo occurred after the expected increase in adipogenesis immediately after injury. Taken together, these results suggest that HA plays a regulatory role during wound repair by controlling dermal adipogenesis. HMW-HA facilitates fat maturation early in the wound repair process, and hyaluronidase action stops this event later in the wound healing process.