Hyaluronan Fragments Research Articles

Enhanced Deposition of Low-Molecular-Weight Hyaluronan in Lungs of Cigarette Smoke–Exposed Mice

Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

Background Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes. Methods mRNA and related protein levels were measured for CD44, PKCδ, PKCɛ, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed. Results CD44, PKCδ, and PKCɛ mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKCɛ levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKCɛ, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKCɛ, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation. General Significance These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.

Cationic conjugated polymers for homogeneous and sensitive fluorescence detection of hyaluronidase

The cationic charged water-soluble polyfluorenes containing 2,1,3-benzothiadiazole (BT) units (P1–3) have been synthesized and characterized. These polymers demonstrate intramolecular energy transfer from the fluorene units to the BT sites when oppositely charged hyaluronan is added due to the formation of electrostatic complexes, followed by a shift in emission color from blue to green or brown. Upon adding hyaluronidase, the hyaluronan is cleaved into fragments. The relatively weak electrostatic interactions of hyaluronan fragments with polyfluorenes keep their main chains separated and energy transfer from the fluorene units to the BT sites is inefficient, and the polyfluorenes recover their blue emissions. The complexes of conjugated polymers/hyaluronan can be utilized as probes for sensitive and facile fluorescence assays for hyaluronidase. The new assay method interfaces with the aggregation and light harvesting properties of conjugated polymers.

Successive changes in hyaluronan (HA) synthesis and fragmentation in the rat post‐ischemic kidney

The precise time‐course of HA accumulation and fragmentation in inflammatory tissues remains uncertain. We examined the expression of the main hyaluronidases (HYAL1 and HYAL2) and HA synthases (HAS1, HAS2 and HAS3) by real‐time PCR in the outer [OSOM] and inner stripes [ISOM] of the renal outer medulla up to Day 14 after ischemia/reperfusion (IR) injury. We also assessed hyaluronidase activity by zymography and HA fragmentation by membrane filtration and pseudo‐ELISA. HAS1 was up‐regulated more than 100‐fold 12 h post‐IR, returning to baseline faster in the ISOM than in the OSOM. HAS2 increased only after a 2‐day delay and remained elevated. The mRNA for HAS3 could not be detected. Both HYAL1 and HYAL2 were strongly but transiently (12‐48 h) repressed in parallel with a reduction in hyaluronidase activity. HA accumulation was detected as early as 12 h and increased steadily thereafter. However, the amount of low molecular weight (MW) HA (i.e., HA fragments) decreased by 60% from baseline to Day 1 in the ISOM. Later, HA fragments amassed in both zones (×100 in OSOM and ×5 in ISOM vs baseline). In summary, within 24 h post‐IR, there is a massive activation of HAS1 combined with a repression of hyaluronidases, leading to a pool of predominantly high MW HA. Significant amounts of HA fragments appear later (Day 7‐14) when HAS2 is induced and HYAL1 and HYAL2 levels are back to baseline. HA fragments are possibly involved in regeneration.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Biomimetic Regeneration of Elastin Matrices Using Hyaluronan and Copper Ion Cues

Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo- and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu(2+)) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu(2+) ions slowed cell proliferation (5.6 +/- 2.3-fold increase over 21 days vs 22.9 +/- 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 +/- 0.4-fold), tropoelastin (4.1 +/- 0.05-fold) and cross-linked matrix elastin (4.2 +/- 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu(2+) ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu(2+) ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu(2+) ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers ( approximately 1 mum diameter). However, 0.01 M of Cu(2+) ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu(2+) ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.

Anti-oxidant inhibition of hyaluronan fragment-induced inflammatory gene expression

BackgroundThe balance between reactive oxygen species (ROS) and endogenous anti-oxidants is important in maintaining healthy tissues. Excessive ROS states occur in diseases such as ARDS and Idiopathic Pulmonary Fibrosis. Redox imbalance breaks down the extracellular matrix component hyaluronan (HA) into fragments that activate innate immune responses and perpetuate tissue injury. HA fragments, via a TLR and NF-κB pathway, induce inflammatory gene expression in macrophages and epithelial cells. NAC and DMSO are potent anti-oxidants which may help balance excess ROS states.MethodsWe evaluated the effect of H2O2, NAC and DMSO on HA fragment induced inflammatory gene expression in alveolar macrophages and epithelial cells.ResultsNAC and DMSO inhibit HA fragment-induced expression of TNF-α and KC protein in alveolar and peritoneal macrophages. NAC and DMSO also show a dose dependent inhibition of IP-10 protein expression, but not IL-8 protein, in alveolar epithelial cells. In addition, H2O2 synergizes with HA fragments to induce inflammatory genes, which are inhibited by NAC. Mechanistically, NAC and DMSO inhibit HA induced gene expression by inhibiting NF-κB activation, but NAC had no influence on HA-fragment-AP-1 mediated gene expression.ConclusionROS play a central role in a pathophysiologic "vicious cycle" of inflammation: tissue injury generates ROS, which fragment the extracellular matrix HA, which in turn synergize with ROS to activate the innate immune system and further promote ROS, HA fragment generation, inflammation, tissue injury and ultimately fibrosis. The anti-oxidants NAC and DMSO, by inhibiting the HA induced inflammatory gene expression, may help re-balance excessive ROS induced inflammation.

Hyaluronan receptors involved in cytokine induction in monocytes

During inflammation, lower molecular weight fragments of hyaluronan accumulate, and this is known to be inflammatory and immune-stimulatory. In diseases such as inflammatory bowel disease, inflammatory cells bind to hyaluronan; however, the cellular response and molecular mechanism of hyaluronan-hyaluronan receptor interactions in mononuclear cells are not well understood. The expression of hyaluronan receptors in peripheral blood mononuclear cells (PBMC) was examined. PBMC were stimulated with lower and higher molecular weight hyaluronan (molecular weight 100-150 kDa and 2700 kDa) and the induction of proinflammatory cytokines (interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1)) was compared by enzyme-linked immunoabsorbant assay (ELISA). Cells were coincubated with various signaling pathway inhibitors. In addition, neutralizing antibodies against CD44 and TLR4 were added and the effects on PBMC were investigated. Finally, mononuclear cells from CD44-null and toll-like receptor 4 (TLR4) mutant mice were both stimulated with lower molecular weight hyaluronan. Among the hyaluronan receptors, TLR4 and CD44 were markedly expressed on PBMC. Hyaluronan-stimulated PBMC enhanced the attachment to the extracellular matrix. Lower molecular weight hyaluronan induced IL-6 and MCP-1 production in PBMC, but high-molecular-weight hyaluronan did not induce IL-6 and MCP-1 production. An anti-CD44 antibody attenuated the induction of both IL-6 and MCP-1 in lower molecular weight hyaluronan-stimulated PBMC. In both TLR4 mutant and CD44-null mice, the induction of IL-6 by lower molecular weight hyaluronan stimulation was decreased. SB203580 completely abolished IL-6 production in both TLR4 mutant and CD44-null mononuclear cells, while PD98059 abolished IL-6 production in CD44-null mononuclear cells. Hyaluronan receptors, CD44 and TLR4, play distinct roles in cytokine induction in hyaluronan-stimulated mononuclear cells.

Hyaluronan Fragments/CD44 Mediate Oxidative Stress–Induced MUC5B Up-Regulation in Airway Epithelium

Mucus hypersecretion with elevated MUC5B mucin production is a pathologic feature in many airway diseases associated with oxidative stress. In the present work, we evaluated MUC5B expression in airways and in primary cultures of normal human bronchial epithelial (NHBE) cells, as well as the mechanisms involved in its regulation. We found that oxidative stress generated by cigarette smoke or reactive oxygen species (ROS) induces MUC5B up-regulation in airway epithelium from smokers and in NHBE cells, respectively. We have previously shown that ROS-induced MUC5AC expression in NHBE cells is dependent on hyaluronan depolymerization and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) activation. Since hyaluronan fragments can activate MAPK through the hyaluronan receptor CD44, and CD44 heterodimerizes with EGFR, we tested whether ROS and/or hyaluronan fragments induce MUC5B mRNA and protein expression through CD44/EGFR. We found that ROS promotes CD44/EGFR interaction, EGFR/MAPK activation, and MUC5B up-regulation that are prevented by blocking CD44 and/or EGFR. These results were mimicked by hyaluronan fragments. In summary, our results show that oxidative stress in vivo (cigarette smoke) or in vitro (ROS) induces MUC5B up-regulation. This ROS-induced MUC5B expression requires CD44 as well as EGFR and MAPK activation. In addition, we also provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and downstream signaling that results in MUC5B up-regulation, suggesting that hyaluronan depolymerization during inflammatory responses could be directly involved in the induction of mucus hypersecretion.

Tumor-Educated Tolerogenic Dendritic Cells Induce CD3ε Down-Regulation and Apoptosis of T Cells through Oxygen-Dependent Pathways

Defects in the CD3/TCR complex and impairment of T cell function are necessary for tumor evasion, but the underlying mechanisms are incompletely understood. We found that culture supernatants from several types of solid tumor cell lines drove human monocytes to become tolerogenic semimature dendritic cells (TDCs). Upon encountering T cells, the TDCs triggered rapid down-regulation of CD3epsilon and TCR-alpha/beta and subsequent apoptosis in autologous T cells. Consistent with these results, accumulation of immunosuppressive DCs coincided with CD3epsilon down-regulation and T cell deletion in cancer nests of human tumors. The impaired T cell function was mediated by factor(s) released by live TDCs after direct interaction with lymphocytes. Also, the TDC-induced effect on T cells was markedly reduced by blocking of NADPH oxidase but not by inhibition of arginase, inducible NO synthase (iNOS), IDO, or IFN-gamma. Moreover, we found that hyaluronan fragments constituted a common factor produced by a variety of human tumor cell lines to induce formation of TDCs. These observations indicate that tumor microenvironments, including hyaluronan fragments derived from cancer cells, educate DCs to adopt a semimature phenotype, which in turn aids tumor immune escape by causing defects in the CD3/TCR complex and deletion of T cells.

The Adenosine A2a Receptor Inhibits Matrix-Induced Inflammation in a Novel Fashion

Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

Highlights From The Literature

Highlights From The Literature

Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing

To determine if oligosaccharides of hyaluronan (o-HA) promotes wound recovery by accelerating angiogenesis and to study the mechanisms by which o-HA stimulates endothelial cell (EC) proliferation. Using hyaluronidase digestion, we prepared a mixture of hyaluronan (HA) fragments sizesd 2 to 10 disaccharides units, and studied their effects on EC growth and migration in mimicking wound recovery in vitro. The effects of o-HA on EC growth in vitro were studied by counting cell numbers. The roles of 2 hyaluronan receptors on EC cells, CD44 and RHAMM (Receptor for HA-Mediated Motility), were studied in initiating signaling cascades, using immunoblot assay. Signal transduction was determined by blocking antibodies to CD44 and RHAMM. An in vitro wound healing model was prepared by scratching the cellular layer of cultured EC, and movement of cells into the denuded area was quantified. o-HA was a strong stimulator to EC proliferation at low concentration 10microg/ml compared with native high molecular weight HA (n-HA) (P < 0.01). Signal transduction may be initiated by o-HA via RHAMM receptor on EC membrane, but not CD44. In the in vitro model, the lesion area was nearly completely recovered when the EC layer was exposed to o-HA 40hrs post-injury, whereas the wound area remained half recovered pretreated with native undegraded large HA and control medium.(P < 0.05 from 24 to 40hrs). Hyaluronan oligosaccharides may play a role in wound healing by increasing angiogenesis. o-HA-RHAMM binding dependent signal transduction pathway may be important in the regulation of angiogenesis associated with EC proliferation.

Hyaluronan Constitutively Regulates Activation of COX-2-mediated Cell Survival Activity in Intestinal Epithelial and Colon Carcinoma Cells

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization

The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages, and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFkappaB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFkappaB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs) localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.

Extracellular Superoxide Dismutase Inhibits Inflammation by Preventing Oxidative Fragmentation of Hyaluronan

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.

Tumor‐educated tolerogenic dendritic cells induce downregulation of CD3ε and apoptosis in T cells via an oxygen‐dependent pathway

Defects in the CD3/TCR complex and impairment of T cell function are necessary for evasion of tumor, but the underlying mechanisms are incompletely understood. We found that culture supernatants from several types of solid tumor cells drived human monocytes to become tolerogenic semimature dendritic cells (TDCs). Upon encountering T cells, the TDCs triggered rapid down‐regulation of CD3ε and TCR‐α/β and subsequent apoptosis in autologous T cells. Consistent with these results, accumulation of immunosuppressive DCs coincided with CD3ε down‐regulation and T cell deletion in cancer nests of human tumors. The impaired T cell function was mediated by factor(s) released by live TDCs after direct interaction with lymphocytes. Also, the TDC‐induced effect on T cells was markedly reduced by blocking of NADPH oxidase but not by inhibition of arginase, iNOS, indoleamine 2,3‐dioxygenase (IDO), or IFN‐γ. Moreover, we found that hyaluronan fragments constituted a common factor produced by a variety of human tumors to induce formation of TDCs. These observations indicate that tumor microenvironments, including hyaluronan fragments derived from cancer cells, educate DCs to adopt a semimature phenotype, which in turn aids tumor immune escape by causing defects in the CD3/TCR complex and deletion of T cells.

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8·102 g mol−1 to 2.5·105 g mol−1 and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme–substrate complex is maximum for HA molar masses ranging from 3·103 to 2·104 g mol−1. Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2·104 g mol−1, are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.

β-defensin 2 release by human keratinocytes following low molecular weight hyaluronan fragments treatment

β-defensin 2 release by human keratinocytes following low molecular weight hyaluronan fragments treatment

Benefits of concurrent delivery of hyaluronan and IGF-1 cues to regeneration of crosslinked elastin matrices by adult rat vascular cells

Elastin, a major component of vascular matrices, critically determines vascular mechanics and maintains the quiescence of smooth muscle cells (SMCs). Attempts to regenerate elastin in elastin-compromised blood vessels using tissue-engineering approaches is limited by the unavailability of elastogenic cues to upregulate poor elastin output and matrix assembly by adult vascular cells. We previously showed that hyaluronan (HA) elastogenically stimulates aortic SMCs, although these effects are highly specific to HA fragment size. The elastogenic response of SMCs can also be modulated with growth factors such as insulin-like growth factor (IGF-1). Here, we evaluate the benefits of concurrent delivery of HA fragments (0.76-2000 kDa) and IGF-1 (500 ng/ml) to elastin synthesis, organization and crosslinking. The study outcomes show that, relative to supplement-free cultures, IGF-1 and long-chain HA/large HA fragments, but not HA oligomers, together induce multifold increases in the synthesis of elastin precursors, structural elastin matrix yields and crosslink densities within cell layers, and encourage elastic fibre formation. These outcomes are not all obtained when either of the cues is provided separately. IGF-1 and large HA fragments (>20 kDa) also together inhibit cell proliferation, a concern in elastin-compromised vessels, where SMC hyperproliferation is common. The results will benefit efforts to provide exogenous or scaffold-based elastogenic cues (IGF-1 + HMW HA/large HA fragments) to enable robust and faithful regeneration of elastin matrix structures in vivo or in vitro. The present outcomes may be used to restore elastin matrix homeostasis in de-elasticized vessels and tissue-engineered constructs that may be grafted as a substitute.