Hyaluronan Fragments Research Articles

Hyaluronic acid cues for functional endothelialization of vascular constructs

Current vascular implant materials insufficiently recruit endothelial cells (ECs) to form a normally functional and confluent endothelium, a key challenge to reinstating vascular homeostasis at the surgical site. Recent studies indicate that hyaluronan (HA), a connective tissue GAG whose biological effects are often dictated by its fragment size, may inherently stimulate endothelialization. We previously showed that ECs respond poorly to large HA fragments (10 kDa < MW < 1 MDa), therefore, we currently sought to comprehensively study the effects of exogenous high molecular weight (>1000 kDa) and oligomeric (0.75-10 kDa) ranges on various phenotypic and functional aspects of cultured ECs. HA-1500 (1500 kDa) was enzymatically digested into oligomers under iteratively defined conditions, until a mixture (HA-o) containing a maximal yield of HA-6-mer and 12-mers (33.3 +/- 2.44% and 39.2 +/- 2.68% w/w, respectively) was obtained. The effects of HA-1500, HA-o and pure HA-6-mers on rat aortic ECs were compared. DNA and tube formation assays revealed HA-o and HA-6-mers to stimulate EC proliferation and EC tube formation (angiogenesis) much more than non-HA controls, while HA-1500 had a significant but more modest effect. Both HA-o and HA-6-mers attenuated platelet adhesion and activation on EC layers, while HA-1500 drastically inhibited the same relative to controls. However, flow cytometry and cytokine array studies found that HA-o incited increased expression levels of EC activation markers (ICAM-1, VCAM-1) and promoted the release of select inflammatory cytokines to a greater degree than HA-1500. These results suggest that HA-o and HA-1500 both provide benefits, although frequently of different kinds, to endothelial cell sustenance, proliferation and normal functionality. Thus, tissue engineering scaffolds containing both these cues in optimized ratios could potentially serve as excellent materials for vascular EC regeneration. This forms the basis of our ongoing studies.

Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4

Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk-B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)-8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll-like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA-triggered activation of IL-8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4-specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP- and cytokine-expression, in part in a TLR4-dependent manner, providing new insights into the relationship between cancer and innate immunity.

Hyaluronan in Tissue Injury and Repair

A hallmark of tissue injury and repair is the turnover of extracellular matrix components. This review focuses on the role of the glycosaminoglycan hyaluronan in tissue injury and repair. Both the synthesis and degradation of extracellular matrix are critical contributors to tissue repair and remodeling. Fragmented hyaluronan accumulates during tissue injury and functions in ways distinct from the native polymer. There is accumulating evidence that hyaluronan degradation products can stimulate the expression of inflammatory genes by a variety of immune cells at the injury site. CD44 is the major cell-surface hyaluronan receptor and is required to clear hyaluronan degradation products produced during lung injury; impaired clearance of hyaluronan results in persistent inflammation. However, hyaluronan fragment stimulation of inflammatory gene expression is not dependent on CD44 in inflammatory macrophages. Instead, hyaluronan fragments utilize both Toll-like receptor (TLR) 4 and TLR2 to stimulate inflammatory genes in macrophages. Hyaluronan also is present on the cell surface of lung alveolar epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. The simple repeating structure of hyaluronan appears to be involved in a number of important aspects of noninfectious tissue injury and repair that are dependent on the size and location of the polymer as well as the interacting cells. Thus, the interactions between the endogenous matrix component hyaluronan and its signaling receptors initiate inflammatory responses, maintain structural cell integrity, and promote recovery from tissue injury.

卵丘細胞に発現するToll Like Receptor (TLR)の活性化はケモカインを分泌させ，それが精子の受精能獲得を誘起する

卵丘細胞に発現するToll Like Receptor (TLR)の活性化はケモカインを分泌させ，それが精子の受精能獲得を誘起する

Degradation of High-Molar-Mass Hyaluronan and Characterization of Fragments

A sample of high-molar mass hyaluronan was oxidized by seven oxidative systems involving hydrogen peroxide, cupric chloride, ascorbic acid, and sodium hypochlorite in different concentrations and combinations. The process of the oxidative degradation of hyaluronan was monitored by rotational viscometry, while the fragments produced were investigated by size-exclusion chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and non-isothermal chemiluminometry. The results obtained imply that the degradation of hyaluronan by these oxidative systems, some of which resemble the chemical combinations present in vivo in the inflamed joint, proceeds predominantly via hydroxyl radicals. The hyaluronan fragmentation occurred randomly and produced species with rather narrow and unimodal distribution of molar mass. Oxidative degradation not only reduces the molecular size of hyaluronan but also modifies its component monosaccharides, generating polymer fragments that may have properties substantially different from those of the original macromolecule.

Extracellular Matrix Fragments as Regulators of Cartilage Metabolism in Health and Disease

It is now firmly established that fragments of extracellular matrix (ECM) macromolecules that arise from the damaged ECM have the ability to regulate chondrocyte metabolism and enhance the degradative processes. Fibronectin fragments (Fn-fs), type II collagen fragments (Col-fs) and hyaluronan fragments (HA-fs) have all been shown to upregulate matrix metalloproteinases and cause extensive damage to the ECM, while Fn-fs have also been shown to enhance anabolic pathways. Since these ECM fragments are elevated in states of cartilage degeneration or in osteoarthritic cartilage, the ECM fragments likely play a role in either initiation or augmentation of degradative and anabolic pathways which collectively dictate the metabolic state of the damaged tissue. Retrospectively, the ability of ECM fragments to serve as the initial sensors of cartilage damage provides the most upstream signaling possible for effective cartilage repair/ remodeling. Newer observations suggest that the perturbation of the matrix to chondrocyte interactions by ECM fragments may be the key initiating event in the catabolic pathways since this may indirectly affect matrix to receptor interaction and downstream signaling. It is conceivable that these fragment pathways either intersect or overlap and involve common players which might constitute common targets for therapeutic intervention.

Apical Oxidative Hyaluronan Degradation Stimulates Airway Ciliary Beating via RHAMM and RON

Hyaluronan (HA) is synthesized in high-molecular-weight form at the apical pole of airway epithelial cells, covering the luminal surface. When human airway epithelial cells grown and redifferentiated at the air-liquid interface (ALI) were exposed to xanthine/xanthine oxidase (X/XO), ciliary beat frequency (CBF) increased. This effect was blocked by superoxide dismutase (SOD) and catalase. Inhibition of hyaluronan synthesis inhibited the CBF response to X/XO, while addition of exogenous HA amplified it. A functionally blocking antibody to the receptor for hyaluronic acid-mediated motility (RHAMM) reduced the CBF response to X/XO. Since RHAMM has no transmembrane domain and thus cannot signal on its own, the association of RHAMM with recepteur d'origine nantais (RON), a member of the hepatocyte growth factor receptor family, was explored. Immunohistochemistry of human airway epithelium showed co-localization of RHAMM and RON at the apex of ciliated cells. Physical association of RHAMM and RON was confirmed with co-immunoprecipitations. Macrophage-stimulating protein (MSP), an agonist of RON, stimulated CBF. Genistein, a nonspecific tyrosine kinase inhibitor, and MSP beta chain (beta-MSP), a specific RON inhibitor, blocked the X/XO-induced CBF increase. HA present in the apical secretions of human airway epithelial cells was shown to degrade upon exposure to X/XO, a process inhibited by SOD. Low-molecular-weight HA fragments stimulated CBF, an effect blocked by anti-RHAMM antibody and genistein. These data suggest that high molecular form HA is broken down by reactive oxygen species to form low-molecular-weight fragments that signal via RHAMM and RON to stimulate CBF.

Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes

AbstractMacrophages (Mφ) in most solid tumors exhibit a distinct immunosuppressive phenotype, but the mechanisms that allow tumor microenvironments to “educate” Mφ are incompletely understood. Here, we report that culture supernatants (TSNs) from several types of tumor cell lines can drive monocytes to become immunosuppressive Mφ. Kinetic experiments revealed that soon after exposure to these TSNs, monocytes began to provoke transient proinflammatory responses and then became refractory to subsequent stimulation. Other TSNs that failed to cause such temporary preactivation did not alter Mφ polarization. Consistent with these results, we observed that the monocytes/Mφ in different areas of human tumor samples exhibited distinct activation patterns. Moreover, we found that hyaluronan fragments constitute a common factor produced by various tumors to induce the formation of immunosuppressive Mφ, and also that upregulation of hyaluronan synthase-2 in tumor cells is correlated with the ability of the cells to cause Mφ dysfunction. These results indicate that soluble factors derived from tumor cells, including hyaluronan fragments, co-opt the normal development of Mφ to dynamically educate the recruited blood monocytes in different niches of a tumor. The malignant cells can thereby avoid initiation of potentially dangerous Mφ functions and create favorable conditions for tumor progression.

ACTIVATION OF TOLL-LIKE RECEPTORS 2 AND 4 ON CUMULUS CELLS OF OVULATED CUMULUS OOCYTE COMPLEXES STIMULATES PRODUCTION OF CYTOKINES/CHEMOKINES THAT CAN INDUCE SPERM CAPACITATION LEADING TO SUCCESSFUL FERTILIZATION

Our microarray analyses revealed that pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) family and related molecules are expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and exhibit functions similar to those in macrophages. Specifically, we showed that when COCs were cultured with the TLR4 ligand bacterial lipopolysaccharide (LPS), the expression of Il-6, Tnfa and Ptgs2 were induced. However, the endogenous ligand for TLR family and its physiological role in ovulated COCs was not analyzed. Recently, it was reported that hyaluronan fragments, but not the high molecular weight hyaluronan polymers, activate TLR2 and TLR4. Since cumulus cells produce a hyaluronan rich matrix during the ovulation process, and because the matrix is broken-down by sperm-secreted hyaluronidase during fertilization, we hypothesized that hyaluronan fragments generated during matrix degradation might act as an endogenous ligand for TLR2 and/or TLR4 on cumulus cells and be required for successful fertilization. To examine this, mouse ovulated COCs were cultured for 2 hr with either LPS or Pam3Cys, ligands for TLR4 or TLR2, respectively. Each ligand increased mRNAs encoding specific cytokines/chemokines: Il6, Mip2, Ccl5 ∼7-fold, ∼10-fold, ∼15-fold, respectively. When ovulated COCs were cultured with hyaluronan fragments or treated with hyaluronidase, the expression of Il6, Mip2, Ccl5 mRNAs also increased with peak levels observed using hyaluronan fragments at 100 μg/ml or hyaluronidase at 1 IU/ml for 2 hr. Anti-TLR2 and anti-TLR4 monoclonal antibodies significantly suppressed hyaluronan fragment- and hyaluronidase- induced expression of these genes. Thus, TLR2/TLR4 on cumulus cells of ovulated COCs are functional and activated by hyaluronan fragments. To determine the role of activated TLR2/TLR4 on cumulus cells in fertilization process, ovulated COCs were cultured with 1x105 sperm in the presence or absence of anti-TLR2 and anti-TLR4 antibodies. Using a Bioplex Protein Array system we identified 17 cytokine and chemokine factors that were secreted by COCs. Secretion of 10 factors, CCL-5, G-CSF, IL-1a, IL-6, IL-9, IL-12 IL-17, MCP-1, MIP-1a, and TNFa was significantly up-regultaed by co-culture with sperm. The addition of anti-TLR2 and anti-TLR4 antibodies significantly suppressed the secretion of CCL-5, G-CSF, MCP-1, MIP-1a, and IL-6, concomitantly with the reduction of fertilization rate to 32 % as compared with that in oocytes without antibodies (75 %). Additionally, Ccr1, Ccr2, Ccr3 and Ccr5 mRNAs that encode CCL5, MCP-1, and MIP-1a receptors were expressed in spermatozoa. Sperm motility was enhanced by specific ligands suggesting that the chemokines secreted from cumulus cells of ovulated COCs induces sperm capacitation and subsequent oocyte penetration by mechanisms dependent on TLR2/4 activation. Supported in part by JSPS- 18688016 (MS), HD-16229, HD07459 (JSR) (platform)

Recognition of Hyaluronan Released in Sterile Injury Involves a Unique Receptor Complex Dependent on Toll-like Receptor 4, CD44, and MD-2

Inflammation under sterile conditions is not well understood despite its importance in trauma and autoimmune disease. To investigate this process we established mouse models of sterile injury and explored the role of hyaluronan in mediating inflammation following injury. The response of cultured monocytes to hyaluronan was different than the response to lipopolysaccharide (LPS) despite both being dependent on Toll-like receptor 4 (TLR4). Cultured cells exposed to hyaluronan showed a pattern of gene induction that mimics the response seen in mouse skin after sterile injury with an increase in molecules such as transforming growth factor-beta2 and matrix metalloproteinase-13. These factors were not induced by LPS despite the mutual dependence of both hyaluronan and LPS on TLR4. Explanation for the unique response to hyaluronan was provided by observations that a lack of TLR4 or CD44 in mice diminished the response to sterile injury, and together with MD-2, was required for responsiveness to hyaluronan in vitro. Thus, a unique complex of TLR4, MD-2, and CD44 recognizes hyaluronan. Immunoprecipitation experiments confirmed the physical association of TLR4 and CD44. Taken together, our results define a previously unknown mechanism for initiation of sterile inflammation that involves recognition of released hyaluronan fragments as an endogenous signal of tissue injury.

The Toll-Like Receptor Adaptor Proteins MyD88 and Mal/TIRAP Contribute to the Inflammatory and Destructive Processes in a Human Model of Rheumatoid Arthritis

The widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-alpha, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA.

The Anti-rheumatic Gold Salt Aurothiomalate Suppresses Interleukin-1β-induced Hyaluronan Accumulation by Blocking HAS1 Transcription and by Acting as a COX-2 Transcriptional Repressor

Gold compounds are among the oldest disease-modifying drugs and are still widely used today for treating rheumatoid arthritis. Despite decades of use, little is known about the mode of action of this class of drugs. Here we have demonstrated that aurothiomalate (AuTM) suppresses hyaluronan accumulation by blocking interleukin (IL)-1beta-induced hyaluronan synthase-1 transcription. We have further demonstrated that, in fibroblast-like synoviocytes (FLSs), AuTM acts as a specific COX-2 transcriptional repressor in that IL-1beta-induced COX-2 transcription is blocked, whereas COX-1 transcription and translation is unaffected. As a consequence, PGE2 levels released by FLS are dose-dependently reduced in cells exposed to AuTM. Of similar importance is the demonstration that AuTM does block NFkappaB-DNA interaction. In addition, two other transcription factors implicated in inflammatory events, namely AP-1 and STAT3, are blocked as well. The effect on NFkappaB likely explains the inhibition of COX-2 as well as that of HAS1, as both are genes that depend on the activation of NFkappaB. Interestingly, AuTM does not interfere with IL-1beta-induced IkappaB alpha degradation, in most cases a prerequisite for subsequent NFkappaB activation. Furthermore, evidence is presented that, in FLS, AuTM blocks NFkappaB-DNA interaction neither by binding to NFkappaB binding sites nor by interacting with activated NFkappaB proteins. Taken together, AuTM treatment of FLS blocks two of the most important proinflammatory events that are associated with rheumatoid arthritis. AuTM blocks the release of PGE2 and prevents the activation of NFkappaB, therefore blocking IL-1beta-induced hyaluronan accumulation and likely a series of other pro-inflammatory NFkappaB-dependent genes.

Ligand-induced Structural Changes of the CD44 Hyaluronan-binding Domain Revealed by NMR

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

The Role of Hyaluronan Degradation Products as Innate Alloimmune Agonists

Dendritic cells (DCs) play a key role in initiating alloimmunity yet the substances that activate them during the host response to transplantation remain elusive. In this study we examined the potential roles of endogenous innate immune agonists in activating dendritic cell-dependent alloimmunity. Using a murine in vitro culture system, we show that 135 KDa fragments of the extracellular matrix glycosaminoglycan hyaluronan induce dendritic cell maturation and initiate alloimmunity. Priming of alloimmunity by hyaluronan-activated DCs was dependent on signaling via TIR-associated protein, a Toll-like receptor (TLR) adaptor downstream of TLRs 2 and 4. However, this effect was independent of alternate TLR adaptors, MyD88 or Trif. Using an in vivo murine transplant model, we show that hyaluronan accumulated during skin transplant rejection. Examination of human lung transplant recipients demonstrated that increased levels of intragraft hyaluronan were associated with bronchiolitis obliterans syndrome. In conclusion, our study suggests that fragments of hyaluronan can act as innate immune agonists that activate alloimmunity.

Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI–TOF mass spectrometric investigations

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl 2 present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.

A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells

Crosslinked gels (hylans) containing long-chain (MW>1×10 6 Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20 kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI–TOF showed the HA digests to contain HA 4–8mers with a 75±0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9±3 amine groups/nm 2 and 0.57±0.44 μg/cm 2, respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell–surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior.

The role of Toll-like receptors in non-infectious lung injury

The role of Toll-like receptors (TLRs) in pathogen recognition has been expeditiously advanced in recent years. However, investigations into the function of TLRs in non-infectious tissue injury have just begun. Previously, we and others have demonstrated that fragmented hyaluronan (HA) accumulates during tissue injury. CD44 is required to clear HA during tissue injury, and impaired clearance of HA results in unremitting inflammation. Additionally, fragmented HA stimulates the expression of inflammatory genes by inflammatory cells at the injury site. Recently, we identified that HA fragments require both TLR2 and TLR4 to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. In a non-infectious lung injury model, mice deficient in both TLR2 and TLR4 show an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis, and decreased survival. Lung epithelial cell overexpression of high molecular mass HA protected mice against acute lung injury and apoptosis, in part through TLR-dependent basal activation of NF-kappaB. The exaggerated injury in TLR2 and TLR4 deficient mice appears to be due to impaired HA-TLR interactions on epithelial cells. These studies identify that host matrix component HA and TLR interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute lung injury.

Hyaluronan fragments: An information-rich system

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [- d-glucuronic acid- β1,3- N-acetyl- d-glucosamine- β1,4-] n . Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell–cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2×10 4 kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous “danger signals”, while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.

Hyaluronan Fragments Act as an Endogenous Danger Signal by Engaging TLR2

Upon tissue injury, high m.w. hyaluronan (HA), a ubiquitously distributed extracellular matrix component, is broken down into lower m.w. (LMW) fragments, which in turn activate an innate immune response. In doing so, LMW HA acts as an endogenous danger signal alerting the immune system of a breach in tissue integrity. In this report, we demonstrate that LMW HA activates the innate immune response via TLR-2 in a MyD88-, IL-1R-associated kinase-, TNFR-associated factor-6-, protein kinase Czeta-, and NF-kappaB-dependent pathway. Furthermore, we show that intact high m.w. HA can inhibit TLR-2 signaling. Finally, we demonstrate that LMW HA can act as an adjuvant promoting Ag-specific T cell responses in vivo in wild-type but not TLR-2(null) mice.

Matrix Regulation of Lung Injury, Inflammation, and Repair: The Role of Innate Immunity

Mechanisms that regulate host defense after noninfectious tissue injury are incompletely understood. Our laboratory is interested in the role of the extracellular matrix glycosaminoglycan hyaluronan in the regulation of lung inflammation and fibrosis. We have identified key roles for two cell surface receptor systems that interact with hyaluronan to control lung inflammation and tissue repair. Hematopoietic CD44 is necessary to clear hyaluronan fragments that are produced after lung injury. Failure to clear hyaluronan fragments leads to unremitting inflammation. However, in the absence of CD44, alveolar macrophages continue to produce chemokines in response to hyaluronan fragments, implicating another receptor system in controlling macrophage effector function. We found that Toll-like receptors 2 and 4 (TLR2 and TLR4) are responsible for macrophage inflammatory gene expression in response to hyaluronan fragments. Although TLR2 and TLR4 initiate the innate immune response in noninfectious inflammation, they have a protective role against lung injury on alveolar epithelial cells.