Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

Cervical hyaluronan biology in pregnancy, parturition and preterm birth.

Cervical hyaluronan (HA) synthesis is robustly induced in late pregnancy in numerous species including women and mice. Recent evidence highlights the diverse and dynamic functions of HA in cervical biology that stem from its expression in the cervical stroma, epithelia and immune cells, changes in HA molecular weight and cell specific expression of HA binding partners. Mice deficient in HA in the lower reproductive tract confirm a structural role of HA to increase spacing and disorganization of fibrillar collagen, though this function is not critical for pregnancy and parturition. In addition, cervical HA depletion via targeted deletion of HA synthase genes, disrupts cell signaling required for the differentiation of epithelia and their mucosal and junctional barrier, resulting in increased susceptibility to ascending infection-mediated preterm birth. Finally the generation of HA disaccharides by bacterial hyaluronidases as made by Group B streptococcus can ligate toll like receptors TLR2/4 thus preventing appropriate inflammatory responses as needed to fight ascending infection and preterm birth. This review summarizes our current understanding of HA's novel and unique roles in cervical remodeling in the process of birth.

Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors.

Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8(+) T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC.

Improving the distribution of Doxil® in the tumor matrix by depletion of tumor hyaluronan.

Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Regulation of cell volume by glycosaminoglycans.

Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra- and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding.

Hyaluronan deficiency in tumor stroma impairs macrophage trafficking and tumor neovascularization.

Despite the importance of stromal cells in tumor progression, our overall understanding of the molecular signals that regulate the complex cellular interactions within tumor stroma is limited. Here, we provide multiple lines of evidence that tumor-associated macrophages (TAM) preferentially traffic to stromal areas formed within tumors in a manner dependent on a hyaluronan (HA)-rich tumor microenvironment. To address the role of stroma-derived HA in macrophage recruitment, we disrupted the HA synthase 2 (Has2) gene in stromal fibroblasts using conditional gene targeting. The Has2 null fibroblasts showed severe impairment in recruiting macrophages when inoculated with tumor cells into nude mice, which shows the contribution of stroma-derived HA in intratumoral macrophage mobilization. Furthermore, a deficiency in stromal HA attenuated tumor angiogenesis and lymphangiogenesis concomitantly with impaired macrophage recruitment. Taken together, our results suggest that stromal HA serves as a microenvironmental signal for the recruitment of TAMs, which are key regulatory cells involved in tumor neovascularization.

Deficiency in the serum-derived hyaluronan-associated protein-hyaluronan complex enhances airway hyperresponsiveness in a murine model of asthma.

BACKGROUND: Serum-derived hyaluronan (HA)-associated proteins (SHAPs), the heavy chains of inter-α-trypsin inhibitor, covalently bind to HA to form the SHAP-HA complex. The SHAP-HA complex is involved in the pathophysiology of inflammatory diseases, including rheumatoid arthritis. We investigated whether this complex is also involved in airway allergy. METHODS: SHAP-HA-deficient (bikunin knockout, KO) mice and wild-type (WT) mice were immunized twice by intraperitoneal injection of ovalbumin (OVA) and exposed to aerosol OVA for 30 min each day for 2 weeks. Twenty-four hours after the final OVA challenge, airway responsiveness to inhaled methacholine (MCh) was measured, and analysis of bronchoalveolar lavage fluid (BALF) and lung histological studies were done. RESULTS: Compared to WT mice, KO mice showed higher airway hyperresponsiveness to inhaled MCh and higher late-phase responses to OVA whereas the early-phase responses were similar. Cell differentials of BALF showed an increased number of macrophages and neutrophils in KO mice. Furthermore, decreased concentrations of soluble tumor necrosis factor receptor-1 (sTNFR1) were found in BALF from KO mice whereas the levels of Th1 and Th2 cytokines were not different from WT mice. Immunochemical study of the lung tissues revealed stronger staining of sTNFR1 in KO than in WT mice. CONCLUSIONS: Our results suggest that in this murine asthma model, the SHAP-HA complex has an inhibitory role in the development of airway hyperresponsiveness and allergic airway inflammation which may be attributed, at least in part, to negative feedback mechanisms exerted by sTNFR1, the shedding of which from the cell surface might also be promoted by the SHAP-HA complex.

Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice.

Changes in the molecular organization of the extracellular matrix are key factors in neuropathology. We investigated aggrecan-based perineuronal nets (PNs) in relation to neurodegeneration and activation of glial cells in a transgenic mouse (Tg2576) model of Alzheimer's disease. The formation of amyloid plaques in the cerebral cortex occurred independently of the area-specific distribution of PNs. Matrix components were only affected in the core of plaques in advanced stages of pathology. PNs remained unchanged in the large marginal zone occupied by reactive astrocytic processes. We conclude that the aggrecan-based extracellular matrix of PNs is not enzymatically altered in peripheral plaque territories and is only removed after neuronal death.

Potential role of high molecular weight hyaluronan in the anti-Candida activity of human oral epithelial cells.

Candida albicans is both a commensal and a pathogen in the oral mucosa. Previous studies have indicated that epithelial cell-associated carbohydrate moiety can inhibit C. albicans growth. In the present study, the mechanisms by which epithelial cells inhibit Candida growth were studied by examining the effect of hyaluronan (HA). A coculture of C. albicans and KB cells or COS-7 cells inhibited in vitro growth of the fungus by 50-87% at an effector-to-target (E:T) ratio of 80:1. Removing extracellular HA by hyaluronidase caused a significant decrease in the anti-Candida activity of the cells. In addition anti-Candida activity was observed at 1 micro g/ml HA (2000 kDa). The antifungal activity of extracellular HA was further studied by transiently transfecting COS-7 cells with human HSA1, HSA2, or HSA3 in order to produce high levels of extracellular HA. All of the transfectants inhibited C. albicans growth in vitro by 51-65% compared to 38% inhibition by the vector control (P<0.05). These results suggest that the anti-Candida activity of epithelial-cells is mediated by extracellular HA.

Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.

Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.

Investigation of hyaluronan function in the mouse through targeted mutagenesis.

It has become increasingly apparent that the high molecular mass glycosaminoglycan, hyaluronan (HA), is required for many morphogenetic processes during vertebrate development. This renewed understanding of the various developmental roles for HA, has come about largely through the advent of gene targeting approaches in the mouse. To date, mutations have been engineered in the enzymes responsible for biosynthesis and degradation and for those proteins that bind to HA within the extracellular matrix and at the cell surface. Collectively, the phenotypes resulting from these mutations demonstrate that HA is critical for normal mammalian embryogenesis and for various processes in postnatal and adult life (Table 1). In this article we will review our progress in understanding the biological functions for HA through targeted mutagenesis of the HA synthase 2 (Has2) and 3 (Has3) genes. Data that has been obtained from a conventional targeted disruption of the Has2 gene, is presented in an accompanying review by Camenisch and McDonald. More specifically, in this review we will provide an overview of the conditional gene targeting strategy being used to create tissue-specific deficiencies in Has2 function, along with our progress in understanding the role for Has3-dependent HA biosynthesis.

Hyaluronan: genetic insights into the complex biology of a simple polysaccharide.

It is appropriate that this review should appear in a volume dedicated to Mert Bernfield. Much of my interest in the cell biology of the extracellular matrix, particularly during development, echoes Mert's pioneering studies. His kind but provocative questioning during meetings is especially missed. The glycosaminoglycan hyaluronan is ubiquitous, and is especially abundant during embryogenesis. Hydrated matrices rich in hyaluronan expand the extracellular space, facilitating cell migration. The viscoelastic properties of hyaluronan are also essential for proper function of cartilage and joints. Recent understanding of hyaluronan biology has benefited from the identification of genes encoding hyaluronan synthases and hyaluronidases, genetic analysis of the roles of hyaluronan during development, elucidation of the biochemical mechanisms of hyaluronan synthesis, and by studies of human genetics and tumors. This review focuses on recent studies utilizing hyaluronan-deficient, gene targeted mice with null alleles for the principal source of hyaluronan during mid-gestation, hyaluronan synthase-2 (has-2).

Hyaluronan reduces migration and proliferation in CHO cells.

Expression of the hyaluronan synthase gene in hyaluronan-deficient CHO cells changed the cell morphology from a spindle shape to a flattened epithelial-type form. Hyaluronan producing CHO cells showed reduced initial cell adhesion, migration, proliferation and density at contact inhibition, but no difference in random migration determined by the Boyden chamber assay. Addition of hyaluronan to the medium of CHO cells reduced migration, proliferation and initial cell adhesion. In contrast, coating the plastic dish with hyaluronan enhanced initial cell adhesion. These results are discussed in the context of the perplexing properties of hyaluronan on cellular functions.

Mutation in csrR global regulator reduces Streptococcus pyogenes internalization.

Transposon (Tn 916) mutagenesis was employed to identify genes in group A streptococcus (GAS) that are involved in bacterial internalization by epithelial cells. One mutant displayed significantly reduced internalization efficiency and was therefore selected for further characterization. The mutant harbored a single Tn 916 insertion in csr, a genetic locus encoding a two-component regulatory system. Mutations in csr were found to derepress hyaluronic acid (HA) capsule synthesis. Since capsule expression has been previously reported to interfere with internalization of GAS, it was possible that the transposon exerted its inhibitory effect either by derepression of capsule synthesis, or by another mechanism. To study the effect of the csr mutation on bacterial internalization, isogenic mutants deficient in either csrR, hasA or both were generated. The hasA mutant adhered to and internalized into HEp-2 cells significantly better than the parent and the csrR mutant strains. The internalization efficiency of the double mutant (csrR(-)/hasA(-)) was reduced by seven-fold compared to that of the hasA mutant. These findings suggest that csrR affects streptococcal entry by modulating capsule expression as well as by another, yet unknown, mechanism.

Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection.

Group A streptococcal (GAS) pharyngitis and the subsequent bacterial colonization of the human throat elicit an immune response that may precipitate acute rheumatic fever in a susceptible host. To study the bacterial determinants that influence throat colonization and induction of humoral immunity, we characterized the behavior of GAS strains in a baboon model. An M-type 3 clinical isolate of GAS typical of strains that cause pharyngitis and invasive infection was recovered from the pharynx of six out of six baboons for at least 6 weeks after oral inoculation. By contrast, an isogenic mutant deficient in M protein failed to colonize most animals or was rapidly cleared. An isogenic mutant deficient in hyaluronic acid capsule colonized five out of six animals, but only persisted in the pharynx for 14-21 days. Colonized animals developed serum antistreptolysin O (SLO) and anti-M protein immunoglobulin (Ig)G. The kinetics of the antibody responses were similar to those seen after human infection. Peak titres increased with the duration of throat carriage. Colonization with GAS prevented recurrent colonization after challenge with the homologous wild-type strain, but not after challenge with a strain of different M protein type. Early clearance of the M protein-deficient strain was associated with increased susceptibility of this strain to phagocytic killing in non-immune serum, whereas clearance of the acapsular strain was associated with increased susceptibility to phagocytic killing in the presence of specific antibody. These studies support critical and distinct effects of the GAS M protein and capsule on throat colonization and induction of humoral immunity in a model that reproduces important features of pharyngeal colonization and immune response following human infection.

Relationship between hyaluronan production and metastatic potential of mouse mammary carcinoma cells.

To investigate the roles of hyaluronan produced by cancer cells in cancer metastasis, the metastatic potential of the highly metastatic mouse mammary carcinoma FM3A HA1 cell line was compared with those of hyaluronan-deficient mutant cells. Five different mutant clones showed markedly reduced hyaluronan production and lacked the ability to form hyaluronan-rich pericellular coats. These mutant clones displayed significant decreases in metastatic ability compared with the parental cells after i.v. injection into syngeneic mice. These results suggested that the decreased hyaluronan production caused not only the lack of matrix formation but also decreased metastatic potential of the cancer cells. Expression of mouse hyaluronan synthase 1 (HAS1) by transfection into HAS- cells defective in hyaluronan synthase activity rescued hyaluronan matrix formation as well as hyaluronan production. Lung metastasis after i.v. injection of HAS1 transfectants was also recovered significantly. The results provide direct evidence for the involvement of hyaluronan in cancer metastasis.

Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium.

1. The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity (Qs) is important for the retention of intra-articular lubricant. The resistance has been attributed in part to extracellular glycosaminoglycans, including hyaluronan and chondroitin sulphates. Increased permeability in joints infused with testicular hyaluronidase, which digests both chondroitin sulphates and hyaluronan, supports this view. In this study the importance of interstitial hyaluronan per se was assessed using leech and Streptomyces hyaluronidases, which degrade only hyaluronan. 2. Ringer solution was infused into the knee joint cavity of anaesthetized rabbits for 30 min, with or without hyaluronidase, after which intra-articular pressure (Pj) was raised and the relation between pressure and outflow determined. 3. Treatment with Streptomyces, leech or testicular hyaluronidases increased the fluid escape rates by similar factors, namely 4- to 6-fold. After Streptomyces hyaluronidase treatment the slope d 8d s/dPj, which at low pressures represents synovial hydraulic conductance, increased from a control of 0.90 +/- 0.20 microl min-1 cmH2O-1 (mean +/- s.e.m. , n = 6) to 4.52 +/- 0.70 microl min-1 cmH2O-1. The slope d 8d s/dPj increased to a similar level after testicular hyaluronidase, namely to 4.14 +/- 1.06 microl min-1 cmH2O-1 (control, 0.54 +/- 0.24 microl min-1 cmH2O-1). Streptomyces and leech hyaluronidases were as effective as testicular hyaluronidase (no statistically significant differences) despite differences in substrate specificity. 4. It was shown using histochemical and immunohistochemical techniques that hyaluronan was removed from the synovium by leech, Streptomyces and testicular hyaluronidases. The binding of antibodies 2-B-6 and 3-B-3 showed that the core proteins of the chondroitin sulphate proteoglycans remained intact after treatment with hyaluronidases, and the binding of 5-D-4 showed that keratan sulphate was unaffected. An azocasein digestion assay confirmed that the hyaluronidase preparations had no significant proteolytic activity. 5. The effect of the hyaluronidases was four times greater than predicted from the low concentration of interstitial hyaluronan and its resistivity. Factors that might amplify the effect of hyaluronan depletion include the matrix-organizing role of hyaluronan, and/or non-uniformity of hyaluronan distribution. It is concluded that interstitial hyaluronan makes a major contribution to synovial hydraulic resistance, but the mechanisms are as yet poorly understood.

The role of hyaluronic acid in joint stability--a hypothesis for hip dysplasia and allied disorders.

The concentration of hyaluronic acid (HA) and proteins in synovial fluids of hip and shoulder joints of a variety of canine breeds has been investigated. In the Australian Kelpie, a working dog with a low incidence of hip dysplasia, shoulder synovial fluid viscosity and HA concentration were higher than in similar joints of Alsatians in which hip dysplasia is relatively common. Moreover, the HA levels and viscosity in shoulder fluids of animals with clinically defined hip dysplasia were substantially lower than in all other breeds studied. On the basis of these findings, we propose that hip dysplasia and other joint abnormalities may arise as a consequence of a deficiency in the levels of HA in synovial fluids.