Molecular Mass Hyaluronan Research Articles

Ovarian stiffness increases with age in the mammalian ovary and depends on collagen and hyaluronan matrices.

Fibrosis is a hallmark of aging tissues which often leads to altered architecture and function. The ovary is the first organ to show overt signs of aging, including increased fibrosis in the ovarian stroma. How this fibrosis affects ovarian biomechanics and the underlying mechanisms are unknown. Using instrumental indentation, we demonstrated a quantitative increase in ovarian stiffness, as evidenced by an increase in Young's modulus, when comparing ovaries from reproductively young (6–12 weeks) and old (14–17 months) mice. This ovarian stiffness was dependent on collagen because ex vivo enzyme‐mediated collagen depletion in ovaries from reproductively old mice restored their collagen content and biomechanical properties to those of young controls. In addition to collagen, we also investigated the role of hyaluronan (HA) in regulating ovarian stiffness. HA is an extracellular matrix glycosaminoglycan that maintains tissue homeostasis, and its loss can change the biomechanical properties of tissues. The total HA content in the ovarian stroma decreased with age, and this was associated with increased hyaluronidase (Hyal1) and decreased hyaluronan synthase (Has3) expression. These gene expression differences were not accompanied by changes in ovarian HA molecular mass distribution. Furthermore, ovaries from mice deficient in HAS3 were stiffer compared to age‐matched WT mice. Our results demonstrate that the ovary becomes stiffer with age and that both collagen and HA matrices are contributing mechanisms regulating ovarian biomechanics. Importantly, the age‐associated increase in collagen and decrease in HA are conserved in the human ovary and may impact follicle development and oocyte quality.

A guide to hyaluronan and related enzymes in breast cancer: biological significance and diagnostic value.

Hyaluronan (HA) is a unique nonsulfated glycosaminoglycan that contributes to breast cancer cells growth and functional properties, including cell migration, invasion, adhesion, as well as tumor-associated angiogenesis in different stages of breast cancer progression and especially metastasis. Latest data show that the levels of HA and/or low molecular mass HA in blood serum and plasma of breast cancer patients may be a useful biomarker for breast cancer prognosis, differential diagnosis, and patients' treatment monitoring. Therefore, the qualitative and quantitative determination of HA in biological samples is an emerging area of research. This review gathers, categorizes, and sums up all the currently used methodologies to analyze HA and HA-related enzymes. The advantages, disadvantages, limitations in use, and the information they provide, are critically considered and discussed. Moreover, emphasis is given to the significance of HA determination in breast cancer, as well as of its related enzymes, for diagnosis and prognosis of this type of cancer.

Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy.

During the development of hypertrophic cardiomyopathy, the heart returns to fetal energy metabolism where cells utilize more glucose instead of fatty acids as a source of energy. Metabolism of glucose can increase synthesis of the extracellular glycosaminoglycan hyaluronan, which has been shown to be involved in the development of cardiac hypertrophy and fibrosis. The aim of this study was to investigate hyaluronan metabolism in cardiac tissue from patients with hypertrophic cardiomyopathy in relation to cardiac growth. NMR and qRT-PCR analysis of human cardiac tissue from hypertrophic cardiomyopathy patients and healthy control hearts showed dysregulated glucose and hyaluronan metabolism in the patients. Gas phase electrophoresis revealed a higher amount of low molecular mass hyaluronan and larger cardiomyocytes in cardiac tissue from patients with hypertrophic cardiomyopathy. Histochemistry showed high concentrations of hyaluronan around individual cardiomyocytes in hearts from hypertrophic cardiomyopathy patients. Experimentally, we could also observe accumulation of low molecular mass hyaluronan in cardiac hypertrophy in a rat model. In conclusion, the development of hypertrophic cardiomyopathy with increased glucose metabolism affected both hyaluronan molecular mass and amount. The process of regulating cardiomyocyte size seems to involve fragmentation of hyaluronan.

Methods for Hyaluronan Molecular Mass Determination by Agarose Gel Electrophoresis.

The average molecular mass of hyaluronan (HA) in most healthy biological fluids and tissues is usually about 6000-8000kDa, but the biosynthetic mechanism results in a polydisperse mixture of sizes. Subsequent enzymatic degradation, or the action of reactive oxygen and nitrogen species, can further increase polydispersity and decrease the average size. Fragmented HA can be a biomarker of inflammation. In addition, reductions in HA size are associated with tissue remodeling and repair processes. Some cell-surface receptor proteins have been reported to have HA-binding affinities that are size specific, and participate in activation of signaling cascades controlling multiple aspects of cell behavior. Here we describe simple agarose gel electrophoresis protocols for the determination of the molecular mass distribution of HA isolated from tissues and fluids.

Hyaluronan in vocal folds and false vocal folds in patients with recurrent respiratory papillomatosis

Background: Hyaluronan (HA) is a glycosaminoglycan with viscoelastic properties necessary for vocal fold (VF) vibration and voice production. Changes in HAs molecular mass, possibly related to human papilloma virus, could affect formation/persistence of recurrent respiratory papillomatosis (RRP). Aims/Objective: Describing mass and localization of HA and localization of HA receptor CD44 in VF and false vocal folds (FVF) in RRP. Materials and Methods: Biopsies from VF and FVF from 24 RRP patients. Twelve were studied with histo-/immunohistochemistry for HA and CD44 in epithelium, stroma and RRP lesions. Twelve samples were analyzed for HA molecular mass distribution with gas-phase-electrophoretic-molecular-mobility-analyzer (GEMMA). Results: Three of 23 stains (VF and FVF combined) showed faint HA staining in the epithelium; there was more extensive staining in the stroma. CD44 was present throughout all areas in FVF and VF, it did not concur with HA. GEMMA analysis revealed very high mass HA (vHMHA) with more varying amounts in VF. Conclusions/Significance: HA was mainly distributed in the stroma. CD44 not binding to HA might explain the non-inflammatory response described in RRP. Possibly crosslinked vHMHA was seen in VF and FVF, with more variable amounts in VF samples. Counteracting HA crosslinking could become a treatment option in RRP.

Fragmented hyaluronan has no alarmin function assessed in arthritis synovial fibroblast and chondrocyte cultures.

Hyaluronan (HA) is a large polymer and an important component of the extracellular matrix. During homeostasis, high molecular mass HA is the predominant form, but upon inflammation, degradation products of HA accumulate. These HA fragments (HA-fs) have been reported to possess pro-inflammatory activities and thus act as alarmins, notifying immune cells of danger via TLR4 and CD44. HA is found in large quantities in synovial joint fluid. In order to reveal a potential role of HA-fs in arthritis pathogenesis, the in vitro effects of HA of various molecular masses (from 1680 kDa to oligosaccharide HA) on synovial fibroblasts and chondrocytes from rheumatoid arthritis patients, and on peripheral blood mononuclear cells from healthy donors, were investigated. TLR4 and CD44 surface expression was confirmed by immunocytochemistry, and cell activation was determined based on cytokine and chemokine production. While the cell types investigated expressed TLR4 and CD44, no increased release of IL-1ß, IL-6, IL-8, IL-10, IL-12 or TNF-α was detected after HA stimulation. Similarly, HA did not enhance activation after priming cells with low doses of LPS or by forming complexes with LPS. Hence, this study does not support the common view of HA-fs being pro-inflammatory mediators and it is not likely that HA-fs generated during arthritis contribute to disease pathogenesis.

A competitive alphascreen assay for detection of hyaluronan

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

Intermediate Molecular Mass Hyaluronan and CD44 Receptor Interactions Enhance Neutrophil Phagocytosis and IL-8 Production via p38- and ERK1/2-MAPK Signalling Pathways.

CD44 is a common leukocyte adhesion molecule expressed on the surface of various cells. Hyaluronan (HA), the natural ligand of CD44, is a simple repeated disaccharide with variable molecular mass that is widely distributed on cell surfaces and the connective tissue matrix. The binding of small molecular mass HA (SMM-HA, MW<80kDa) to CD44 on immune-related cells elicits cell proliferation, differentiation, and cytokine production. However, the effects and molecular basis of intermediate molecular mass HA (IMM-HA, MW≈500kDa)-CD44 interactions on polymorphonuclear neutrophil (PMN) functions have not been elucidated. We hypothesised that IMM-HA would potentiate immune functions as well as SMM-HA. In the present study, we demonstrated IMM-HA and CD44 interactions enhanced normal PMN phagocytosis and IL-8 production compared to those with LPS or anti-CD45 treatment via F-actin cytoskeleton polymerization and subsequent ERK1/2- and p38-MAPK phosphorylation. Antibody-based inhibition of CD44 did not affect PMN function; however, F-actin aggregation was induced without MAPK phosphorylation. Enhanced PMN function via IMM-HA was determined to be CD44-dependent since this effect was abolished in DMSO-induced CD44(-) PMN-like cells obtained from HL-60 cells. In conclusion, we demonstrated that IMM-HA and CD44 interactions on PMNs potently elicit F-actin cytoskeleton polymerization and p38- and ERK1/2-MAPK phosphorylation to enhance PMN function.

Effect of hyaluronan on osteoclast differentiation in mouse bone marrow-derived cells cocultured with primary osteoblasts

Glycosaminoglycans (GAG) are major components of the extracellular matrix present in many tissues. In bone, hyaluronan (HA) accounts for 4−7% of total GAG. GAGs have both structural and functional roles in the regulation of biological processes including cellular growth, migration, and differentiation. GAGs also enhance the biological activity of bone morphogenetic proteins (BMPs). HA hydrogel-delivered BMP-2 precomplexed with dermatan sulfate or heparin can induce bone formation in vivo. 8 Furthermore, biglycan regulates osteoclast differentiation through its effect on osteoblasts and their precursors. HA is an unsulfated polymer of repeating D-glucuronic acid and N-acetylglucosamine disaccharide units which regulates cell proliferation, cell motility, and tissue repair. It is a widely used as a diseasemodifying drug for reducing pain associated with osteoarthritis. Evidence for the efficacy of HA in reducing pain has been demonstrated in animal experimental models, in vitro experiments, 21 and clinical trials. Several studies have focused on the possibility that bone regeneration could be induced by the presence of a HA scaffold. 23, 24 For example, HA enhanced BMP-2 induction of osteoblastic differentiation in osteoblast-like cells. 25, 26 The properties of HA differ depending on the molecular mass of the polymer. 27, 28 Huang et al. reported that low molecular mass HA (60 kDa) increased cell growth and osteocalcin mRNA expression, while high molecular mass HA (900 or 2300 kDa) stimulated ALP activity and cell mineralization in rat calvarial osteoblasts. Sasaki et al. applied high molecular mass HA (1900 kDa) to bone wounds following bone marrow ablation and observed maintenance of osteoinductive growth factors within the local environment and accelerated new bone formation via mesenchymal cell differentiation within the wound. However, Ariyoshi et al. reported that low molecular mass HA (<8 kDa) elevated tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation, while high molecular mass HA (2500 kDa) down-regulated differentiation of osteoclast-like celEffect of hyaluronan on osteoclast differentiation in mouse bone marrow-derived cells cocultured with primary osteoblasts

Hyaluronan stimulates ex vivo B lymphocyte chemotaxis and cytokine production in a murine model of fungal allergic asthma

Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive eosinophilic and lymphocytic inflammation with associated changes in the extracellular matrix (ECM) resulting in airway wall remodeling. Hyaluronan (HA) is a nonsulfated glycosaminoglycan ECM component that functions as a structural cushion in its high molecular mass (HMM) but has been implicated in metastasis and other disease processes when it is degraded to smaller fragments. However, relatively little is known about the role HA in mediating inflammatory responses in allergy and asthma. In the present study, we used a murine Aspergillus fumigatus inhalational model to mimic human disease. After observing in vivo that a robust B cell recruitment followed a massive eosinophilic egress to the lumen of the allergic lung and corresponded with the detection of low molecular mass HA (LMM HA), we examined the effect of HA on B cell chemotaxis and cytokine production in the ex vivo studies. We found that LMM HA functioned through a CD44-mediated mechanism to elicit chemotaxis of B lymphocytes, while high molecular mass HA (HMM HA) had little effect. LMM HA, but not HMM HA, also elicited the production of IL-10 and TGF-β1 in these cells. Taken together, these findings demonstrate a critical role for ECM components in mediating leukocyte migration and function which are critical to the maintenance of allergic inflammatory responses.

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M⩽20kDa, M∼20 to 60kDa, and M∼60 to 110kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M⩾110kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.

Hyaluronan Breakdown Contributes to Immune Defense against Group A Streptococcus

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.

Changes in epidermal hyaluronan metabolism following UVB irradiation

Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation, and have shown different biological activities depending on its molecular mass. While many studies have shown changes in the amount of HA after UVB irradiation, molecular mass change remains to be elucidated. To investigate the change in the molecular mass of HA after UVB irradiation in mouse epidermis. The mice were irradiated with a single dose of UVB (0.15J/cm(2)). The amount of HA was examined using HABP sandwich assay. The molecular mass distribution was estimated by Sephacryl S-1000 chromatography. Has and Hyal mRNA expressions were detected by real-time PCR. On day 2 after UVB irradiation, both the amount of HA and the up-regulation of Has3 mRNA expression reached their maximum. The average HA molecular mass was about 1000 kDa, a level similar to that of the non-irradiated epidermis. On day 3, the average HA molecular mass drastically decreased to 100 kDa, while Hyal1, Hyal2, and Hyal3 mRNA expressions slightly increased. The amount of HA, however, remained high. On days 4 and 5, the amount of HA gradually decreased, but the molecular mass of HA remained low. A drastic reduction of the HA molecular mass after UVB irradiation was confirmed. UVB irradiation elicits remarkable changes in the molecular mass of HA, as well as amount. These qualitative and quantitative changes of HA might play an important role in UVB-induced cell proliferation and differentiation. Further study will be required to resolve the mechanism of HA degradation in the epidermis.

Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000kDa. A sample load of 2.5μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.

Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22kDa at sample loads of 0.5μg (for polyacrylamide) to 2.5μg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1–IGD–G2 domains) to a 150-kDa HA standard.

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor-2 (FGF-2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF-2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF-2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF-2-stimulated migration of MPDL22 cells. Moreover, in response to FGF-2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)-1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF-2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti-CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF-2. Furthermore, an anti-CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF-2-induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF-2-mediated cell motility of PDL cells, suggesting that FGF-2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production.

Reactive Oxygen Species and Hyaluronidase 2 Regulate Airway Epithelial Hyaluronan Fragmentation

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

The enzymatic degradation of hyaluronan is associated with disease progression in experimental pulmonary hypertension

Hyaluronan (HA) degradation fragments have been linked to inflammation in a wide range of lung diseases. In idiopathic pulmonary arterial hypertension, HA accumulation has been associated with advanced disease. In this study, we investigated the potential role of HA degradation in the early stages of disease by examining HA distribution, molecular mass, synthesis, and enzymatic degradation at different stages of disease progression in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). At 28 days post-MCT, severe PH was associated with increased total lung HA (P = 0.04). In contrast, a significant decrease in total lung HA was observed on day 10, before the onset of PH (P = 0.02). Molecular mass analysis revealed a loss of high molecular mass (HMM) HA at 10 and 24 days post-MCT, followed by an increase in HMM HA at 28 days. Expression of HA synthase 2 (HAS2) was elevated in MCT-challenged animals at 24 and 28 days, consistent with increased synthesis of HMM HA. Analysis by Morgan Elson assay and zymography demonstrated increased hyaluronidase-1 activity in the lungs of MCT-challenged rats, indicating that the observed increases in HAS2 expression and HA synthesis were counterbalanced, in part, by enhanced degradation. The present data demonstrate that, in the MCT model, early-stage PH is associated with enhanced hyaluronidase-1 activity, while both degradation and synthesis are increased at later stages. Thus an early increase in the generation of proinflammatory HA fragments may play a role in the onset and progression of pulmonary arterial hypertension.

Hyaluronan Accumulation Is Elevated in Cultures of Low Density Lipoprotein Receptor-deficient Cells and Is Altered by Manipulation of Cell Cholesterol Content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Hyaluronan Binding to Link Module of TSG-6 and to G1 Domain of Aggrecan Is Differently Regulated by pH

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 to HA was maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The K(D) determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1.