Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.

BackgroundPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and the number of PDAC-related deaths is rising. Recently the tumour stroma and in particular one of its main components, hyaluronan (HA), have attracted considerable attention as intravenous hyaluronidase treatment together with conventional chemotherapy considerably prolonged survival in HA-rich PDA patients. We therefore wanted to investigate the prognostic significance of HA deposition in PDA using both antibodies to HA and hyaluronan binding protein (HABP).Material and methodsTissue microarrays of PDAs of 184 patients and pancreatic xenografts tumours were immunohistochemically (IHC) stained for HA using either biotinylated hyaluronic acid binding protein (HABP) or anti-HA antibody.ResultsThe pattern of staining with HABP differed significantly from that with antibody IHC. Antibody staining was found both within cancer cells and in the extracellular matrix and staining could not be eliminated by hyaluronidase predigestion of the tissue sections. In contrast, HABP staining was generally confined to the extracellular matrix and was completely abolished by hyaluronidase pretreatment. HA positivity as determined by HABP was associated with larger primary tumours (p = 0.046). There were no correlations between overall survival, disease-free survival and HA expression.ConclusionPresence of HA alone is not of prognostic importance in PDAC, and IHC with utilization of antibody detection shows no reliable staining pattern and should not be applied for HA IHC.

OBJECTIVES:Binding of mononuclear leukocytes to hyaluronan cable structures is a well-known pathomechanism in several chronic inflammatory diseases, but has not yet described for chronic oral inflammations. The aim of this study was to evaluate if and how binding of mononuclear leukocytes to pathologic hyaluronan cable structures can be induced in human gingival fibroblasts.MATERIAL AND METHODS:Experiments were performed with human gingival fibroblasts and peripheral blood mononuclear cells (PBMCs) from three healthy blood donors. Gingival fibroblasts were stimulated with (1) tunicamycin, (2) polyinosinic/polycytidylic acid (Poly:IC), and (3) lipopolysaccharides (LPS) to simulate (1) ER stress and (2) viral and (3) bacterial infections, respectively. Fibroblasts were then co-incubated with PBMCs, and the number of bound and fluorescently labeled PBMCs was assessed using a fluorescence reader and microscopy. For data analysis, a linear mixed model was used.RESULTS:Hyaluronan-mediated binding of PBMCs to gingival fibroblasts was increased by tunicamycin and Poly(I:C) but not by LPS. Hyaluronidase treatment and co-incubation with hyaluronan transport inhibitors reduced this binding.CONCLUSIONS:Results suggest that hyaluronan-mediated binding of blood cells might play a role in oral inflammations. A potential superior role of viruses needs to be confirmed in further clinical studies.CLINICAL RELEVANCE:The linkage between pathological hyaluronan matrices and oral infections opens up potential applications of hyaluronan transport inhibitors in the treatment of chronic oral inflammations.

The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation.

Rheumatoid arthritis an antigen-driven autoimmune disease and the eliciting antigens are unknown. They should be present in immune complexes of synovial fluids. The immune complexes were isolated from synovial fluids of patients with rheumatoid arthritis and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immune complexes contained IgG as major protein. The antibodies dissociated at pH 2.5 and self-aggregated upon neutralization. In ELISA assays, they had the highest specificity to homologous IgG, less to normal IgG and lowest of heterologous arthritic IgG confirming earlier results that altered IgG structures were antigenic. Sequencing of the Fc-regions revealed mutations in all patients analyzed. Therefore, mutations within the Fc-regions of IgG could be antigens in rheumatoid arthritis. We propose that methods should be developed to prove this hypothesis.

BACKGROUND:We have shown previously that some calcium antagonists inhibit hyaluronan export, loss of proteoglycans, and degradation of collagen from osteoarthritic cartilage. Clinically approved calcium antagonists normally are prescribed for cardiac arrhythmia. In the present study, we compared the effect of these drugs on osteoarthritic patients which had received no medication and patients which were also diagnosed for cardiac arrhythmias and were treated with calcium antagonists. The effects and the side effects of the used drugs were analyzed.METHOD:We used the Lequesne questionnaire to examine patients with osteoarthritis (212 patients, control group receiving no calcium antagonists) and patients with cardiac arrhythmia and osteoarthritis (188 patients treated with various calcium antagonists). The answers of the questionnaires were transformed into the Lequesne scoring system quantifying the severity of the disease. The Lequesne score is a standardized questionnaire focused on osteoarthritis. It is a 24-scale questionary in which low scores indicate low functional activity.RESULTS:The data showed that the mean score of the control group (6.2) was higher than the treated group (5.2), the drugs differed in their efficiency. Verapamil had a slightly worse score and Azupamil, Escor, Felodipine, and Nifedipine showed no alteration. Adalat, Amlodipine, Carmen, Nitrendipin, and Norvasc lead to an improvement.CONCLUSION:These results suggest that inhibition of hyaluronan export may have a beneficial effect on human osteoarthritis.

ABSTRACTIn previous publications, we showed that extracellular glycosaminoglycans reduced the membrane potential, caused cell blebbing and swelling and decreased the intracellular pH independently of cell surface receptors. These phenomena were explained by Donnan effects. The effects were so large that they could not be attributed to glycosaminoglycans in solution. Therefore, we tested the hypothesis that glycosaminoglycans were concentrated on the cell membrane and analysed the mechanism of adsorption by fluorescent hyaluronan, chondroitin sulphate and heparin. The influence of the CD44 receptor was evaluated by comparing CD44 expressing human fibroblasts with CD44 deficient HEK cells. Higher amounts of glycosaminoglycans adsorbed to fibroblasts than to HEK cells. When the membrane potential was annihilated by substituting NaCl by KCl in the medium, adsorption was reduced and intracellular pH decrease was abolished. To eliminate other cellular interfering factors, potential‐dependent adsorption was demonstrated for hyaluronan which adsorbed to inert gold foils in physiological salt concentrations at pH 7.2 and surface potentials up to 120 mV. From these results, we conclude that large cellular Donnan effects of glycosaminoglycans results from receptor mediated, hydrophobic and ionic adsorption to cell surfaces. J. Cell. Biochem. 115: 1334–1341, 2014. © 2014 Wiley Periodicals, Inc.

The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico‐chemical properties of intra‐ and extracellular components. We analyzed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF‐AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate, or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding. J. Cell. Physiol. 228: 2071–2075, 2013. © 2013 Wiley Periodicals, Inc.

Hyaluronan is overproduced in many diseases including metastasis, inflammation or ischemia, but there is no drug to attenuate hyaluronan production. Hyaluronan is exported from fibroblasts by the multidrug resistance associated protein 5 (MRP5) which is inhibited by the plant phenols curcumin or xanthohumol. We performed virtual docking and chemical synthesis of analogues to optimize the inhibitors. The AutoDock software was used to identify the binding cavity within the open conformation of MRP5. Inhibitory plant phenols bound to the ATP binding site between the two nucleotide binding domains NBD1 and NBD2. This binding cavity was chosen to screen about 120 derivatives and analogues. The superior hyaluronan export inhibitor was 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (hylin). It inhibited hyaluronan export from fibroblasts with an IC50 of 4.9 μM. Hylin is a minor component in natural curcumin preparations and has previously been described as anti-metastatic and anti-inflammatory. Since curcumin itself is unstable under physiological conditions, the active component for many cell biological and pharmaceutical effects of natural curcumin preparations could be hylin that acts by hyaluronan export inhibition.

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying K-ir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux.

The tumor suppressor disorder neurofibromatosis type 1 (NF1) is associated with development of multiple neurofibromas which may grow intraneurally as plexiform neurofibromas (PNF) or intracutaneously (CNF). Upon surgery neurofibromas may show prominent swelling hindering skin‐edge approximation. To assess whether the water binding glycosaminoglycan hyaluronan is involved in intra‐operative swelling, 51 neurofibromas from 33 NF1‐patients were investigated. Hyaluronan was histologically demonstrated and was quantified by ELISA. Molecular weight of hyaluronan was determined by gel filtration. Further, hyaluronan content was measured in cultivated Schwann cells and fibroblasts. Clinically, 67% of PNF were associated with moderate or severe intra‐operative swelling, whereas only 36% of CNF showed this feature. Significantly higher levels of hyaluronan content were found in PNF compared to CNF (P < 0.05). Mast cell density did not correlate with any of the parameters. Molecular weight of hyaluronan in PNF and CNF ranged from higher than 106 Da to approximately 105 Da. Fibroblasts produced less hyaluronan than Schwann cells. The findings support the view that hyaluronan plays an important role in intra‐operative swelling in neurofibroma surgery.

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2‐(2‐acetamido‐3,5‐dihydroxyphenoxy)‐5‐aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508‐CFTR. It also enhanced the plasma membrane concentration of the ΔF508‐CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508‐CFTR from intracellular degradation and activated its export function. J. Cell. Biochem. 113: 156–164, 2012. © 2011 Wiley Periodicals, Inc.

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. J. Cell. Biochem. 113: 340348, 2012. (C) 2011 Wiley Periodicals, Inc.

Scope: An early reaction in osteoarthritic chondrocytes is hyaluronan overproduction followed by proteoglycan loss and collagen degradation. We recently found that hyaluronan is exported by the ATP-binding cassette transporter multidrug resistance associated protein 5 (MRP5) in competition with cGMP and that some phosphodiesterase 5 inhibitors also inhibited hyaluronan export. These inhibitors also prevented osteoarthritic reactions in cartilage. In an effort to identify the improved inhibitors directed primarily toward MRP5, we analyzed the flavonoidsMethods and results: Prenylflavonoids from hop xanthohumol, isoxanthohumol and 8-prenylnaringenin inhibited MRP5 export at lower concentrations than phosphodiesterase 5 activity. They were analyzed for their effect on IL-induced osteoarthritic reactions in bovine chondrocytes. Xanthohumol was the superior compound to inhibit hyaluronan export, as well as proteoglycan and collagen loss. It also prevented the shedding of metalloproteases into the culture medium. It directly inhibited MRP5, because it reduced the export of the MRP5 substrate fluorescein immediately and did not influence the hyaluronan synthase activity.Conclusions: Xanthohumol may be a natural compound to prevent hyaluronan overproduction and subsequent reactions in osteoarthritis.

Objectives: Hyaluronan, a major water binding component of the extracellular matrix, is synthesised within the cytosol and exported across the plasma membrane by the ABC-transporter MRP5 in fibroblasts. Although its synthesis is vital for embryogenesis, MRP5-deficient mice are without phenotype, suggesting that another transporter had substituted for the MRP5 protein. Thus, we searched for a compensatory exporter in fibroblasts from MRP5 deficient mice and found that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was upregulated. Methods: Hyaluronan export was measured in cell culture. The CFTR transporter was knocked out using si-RNA. Blockers of the ABC-transporter family were used to ascertain the hyaluronan transport capabilities functionally. Results: CFTR specific siRNA inhibited hyaluronan export. The tetrasaccharide was exported in undegraded form only from normal human epithelial cells and not from human epithelial cells carrying ΔF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR172 reduced hyaluronan export from CFTR-expressing mouse fibroblasts and from human breast cancer cell lines. Bronchial secretions from patients with cystic fibrosis that consist mainly of necrotic epithelia contained at least 40-fold higher concentration of hyaluronan than secretions from patients with acute bronchitis. Conclusions: CFTR transports hyaluronan across the plasma membrane of epithelial cells and this transport mechanism is defective in cystic fibrosis patients.

The membrane potential is mainly maintained by the K + concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high‐molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration‐dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high‐molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high‐molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high‐molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism. J. Cell. Biochem. 111: 858–864, 2010. © 2010 Wiley‐Liss, Inc.

BACKGROUND:Osteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation.METHODS:To test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1alpha to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil.RESULTS:These drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation.CONCLUSION:Inhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage.

When secreted from malignant cells, hyaluronan facilitates tumor invasion and metastasis, as inhibition of its export by zaprinast inhibited metastasis formation in mice. However, the precise steps of the metastatic cascade, which were influenced by zaprinast, have not been identified as yet. Here we analyzed the cell biological effects of the inhibitor on three human melanoma cell lines that differed in their hyaluronan production and their metastatic capability when xenografted into SCID mice. We measured the influence of zaprinast on cellular hyaluronan export, surface coat formation, proliferation, random migration, colony formation in soft agar, adhesion, and transepithelial resistance. Concentrations of zaprinast not affecting cell proliferation, adhesion and transepithelial resistance, nevertheless reduced hyaluronan export by 50%, surface coat formation, random migration, and colony formation in soft agar. These results indicate that hyaluronan enhances metastasis formation primarily in those steps of the metastatic cascade, which involves tumor cell migration. J. Cell. Biochem. 105: 1260–1266, 2008. © 2008 Wiley‐Liss, Inc.