hymecromone and Inflammation

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.

Topics: Cadherins; Cells, Cultured; Electric Impedance; Female; Gene Expression Regulation; Humans; Hyaluronic Acid; Hymecromone; Inflammation; Lectins, C-Type; Lentivirus; Male; Permeability; Respiratory Mucosa; rho-Associated Kinases; Signal Transduction; Smoking; Transduction, Genetic

4-methylumbelliferone (4MU) is a well-known hyaluronic acid synthesis inhibitor and an approved drug for the treatment of cholestasis. In animal models, 4MU decreases inflammation, reduces fibrosis, and lowers body weight, serum cholesterol, and insulin resistance. It also inhibits tumor progression and metastasis. The broad spectrum of effects suggests multiple and yet unknown targets of 4MU. Aiming at 4MU target deconvolution, we have analyzed publicly available data bases, including: 1. Small molecule library Bio Assay screening (PubChemBioAssay); 2. GO pathway databases screening; 3. Protein Atlas Database. We also performed comparative liver transcriptome analysis of mice on normal diet and mice fed with 4MU for two weeks. Potential targets of 4MU public data base analysis fall into two big groups, enzymes and transcription factors (TFs), including 13 members of the nuclear receptor superfamily regulating lipid and carbohydrate metabolism. Transcriptome analysis revealed changes in the expression of genes involved in bile acid metabolism, gluconeogenesis, and immune response. It was found that 4MU feeding decreased the accumulation of the glycogen granules in the liver. Thus, 4MU has multiple targets and can regulate cell metabolism by modulating signaling via nuclear receptors.

Topics: Animals; Hymecromone; Inflammation; Lipid Metabolism; Liver; Mice; Signal Transduction; Transcriptome

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1β, (IL-1β), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1β and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Astrocytes; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Female; Hyaluronic Acid; Hymecromone; Inflammation; Interleukin-10; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Male; MAP Kinase Signaling System; NF-kappa B; Oxylipins; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Tandem Mass Spectrometry; Tumor Necrosis Factor-alpha

Hyaluronan is a critical component of articular cartilage and partially helps retain aggrecan within the extracellular matrix of this tissue. During osteoarthritis, hyaluronan and aggrecan loss are an early sign of tissue damage. However, our recent attempts to mimic hyaluronan loss with the hyaluronan inhibitor 4-methylumbelliferone (4MU) did not exacerbate arthritis-like features of

Topics: Acetylglucosamine; Acylation; Adenylate Kinase; Animals; Cartilage, Articular; Cattle; Cell Hypoxia; Chondrocytes; Cytoprotection; Deoxyglucose; Dichloroacetic Acid; Energy Metabolism; Female; Glycolysis; Humans; Hyaluronan Synthases; Hymecromone; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-1beta; Male; Matrix Metalloproteinase 13; Middle Aged; Phenotype; Phosphorylation; Signal Transduction

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.

Topics: Animals; Autoimmune Diseases; Diabetes Mellitus, Type 1; Extracellular Matrix; Humans; Hyaluronic Acid; Hydrogels; Hymecromone; Inflammation; Islets of Langerhans; Mice; Microscopy, Atomic Force

Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS.

Topics: Acute Lung Injury; Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Glucuronosyltransferase; Hyaluronan Synthases; Hyaluronic Acid; Hymecromone; Inflammation; Lipopolysaccharides; Lung; Mice; Mice, Inbred C57BL; Pneumonia; Respiratory Distress Syndrome; RNA, Messenger; Spleen

Ischaemia-reperfusion injury (IRI) to the kidney is a complex pathophysiological process that leads to acute renal failure and chronic dysfunction in renal allografts. It was previously demonstrated that during IRI, hyaluronan (HA) accumulates in the cortical and external medullary interstitium along with an increased expression of its main receptor, CD44, on inflammatory and tubular cells. The HA-CD44 pair may be involved in persistent post-ischaemic inflammation. Thus, we sought to determine the role of HA in the pathophysiology of ischaemia-reperfusion (IR) by preventing its accumulation in post-ischaemic kidney.. C57BL/6 mice received a diet containing 4-methylumbelliferone (4-MU), a potent HA synthesis inhibitor. At the end of the treatment, unilateral renal IR was induced and mice were euthanized 48 h or 30 days post-IR.. 4-MU treatment for 14 weeks reduced the plasma HA level and intra-renal HA content at 48 h post-IR, as well as CD44 expression, creatininemia and histopathological lesions. Moreover, inflammation was significantly attenuated and proliferation was reduced in animals treated with 4-MU. In addition, 4-MU-treated mice had a significantly reduced expression of α-SMA and collagen types I and III, i.e. less renal fibrosis, 30 days after IR compared with untreated mice.. Our results demonstrate that HA plays a significant role in the pathogenesis of IRI, perhaps in part through reduced expression of CD44. The suppression of HA accumulation during IR may protect renal function against ischaemic insults.

Topics: Acute Kidney Injury; Animals; Disease Models, Animal; Hyaluronic Acid; Hymecromone; Indicators and Reagents; Inflammation; Kidney Function Tests; Male; Mice; Mice, Inbred C57BL; Reperfusion Injury

A number of 7-hydroxycoumarins have been synthesised by Pechmann cyclisation using differently substituted resorcinols employing perchloric acid as the condensing agent. All the compounds have been characterised by analytical and spectroscopic methods. The anti-inflammatory properties were tested with LPS-induced inflammation in J774 macrophages. Expression of iNOS and COX-2 was determined by Western blot, NO by nitrite assay and IL-6 by ELISA analyses. Fifteen of the tested 7-hydroxycoumarins also inhibited IL-6 production but none of them had any major inhibitory effect on COX-2 expression.

Topics: Animals; Anti-Inflammatory Agents; Blotting, Western; Cell Line; Crystallography, X-Ray; Cyclooxygenase 2; Enzyme-Linked Immunosorbent Assay; Inflammation; Interleukin-6; Lipopolysaccharides; Mice; Nitric Oxide Synthase Type II; Structure-Activity Relationship; Umbelliferones

Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2.

Topics: Aorta; Apoptosis; Cell Movement; Cell Proliferation; Glucose; Glycoproteins; Glycosaminoglycans; Humans; Hyaluronan Receptors; Hyaluronic Acid; Hymecromone; Inflammation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Toll-Like Receptor 4

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature