Heparin and calreticulin interact on the surface of early G0/G1 dividing rat mesangial cells to regulate hyperglycemic glucose-induced responses.

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6 h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at ∼70 kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division.

Monocyte adhesive hyaluronan matrix induced by hyperglycemia in diabetic lung injuries.

Infiltrated pre-inflammatory monocytes and macrophages have important roles in the induction of diabetic lung injuries, but the mechanism mediating their infiltration is still unclear. Here, we showed that airway smooth muscle cells (SMCs) activated monocyte adhesion in response to hyperglycemic glucose (25.6 mM) by significantly increasing hyaluronan (HA) in the cell matrix, with concurrent 2- to 4-fold increases in adhesion of U937 monocytic-leukemic cells. The HA-based structures were attributed directly to the high-glucose and not to increased extracellular osmolality, and they required growth stimulation of SMCs by serum. Treatment of SMCs with heparin in high-glucose induces synthesis of a much larger HA matrix, consistent with our observations in the glomerular SMCs. Further, we observed increases in tumor necrosis factor-stimulated gene-6 (TSG-6) expression in high-glucose and high-glucose plus heparin cultures, and the heavy chain (HC)-modified HA structures existed on the monocyte-adhesive cable structures in high-glucose and in high-glucose plus heparin-treated SMC cultures. Interestingly, these HC-modified HA structures were unevenly distributed along the HA cables. Further, the in vitro assay with recombinant human TSG-6 and the HA14 oligo showed that heparin has no inhibitory activity on the TSG-6-induced HC-transfer to HA, consistent with the results from SMC cultures. These results support the hypothesis that hyperglycemia in airway smooth muscle induces the synthesis of a HA matrix that recruits inflammatory cells and establishes a chronic inflammatory process and fibrosis that lead to diabetic lung injuries.

Transforming Growth Factor-ß Receptor-Mediated, p38 Mitogen-Activated Protein Kinase-Dependent Signaling Drives Enhanced Myofibroblast Differentiation during Skin Wound Healing in Mice Lacking Hyaluronan Synthases 1 and 3.

Normal myofibroblast differentiation is critical for proper skin wound healing. Neoexpression of α-smooth muscle actin (α-SMA), a marker for myofibroblast differentiation, is driven by transforming growth factor (TGF)-ß receptor-mediated signaling. Hyaluronan and its three synthesizing enzymes, hyaluronan synthases (Has 1, 2, and 3), also participate in this process. Closure of skin wounds is significantly accelerated in Has1/3 double-knockout (Has1/3-null) mice. Herein, TGF-ß activity and dermal collagen maturation were increased in Has1/3-null healing skin. Cultures of primary skin fibroblasts isolated from Has1/3-null mice had higher levels of TGF-ß activity, α-SMA expression, and phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182, compared with wild-type fibroblasts. p38α mitogen-activated protein kinase was a necessary element in a noncanonical TGF-ß receptor signaling pathway driving α-SMA expression in Has1/3-null fibroblasts. Myocardin-related transcription factor (MRTF), a cofactor that binds to the transcription factor serum response factor (SRF), was also critical. Nuclear localization of MRTF was increased, and MRTF binding to SRF was enhanced in Has1/3-null fibroblasts. Inhibition of MRTF or SRF expression by RNA interference suppresses α-SMA expression at baseline and diminished its overexpression in Has1/3-null fibroblasts. Interestingly, total matrix metalloproteinase activity was increased in healing skin and fibroblasts from Has1/3-null mice, possibly explaining the increased TGF-ß activation.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

Heparin inhibits proinflammatory and promotes anti-inflammatory macrophage polarization under hyperglycemic stress.

Monocytes are rapidly recruited to sites of diabetic complications and differentiate into macrophages. Previously, we showed that rat kidney mesangial cells dividing during hyperglycemic stress abnormally synthesize hyaluronan (HA) in intracellular compartments. This initiates a stress response, resulting in an extracellular HA matrix after division that recruits inflammatory cells. Cell-cell communication among macrophages that are recruited into the glomeruli and the damaged rat mesangial cells leads to diabetic nephropathy, fibrosis, and proteinurea, which are inhibited in heparin-treated diabetic rats. In this study, we found that murine bone marrow-derived macrophages (BMDMs) and a human leukemic cell line, U937 cells, dividing in hyperglycemia also accumulate intracellular HA and that heparin inhibits the HA accumulation. Both cell types expressed increased levels of proinflammatory markers: inducible nitric-oxide synthase and tumor necrosis factor-α, when cultured under hyperglycemic stress, which was inhibited by heparin. Furthermore, the abnormal intracellular HA was also observed in peripheral blood monocytes derived from three different hyperglycemic diabetic mouse models: streptozotocin-treated, high-fat fed, and Ins2Akita. Moreover, peripheral blood monocytes in humans with type 2 diabetes and poorly controlled blood glucose levels (hemoglobin A1c (HbA1c) levels of >7) also had intracellular HA, whereas those with HbA1c of <7, did not. Of note, heparin increased the anti-inflammatory markers arginase 1 and interleukin-10 in murine BMDMs. We conclude that heparin treatment of high glucose-exposed dividing BMDMs promotes an anti-inflammatory tissue-repair phenotype in these cells.

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2-AS1.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO- group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

Anti-inflammatory protein TNFα-stimulated gene-6 (TSG-6) reduces inflammatory response after brain injury in mice.

BACKGROUND: Current research suggests that the glial scar surrounding penetrating brain injuries is instrumental in preserving the surrounding uninjured tissue by limiting the inflammatory response to the injury site. We recently showed that tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a well-established anti-inflammatory molecule, is present within the glial scar. In the present study we investigated the role of TSG-6 within the glial scar using TSG-6 null and littermate control mice subjected to penetrating brain injuries. RESULTS: Our findings show that mice lacking TSG-6 present a more severe inflammatory response after injury, which was correlated with an enlarged area of astrogliosis beyond the injury site. CONCLUSION: Our data provides evidence that TSG-6 has an anti-inflammatory role within the glial scar.

Hyaluronidase-2 Regulates RhoA Signaling, Myofibroblast Contractility, and Other Key Profibrotic Myofibroblast Functions.

Hyaluronidase (HYAL)-2 is a weak, acid-active, hyaluronan-degrading enzyme broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive; thus the mechanisms through which HYAL2 dysregulation influences pathobiology are unclear. Recently, nonenzymatic HYAL2 functions have been described, and nuclear HYAL2 has been shown to influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts, HYAL2 interacts with the GTPase-signaling small molecule ras homolog family member A (RhoA). Transforming growth factor beta 1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic relocalization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences profibrotic myofibroblast functions, including myosin light-chain kinase-mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor and matrix metalloproteinase-2 expression. These data demonstrate that, in certain biological contexts, the nonenzymatic effects of HYAL2 are crucial in orchestrating RhoA signaling and downstream pathways that are important for full profibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2.

FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.

Cancer initiating cells (CICs) drive tumor formation and drug-resistance, but how they develop drug-resistance characteristics is not well understood. In this study, we demonstrate that chemotherapeutic agent FOLFOX, commonly used for drug-resistant/metastatic colorectal cancer (CRC) treatment, induces overexpression of CD44v6, MDR1, and oncogenic transcription/translation factor Y-box-binding protein-1 (YB-1). Our study revealed that CD44v6, a receptor for hyaluronan, increased the YB-1 expression through PGE2/EP1-mTOR pathway. Deleting CD44v6, and YB-1 by the CRISPR/Cas9 system attenuates the in vitro and in vivo tumor growth of CICs from FOLFOX resistant cells. The results of DNA:CD44v6 immunoprecipitated complexes by ChIP (chromatin-immunoprecipitation) assay showed that CD44v6 maintained the stemness traits by promoting several antiapoptotic and stemness genes, including cyclin-D1, BCL2, FZD1, GINS-1, and MMP9. Further, computer-based analysis of the clones obtained from the DNA:CD44v6 complex revealed the presence of various consensus binding sites for core stemness-associated transcription factors "CTOS" (c-Myc, TWIST1, OCT4, and SOX2). Simultaneous expressions of CD44v6 and CTOS in CD44v6 knockout CICs reverted differentiated CD44v6-knockout CICs into CICs. Finally, this study for the first time describes a positive feedback loop that couples YB-1 induction and CD44 alternative splicing to sustain the MDR1 and CD44v6 expressions, and CD44v6 is required for the reversion of differentiated tumor cells into CICs.

The journey of hyaluronan research in the Journal of Biological Chemistry.

Hyaluronan has a very simple structure. It is a linear glycosaminoglycan composed of disaccharide units of GlcNAc and d-glucuronic acid with alternating ß-1,4 and ß-1,3 glycosidic bonds that can be repeated 20,000 or more times, a molecular mass >8 million Da, and a length >20 µm. However, it has a very complex biology. It is a major, ubiquitous component of extracellular matrices involved in everything from fertilization, development, inflammations, to cancer. This JBC Review highlights some of these processes that were initiated through publications in the Journal of Biological Chemistry.

Heparin affects cytosolic glucose responses of hyperglycemic dividing mesangial cells.

Mesangial expansion underlies diabetic nephropathy, leading to sclerosis and renal failure. The glycosaminoglycan heparin inhibits mesangial cell growth, but the molecular mechanism is unclear. Here, rat mesangial cells (RMCs) were growth-arrested in the G0/G1 phase of cell division, stimulated to divide in normal glucose (5.6 mm) or high glucose (25.6 mm) with or without heparin, and analyzed for glucose uptake. We observed that RMCs entering the G1 phase in normal glucose with or without heparin rapidly cease glucose uptake. RMCs entering G1 in high glucose sustained glucose uptake for the first 3 h, and high-glucose exposure of RMCs only in the first 8 h of G1 induced the formation of an extracellular monocyte-adhesive hyaluronan matrix after cell division was completed. Moreover, a low heparin concentration under high-glucose conditions blocked glucose uptake by 1 h into G1 Of note, glucose transporter 4 (glut4) localized on the RMC surface at G0/G1 and was internalized into G1 cells under normal glucose conditions with or without heparin within 30 min. We also noted that, under high-glucose conditions, glut4 remained on the RMC surface for at least 2 h into G1 and was internalized by 4 h without heparin and within 1 h with heparin. These results provide evidence that the influx of glucose in hyperglycemic dividing RMCs initiates intermediate glucose metabolism, leading to increased cytosolic UDP sugars, and induces abnormal intracellular hyaluronan synthesis during the S phase of cell division.

Transforming growth factor ß1 (TGFß1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts.

The appearance of myofibroblasts is generally thought to be the underlying cause of the fibrotic changes that underlie idiopathic pulmonary fibrosis. However, the cellular/molecular mechanisms that account for the fibroblast-myofibroblast differentiation/activation in idiopathic pulmonary fibrosis remain poorly understood. We investigated the functional role of hyaluronan receptor CD44V6 (CD44 containing variable exon 6 (v6)) for differentiation of lung fibroblast to myofibroblast phenotype. Increased hyaluronan synthesis and CD44 expression have been detected in numerous fibrotic organs. Previously, we found that the TGFß1/CD44V6 pathway is important in lung myofibroblast collagen-1 and α-smooth-muscle actin synthesis. Because increased EGR1 (early growth response-1) expression has been shown to appear very early and nearly coincident with the expression of CD44V6 found after TGFß1 treatment, we investigated the mechanism(s) of regulation of CD44V6 expression in lung fibroblasts by TGFß1. TGFß1-mediated CD44V6 up-regulation was initiated through EGR1 via ERK-regulated transcriptional activation. We showed that TGFß1-induced CD44V6 expression is through EGR1-mediated AP-1 (activator protein-1) activity and that the EGR1- and AP-1-binding sites in the CD44v6 promoter account for its responsiveness to TGFß1 in lung fibroblasts. We also identified a positive-feedback loop in which ERK/EGR1 signaling promotes CD44V6 splicing and found that CD44V6 then sustains ERK signaling, which is important for AP-1 activity in lung fibroblasts. Furthermore, we identified that HAS2-produced hyaluronan is required for CD44V6 and TGFßRI co-localization and subsequent CD44V6/ERK1/EGR1 signaling. These results demonstrate a novel positive-feedback loop that links the myofibroblast phenotype to TGFß1-stimulated CD44V6/ERK/EGR1 signaling.

Transforming growth factor ß1 (TGFß1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFß1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-ß-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFß1/TGFßRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

BACKGROUND AIMS: Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). METHODS: Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (PCTP) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. RESULTS: Mean [Cell], [CTP] and PCTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm2; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. CONCLUSIONS: The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies.

Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC-HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors--CD44, TLR2, TLR4--were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC-HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC-HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. Significance statement: Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation of the periventricular white matter, which results in arrested maturation of OPCs and myelination failure. HA is a major component of the extracellular matrix of the brain, which regulates inflammation through CD44 and TLR2/4 receptors. Here, we show two mechanism-based strategies that effectively enhanced myelination and neurological recovery in preterm rabbit model of IVH. First, degrading HA by hyaluronidase treatment reduced CD44 and TLR4 expression, proinflammatory cytokines, and microglial infiltration, as well as promoted oligodendrocyte maturation and myelination. Second, intraventricular injection of HA oligosaccharide reduced inflammation and enhanced myelination, conceivably by depleting HC-HA levels.

Hyaluronan Rafts on Airway Epithelial Cells.

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan (HA) cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables but occur in distinct sheets or rafts that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has two heavy chains (HC1 and HC2) on its chondroitin sulfate chain. The transesterification transfer of HCs from chondroitin sulfate to HA is mediated by tumor necrosis factor-induced gene 6 (TSG-6), which is up-regulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage of naïve mice and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Is Constitutively Expressed in Adult Central Nervous System (CNS) and Associated with Astrocyte-mediated Glial Scar Formation following Spinal Cord Injury.

Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) binds to hyaluronan and can reorganize/stabilize its structure, also enhancing the binding of this glycosaminoglycan to its cell surface receptor, CD44. TSG-6 is rapidly up-regulated in response to inflammatory cytokines protecting tissues from the damaging effects of inflammation. Despite TSG-6 treatment having been shown to improve outcomes in an experimental model of traumatic brain injury, TSG-6 expression has not been extensively studied in the central nervous system (CNS). We hereby analyzed the expression profile of TSG-6 in the developing CNS and following injury. We show that TSG-6 is expressed in the rat CNS by GFAP(+) and CD44(+) astrocytes, solely in the mature brain and spinal cord, and is not present during the development of the CNS. TSG-6(-/-) mice present a reduced number of GFAP(+) astrocytes when compared with the littermate TSG-6(+/-) mice. TSG-6 expression is drastically up-regulated after injury, and the TSG-6 protein is present within the glial scar, potentially coordinating and stabilizing the formation of this hyaluronan-rich matrix. This study shows that TSG-6 is expressed in the CNS, suggesting a role for TSG-6 in astrocyte activation and tissue repair. We hypothesize that within this context TSG-6 could participate in the formation of the glial scar and confer anti-inflammatory properties. Further studies are required to elucidate the therapeutic potential of targeting TSG-6 after CNS injury to promote its protective effects while reducing the inhibitory properties of the glial scar in axon regeneration.

The Responses of Hyperglycemic Dividing Mesangial Cells to Heparin Are Mediated by the Non-reducing Terminal Trisaccharide.

Our previous studies showed: (i) that growth-arrested G0/G1 rat mesangial cells stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy and the cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division; and (ii) that heparin inhibits the intracellular hyaluronan and autophagy responses, but after completing division, induces hyaluronan synthesis at the plasma membrane with the formation of a larger monocyte-adhesive hyaluronan matrix. This study shows: (i) that the non-terminal trisaccharide of heparin is sufficient to initiate the same responses as intact heparin, (ii) that a fully sulfated tetrasaccharide isolated from bacterial heparin lyase 1 digests of heparin that contains a Δ-2S-iduronate on the non-reducing end does not initiate the same responses as intact heparin, and (iii) that removal of the Δ-2S-iduronate to expose the fully sulfated trisaccharide (GlcNS(6S)-IdoUA(2S)-GlcNS(6S)) does initiate the same responses as intact heparin. These results provide evidence that mammalian heparanase digestion of heparin and heparan sulfate exposes a cryptic motif on the non-reducing termini that is recognized by a receptor on dividing cells.

Hyaluronan regulates bone morphogenetic protein-7-dependent prevention and reversal of myofibroblast phenotype.

Hyaluronan (HA) promotes transforming growth factor (TGF)-ß1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-ß1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-ß1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-ß1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-ß1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na(+)/H(+) exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-ß1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology.