Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice.

Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.

Structure, substrate recognition and initiation of hyaluronan synthase.

Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.

KIAA1429/VIRMA promotes breast cancer progression by m6 A-dependent cytosolic HAS2 stabilization.

N6 -methyladenosine (m6 A), the most abundant internal modification in eukaryotic mRNA, plays important roles in many physiological and pathological processes, including the development and progression of cancer. RNA modification by m6 A is regulated by methyltransferases, demethylases, and m6 A-binding proteins that function in large part by regulating mRNA expression and function. Here, we investigate the expression of m6 A regulatory proteins in breast cancer. We find that expression of KIAA1429/VIRMA, a component of the m6 A methyltransferase complex, is upregulated in breast cancer tissue and correlates positively with poor survival. KIAA1429/VIRMA is mislocalized to the cytosol of breast cancer tissues and cell lines, and shRNA-mediated knockdown inhibits breast cancer cell proliferation, migration, and invasion. Mechanistically, KIAA1429/VIRMA is shown to bind to the m6 A-dependent RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), leading to recruitment and stabilization of m6 A-modified hyaluronan synthase 2 (HAS2) mRNA. HAS2 mRNA and KIAA1429/VIRMA mRNA levels correlate positively in breast cancer tissues, suggesting that the KIAA1429/VIRMA-IGF2BP3-HAS2 axis promotes breast cancer growth and contributes to poor prognosis.

Human TMEM2 is not a catalytic hyaluronidase, but a regulator of hyaluronan metabolism via HYBID (KIAA1199/CEMIP) and HAS2 expression.

Cutaneous hyaluronan (HA) is depolymerized to intermediate sizes in the extracellular matrix, and further fragmented in the regional lymph nodes. Previously, we showed that the HA-binding protein involved in HA depolymerization (HYBID), also known as KIAA1199/CEMIP, is responsible for the first step of HA depolymerization. Recently, mouse transmembrane 2 (mTMEM2) with high structural similarity to HYBID was proposed to be a membrane-bound hyaluronidase. However, we showed that the knockdown of human TMEM2 (hTMEM2) conversely promoted HA depolymerization in normal human dermal fibroblasts (NHDFs). Therefore, we examined the HA-degrading activity and function of hTMEM2 using HEK293T cells. We found that human HYBID and mTMEM2, but not hTMEM2, degraded extracellular HA, indicating that hTMEM2 does not function as a catalytic hyaluronidase. Analysis of the HA-degrading activity of chimeric TMEM2 in HEK293T cells suggested the importance of the mouse GG domain. Therefore, we focused on the amino acid residues that are conserved in active mouse and human HYBID and mTMEM2 but are substituted in hTMEM2. The HA-degrading activity of mTMEM2 was abolished when its His248 and Ala303 were simultaneously replaced by the corresponding residues of inactive hTMEM2 (Asn248 and Phe303). In NHDFs, enhancement of hTMEM2 expression by proinflammatory cytokines decreased HYBID expression and increased hyaluronan synthase 2-dependent HA production. The effects of proinflammatory cytokines were abrogated by hTMEM2 knockdown. A decreased HYBID expression by interleukin-1ß and transforming growth factor-ß was canceled by hTMEM2 knockdown. In conclusion, these results indicate that hTMEM2 is not a catalytic hyaluronidase, but a regulator of HA metabolism.

Melatonin inhibits the stemness of head and neck squamous cell carcinoma by modulating HA synthesis via the FOSL1/HAS3 axis.

Hyaluronic acid (HA) is a glycosaminoglycan and the main component of the extracellular matrix (ECM), which has been reported to interact with its receptor CD44 to play critical roles in the self-renewal and maintenance of cancer stem cells (CSCs) of multiple malignancies. Melatonin is a neuroendocrine hormone with pleiotropic antitumor properties. However, whether melatonin could regulate HA accumulation in the ECM to modulate the stemness of head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we found that melatonin suppressed CSC-related markers, such as CD44, of HNSCC cells and decreased the tumor-initiating frequency of CSCs in vivo. In addition, melatonin modulated HA synthesis of HNSCC cells by downregulating the expression of hyaluronan synthase 3 (HAS3). Further study showed that the Fos-like 1 (FOSL1)/HAS3 axis mediated the inhibitory effects of melatonin on HA accumulation and stemness of HNSCC in a receptor-independent manner. Taken together, melatonin modulated HA synthesis through the FOSL1/HAS3 axis to inhibit the stemness of HNSCC cells, which elucidates the effect of melatonin on the ECM and provides a novel perspective on melatonin in HNSCC treatment.

Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases.

Hyaluronan (HA), the essential [-3-GlcNAc-1-ß-4-GlcA-1-ß-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by "HA synthase" (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature. HASs are fascinating enzymes that polymerize two different UDP-activated sugars via different glycosidic linkages. Therefore, these catalysts were the first examples to break the "one enzyme/one sugar transferred" dogma. Three distinct types of these bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known. Based on biochemical and structural work, we present an updated classification system. Class I membrane-integrated HASs employ a processive chain elongation mechanism and secrete HA across the plasma membrane. This complex operation is accomplished by functionally integrating a cytosolic catalytic domain with a channel-forming transmembrane region. Class I enzymes, containing a single GT family-2 (GT-2) module that adds both monosaccharide units to the nascent chain, are further subdivided into two groups that construct the polymer with opposite molecular directionalities: Class I-R and I-NR elongate the HA polysaccharide at either the reducing or the non-reducing end, respectively. In contrast, Class II HASs are membrane-associated peripheral synthases with a non-processive, non-reducing end elongation mechanism using two independent GT-2 modules (one for each type of monosaccharide) and require a separate secretion system for HA export. We discuss recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.

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.

SMC-Derived Hyaluronan Modulates Vascular SMC Phenotype in Murine Atherosclerosis.

[Figure: see text].

Novel ß1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant ß1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-ß-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, ß1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by ß1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial ß1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.

Evaluation of transmembrane domain deletions on hyaluronic acid polymerization of hyaluronan synthase isolated from Streptococcus equisimilis group G.

The membrane enzyme of hyaluronan synthase (HAS) is the key enzyme in hyaluronic acid (HA) biosynthesis by coupling UDP-sugars. Prior studies proposed the C-terminus region of HAS enzyme mediates the production rate and molecular weight of HA. The current study describes the isolation and characterizations of a transmembrane HAS enzyme isolated from Streptococcus equisimilis Group G (GGS-HAS) in vitro. The effect of transmembrane domains (TMDs) on HA productivity was determined and the shortest active variant was also identified by recombinant expression of full-length and five truncated forms of GGS-HAS in Escherichia coli. We found that the GGS-HAS enzyme is longer than that of S. equisimilis group C (GCS-HAS) which includes three more residues (LER) at the C-terminus region (positions 418-420) and also one-point mutation at position 120 (E120D). Amino acid sequence alignment demonstrated 98% and 71% identity of GGS-HAS with that of S. equisimilis Group C and S. pyogenes Group A, respectively. The in vitro productivity of the full-length enzyme was 35.57 µg/nmol, however, extended TMD deletions led to a reduction in the HA productivity. The HAS-123 variant showed the highest activity among the truncated forms, indicating the essential role of first, second, and third TMDs for the full activity. Despite a decline in activity, the intracellular variant can still mediate the binding and polymerization of HA without any need for TMDs. This significant finding suggests that the intracellular domain is the core for HA biosynthesis in the enzyme and other domains are probably involved in other attributes including the enzyme kinetics that affect the size distribution of the polymer. However, more investigations on the recombinant forms are still needed to confirm clearly the role of each transmembrane domain on these properties.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars.

Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review.

Loss of versican and production of hyaluronan in lung epithelial cells are associated with airway inflammation during RSV infection.

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however, whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production, we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) coculture model. RSV infection of BEC/HLF cocultures led to decreased hyaluronidase expression by HLFs, increased accumulation of HA, and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however, BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared with control mice, which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared with SPC-Cre(-) RSV-infected littermates. Taken together, these data demonstrate that altered extracellular matrix accumulation of HA occurs following RSV infection and may contribute to airway inflammation. In addition, loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment.

Activin A promotes hyaluronan production and upregulates versican expression in human granulosa cells†.

Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.

Small fragments of hyaluronan are increased in individuals with obesity and contribute to low-grade inflammation through TLR-mediated activation of innate immune cells.

BACKGROUND AND AIM: Extracellular matrix (ECM) components released during excessive fat mass expansion are considered potential endogenous danger/alarm signals contributing to innate immune system activation. The aim of the current study was to specifically measure plasma levels of low molecular weight (LMW) hyaluronan (HA) and to evaluate its role as pro-inflammatory damage-associated molecular pattern (DAMP) on leukocyte response in the context of human obesity. SUBJECTS AND METHODS: Participants were selected according to their body mass index (BMI, kg/m2) as non-obese (BMI < 29.9, n = 18) and obese (BMI > 29.9, n = 33). Plasma samples were size-dependent fractionated using ion-exchange chromatography to specifically obtain LMW HA fractions that were subsequently quantified by ELISA. Cell incubation experiments with synthetic HA molecules were performed on freshly Ficoll-isolated neutrophils (PMN) and peripheral blood monocytes (PBMC). Leukocyte and adipose tissue gene expression was assessed by real-time PCR and NF-κB activation by western blot. Plasma cytokine levels were measured by fluorescent bead-based (Luminex) immunoassay. RESULTS: We observed a statistically significant increase in the circulating levels of HA fragments of LMW in individuals with obesity which were consistent with significant up-regulated expression of the LMW HA synthesizing enzyme hyaluronan synthase-1 (HAS-1) in obese adipose tissue. Gene expression assessment of HA receptors revealed up-regulated levels for TLR2 in both obese PMN and PBMC. Synthetic HA molecules of different sizes were tested on leukocytes from healthy donors. LMW HA fragments (15-40 kDa) and not those from intermediate molecular sizes (75-350 kDa) induced a significant up-regulation of the expression of major pro-inflammatory cytokines such as IL-1ß, MCP-1 and IL-8 in PBMC. Importantly, LMW HA was able to induce the phosphorylation of IKK α/ß complex supporting its pro-inflammatory role through NF-κB activation. CONCLUSION: Circulating LMW HA molecules are elevated in obesity and may play an important role in triggering low-grade inflammation and the development of metabolic complications.

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

Glycosylated 4-methylumbelliferone as a targeted therapy for hepatocellular carcinoma.

BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.

CPAP-induced airway hyper-reactivity in mice is modulated by hyaluronan synthase-3.

BACKGROUND: Continuous positive airway pressure (CPAP) is a primary mode of respiratory support for preterm infants. Animal studies have shown long-term detrimental effects on lung/airway development, particularly airway (AW) hyper-reactivity, as an unfortunate consequence of neonatal CPAP. Since the hyaluronan (HA) synthesizing enzyme hyaluronan synthase-3 (HAS3) is involved in various adult pulmonary disorders, the present study used a neonatal mouse model to investigate the role of HAS3 in CPAP-induced AW hyper-reactivity. METHODS: Male and female neonatal mice were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for 7 days. At postnatal day 21 (2 weeks after CPAP ended), airway (AW) hyper-reactivity and HAS3 expression were assessed with and without in vitro HAS3 siRNA treatment. RESULTS: MRIs of 3-day-old mice confirmed that CPAP increased lung volume with incrementing inflation pressures. CPAP increased AW reactivity in both male and female mice, which was associated with increased airway smooth muscle and epithelial HAS3 immunoreactivity. CPAP did not affect HA accumulation, but HAS3 siRNA reversed CPAP-induced AW hyper-reactivity and reduced HAS3 expression. CONCLUSIONS: These data in mice implicate a role for HAS3 in long-term effects of CPAP in the developing airway in the context of preterm birth and CPAP therapy. IMPACT: Neonatal CPAP increases airway smooth muscle and epithelial HAS3 expression in mice. CPAP-induced airway hyper-reactivity is modulated by HAS3. These data enhance our understanding of the role mechanical forces play on lung development. These data are a significance step toward understanding CPAP effects on developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.

Endothelial Hyaluronan Synthase 3 Augments Postischemic Arteriogenesis Through CD44/eNOS Signaling.

Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 HAS isoenzymes (hyaluronan synthases 1-3) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3-deficient (Has3-KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3-KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT (protein kinase B)-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3-KO thigh muscles. Endothelial-specific Has3-KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3-deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

OBJECTIVE: The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. CONCLUSIONS: Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.