Expression Of Hyaluronan Synthase Research Articles

Development and validation of a novel phonomimetic bioreactor.

Vocal fold fibroblasts (VFF) constitute the main cell type of the vocal fold’s lamina propria, produce the extracellular matrix and thereby determine the tissue characteristics. To study VFF behavior under in vitro conditions it is important to mimic the dynamic environment of the in vivo state. The aim of our study was to develop and validate a novel phonomimetic bioreactor system mainly based on commercially available components. The use of cell culture dishes with flexible silicone bottoms in combination with a suitable loudspeaker made it possible to expose the cells to various kinds of phonatory stimuli. The fundamental vibration characteristics of silicone membranes were investigated with and without cell culture medium by laser Doppler vibrometry. Human VFF were seeded in flexible-bottomed plates and placed in a custom-made housing containing a loudspeaker. After the cells were exposed to a predefined audio stimulation protocol, cell viability was assessed and gene as well as protein expression levels were compared to static controls. Laser Doppler vibrometry revealed that addition of cell culture medium changed the resonance frequencies of vibrating membranes. Gene expression of hyaluronan synthase 2, collagen III, fibronectin and TGFβ-1 was significantly upregulated in VFF exposed to vibration, compared to static control. Vibration also significantly upregulated collagen I gene and protein expression. We present a new type of phonomimetic bioreactor. Compared to previous models, our device is easy to assemble and cost-effective, yet can provide a wide spectrum of phonatory stimuli based on the entire dynamic range of the human voice. Gene expression data of VFF cultured in our phonomimetic bioreactor show a significant effect of vibration on ECM metabolism, which illustrates the efficacy of our device.

HAS2-AS1 is a novel LH/hCG target gene regulating HAS2 expression and enhancing cumulus cells migration

BackgroundThe cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary.ResultsHAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells.ConclusionsOur results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration.

Estrogen-Mediated MicroRNA-101-3p Expression Represses Hyaluronan Synthase 2 in Synovial Fibroblasts From Idiopathic Condylar Resorption Patients

Idiopathic condylar resorption (ICR) is an aggressive form of osteoarthritis that is frequently observed in adolescent female patients. We hypothesized that an estrogen-mediated pathway may contribute to ICR development. An enzyme-linked immunosorbent assay was used to detect the levels of estradiol (E2) and hyaluronan in synovial fluid. Immunohistochemistry, real-time polymerase chain reaction, and Western blotting were used to detect the expression of microRNAs (miRNAs) and related genes after transfection of miRNA-101-3p mimics, inhibitor, or short interfering RNA into synovial fibroblasts.Dual-luciferase activity was determined to identify the direct effect of miRNA-101-3p on hyaluronan synthase 2 (HAS2). Linear regression analysis, the nonparametric Mann-Whitney U test, the Student t test, and 1-way analysis of variance were carried out to analyze the results of each group. The relationship between hyaluronan and E2 was negatively correlated in synovial fluid (Pearson r=-0.3179, P=.0230). Among the screened miRNAs, miRNA-101-3p was the most overexpressed in ICR. E2 mostly upregulated the expression of miRNA-101-3p at a dose of 10nmol/L 12hours after transfection in synovial fibroblasts of patients with ICR. However, E2 induction of miRNA-101-3p expression was significantly repressed by estrogen receptor α interference (P = 0.0286).The dual-luciferase assay showed that miRNA-101-3p regulated the expression of HAS2 by directly targeting its 3' untranslated region. We speculate that E2 regulates HAS2 expression by targeting miRNA-101-3p in synovial fibroblasts of patients with ICR. Thus, the E2-miRNA-101-3p-HAS2 pathway might play an important role in the pathogenesis of ICR.

The plant alkaloid conophylline inhibits matrix formation of fibroblasts

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor β (TGFβ), which promotes collagen synthesis, but not in cells not treated with TGFβ. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFβ-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFβ-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

Expression of hyaluronan synthases upregulated by thyroid hormone is involved in intestinal stem cell development during Xenopus laevis metamorphosis.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history

Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE2 were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.

Higher titer hyaluronic acid production in recombinant Lactococcus lactis

Hyaluronic acid (HA) is a natural biopolymer and has long been attracting the attention of biotechnology industry due to its various biological functions. HA production with natural producer Streptococcus equi subsp. zooepidemicus has not been preferred because it has many drawbacks due to its pathogenicity. Therefore, in the present study, Streptococcal hyaluronan synthase gene (hasA) was introduced and expressed in Lactococcus lactis, through the auto inducible NICE system and the effect of nisin amount on the production of HA was examined. Newly constructed plasmid was transformed into L. lactis CES15, produced 6.09 g/l HA in static flask culture after three hours of induction period with initial 7.5 ng/ml nisin concentration within total six hours of incubation. The highest HA titer value ever was reported for recombinant HA-producing L. lactis by examining the effect of initial nisin concentration. We have shown that initial nisin concentration, which used to initiate the auto-inducing mechanism of NICE system and consequently hyaluronan synthase expression, has a direct and significant effect on the produced HA amount. Recently constructed recombinant L. lactis CES15 strain provide significant advantages for industrial HA production than those in literature in terms of production time, energy demand, carbon usage, and safety status.

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

Abstract 4037: Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells

Abstract Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway. Citation Format: Brenda G. Trevizo-Barresi, JoEllen Welsh. Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4037.

Loss of Insulin Actions on Arterial Smooth Muscle Cells May Enhance Unstable Atherosclerotic Plaque Formation

Formation of unstable atherosclerotic plaque, a major cause of cardiovascular mortality, is accelerated in insulin resistance and diabetes. Previous studies have suggested that endothelial dysfunction and systemic hyperlipidemia due to loss of insulin actions can accelerated its formation. However, the mechanism for insulin resistance to accelerate the various pathologies of unstable plaque, such as decreases in VSMC and extra cellular matrix (ECM) and increased inflammatory cells, have not been defined. Since insulin actions on the vascular wall are diminished in insulin resistant states, we assessed the loss of insulin action in VSMC and its effect on the formation of atherosclerotic plaque. Insulin receptor (IR) was specifically deleted from VSMC by breeding SM22 a promoter Cre and IR Flox mice to produce SMIRKO mice which were mated with ApoE-/- mice to generate SMIRKO/ ApoE-/- mice. Cultured VSMC from SMIRKO/ApoE-/- mice showed decreased insulin signaling and stimulated proliferation by >50%, but not to PDGF. Weight, blood pressure, glucose, and systemic insulin tolerance were comparable between ApoE-/- and SMIRKO/ApoE-/- mice; which were fed with normal chow to mimic pathophysiological hyperlipidemia encountered clinically. Surprisingly, SMIRKO/ ApoE-/- mice developed significantly more atherosclerotic aortic plaques (Sudan IV staining for lipid content) compared to ApoE-/- mice with parallel increases of macrophage content and inflammatory cytokines such as IL18 and ICAM1. In contrast, SMC as measured by SM22α staining was decreased with elevation of necrotic plaques (p<0.05). Further, proliferative index of VSMC as measured by Ki67 staining and expression of hyaluronan synthase-2 were decreased. Thus, surprisingly, the loss of insulin’s specific actions on VSMC to increase proliferation increased inflammatory cell influx as observed in insulin resistance, may accelerated formation of unstable plaques in diabetes. Disclosure Q. Li: None. J. Fu: None. K. Park: None. C. Rask-Madsen: Research Support; Self; Novo Nordisk A/S. G.L. King: Research Support; Self; Sanofi-Aventis.

Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas

BackgroundDiffusely infiltrating astrocytomas originate from astrocytic glial cells or their precursor cells and are the most common type of brain tumors in adults. In this retrospective study, we investigated the content of hyaluronan, its cell surface receptor, CD44 and the expression of hyaluronan metabolizing enzymes, in these aggressive tumors. Hyaluronan is the main component of extracellular matrix in the brain. In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential.MethodsOur material consisted of 163 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, hyaluronan synthases 1–3 (HAS1–3) and hyaluronidase 2 (HYAL2). The immunostaining results were compared with χ2 –test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression.ResultsHyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade or any other clinicopathological parameters whereas high HAS2 staining intensity was observed in IDH1 negative tumors (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity correlated with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). In the Cox regression analysis HAS2 expression turned out to be a significant independent prognostic factor (p = 0.008).ConclusionsThis study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas.

Switching-Off Adora2b in Vascular Smooth Muscle Cells Halts the Development of Pulmonary Hypertension.

Background: Pulmonary hypertension (PH) is a devastating and progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and remodeling of the lung vasculature. Adenosine signaling through the ADORA2B receptor has previously been implicated in disease progression and tissue remodeling in chronic lung disease. In experimental models of PH associated with chronic lung injury, pharmacological or genetic inhibition of ADORA2B improved markers of chronic lung injury and hallmarks of PH. However, the contribution of ADORA2B expression in the PASMC was not fully evaluated.Hypothesis: We hypothesized that adenosine signaling through the ADORA2B receptor in PASMC mediates the development of PH.Methods: PASMCs from controls and patients with idiopathic pulmonary arterial hypertension (iPAH) were characterized for expression levels of all adenosine receptors. Next, we evaluated the development of PH in ADORA2Bf/f-Transgelin (Tagln)cre mice. These mice or adequate controls were exposed to a combination of SUGEN (SU5416, 20 mg/kg/b.w. IP) and hypoxia (10% O2) for 28 days (HX-SU) or to chronic low doses of bleomycin (BLM, 0.035U/kg/b.w. IP). Cardiovascular readouts including right ventricle systolic pressures (RVSPs), Fulton indices and vascular remodeling were determined. Using PASMCs we identified ADORA2B-dependent mediators involved in vascular remodeling. These mediators: IL-6, hyaluronan synthase 2 (HAS2) and tissue transglutaminase (Tgm2) were determined by RT-PCR and validated in our HX-SU and BLM models.Results: Increased levels of ADORA2B were observed in PASMC from iPAH patients. ADORA2Bf/f-Taglncre mice were protected from the development of PH following HX-SU or BLM exposure. In the BLM model of PH, ADORA2Bf/f- Taglncre mice were not protected from the development of fibrosis. Increased expression of IL-6, HAS2 and Tgm2 was observed in PASMC in an ADORA2B-dependent manner. These mediators were also reduced in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM.Conclusions: Our studies revealed ADORA2B-dependent increased levels of IL-6, hyaluronan and Tgm2 in PASMC, consistent with reduced levels in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Taken together, our data indicates that ADORA2B on PASMC mediates the development of PH through the induction of IL-6, hyaluronan and Tgm2. These studies point at ADORA2B as a therapeutic target to treat PH.

Extracellular ATP activates hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling, and MAPK pathways

Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6 h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.

The fasciacytes: A new cell devoted to fascial gliding regulation.

Hyaluronan occurs between deep fascia and muscle, facilitating gliding between these two structures, and also within the loose connective tissue of the fascia, guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also promotes the functions of the deep fascia. In this study a new class of cells in fasciae is identified, which we have termed fasciacytes, devoted to producing the hyaluronan-rich extracellular matrix. Synthesis of the hyaluronan-rich matrix by these new cells was demonstrated by Alcian Blue staining, anti-HABP (hyaluronic acid binding protein) immunohistochemistry, and transmission electron microscopy. Expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was detected and quantified using real time RT-PCR. This new cell type has some features similar to fibroblasts: they are positive for the fibroblast marker vimentin and negative for CD68, a marker for the monocyte-macrophage lineage. However, they have morphological features distinct from classical fibroblasts and they express the marker for chondroid metaplasia, S-100A4. The authors suggest that these cells represent a new cell type devoted to the production of hyaluronan. Since hyaluronan is essential for fascial gliding, regulation of these cells could affect the functions of fasciae so they could be implicated in myofascial pain. Clin. Anat. 31:667-676, 2018. © 2018 Wiley Periodicals, Inc.

Changes in Hepatic Expression of Hyaluronan Synthases (HAS2 and HAS3) After Treatment with Steptozotocin in Channel Catfish

Streptozotocin (STZ) is commonly used experimentally to induce hyperglycemia in rodents. As an alternative model organism for investigating human obesity, channel catfish show a genetic predisposition toward an accelerated growth rate resulting in a phenotype similar to obese humans. In our previous studies, STZ treatment resulted in hypoglycemia and abnormal liver morphology in channel catfish, indicating that STZ treatment possibly induces liver damage. The mechanisms involved in the STZ‐induced hypoglycemic and tissue damage have never been examined. Another noted marker for liver damage stems from hyaluronan synthases (HAS), which have been associated with tissue damage in both humans and rodents. We hypothesized the expression of HAS mRNA would change in response to liver damage induced by STZ in channel catfish. Juvenile channel catfish were treated with 0, 3.6, 36, 180, or 360 mg STZ/kg body weight (n=4 fish/treatment) on day 0. Liver samples were collected on day 7. Hepatic expression of HAS2 and HAS3 mRNA was examined, using real‐time PCR. Expression of HAS2 and HAS3 mRNA decreased in response to an increased dose of STZ (p < 0.05 and p < 0.10, respectively). These results suggest STZ induces severe liver damage that down regulates the expression of HAS2 and HAS3 mRNA. However, the effects of STZ on gene expression appear to be global, and the tissue damage induced by STZ is not reversible by changes in the amount or activity of HAS.Support or Funding InformationThis research was funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS‐1238831). It was also supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating The Effect of Melatonin on HAS2, and PGR expression, as well as Cumulus Expansion, and Fertility Potential in Mice.

Objective Infertility is a worldwide health problem which affects approximately 15% of sexually active couples. One of the factors influencing the fertility is melatonin. Also, protection of oocytes and embryos from oxidative stress inducing chemicals in the culture medium is important. The aim of the present study was to investigate if melatonin could regulate hyaluronan synthase-2 (HAS2 ) and Progesterone receptor (PGR ) expressions in the cumulus cells of mice oocytes and provide an in vitro fertilization (IVF) approach. Materials and Methods In this experimental study, for this purpose, 30 adult female mice and 15 adult male mice were used. The female mice were superovulated using 10 U of pregnant mare serum gonadotropin (PMSG) and 24 hours later, 10 U of human chorionic gonadotropin (hCG) were injected. Next, cumulus oocyte complexes (COCs) were collected from the oviducts of the female mice by using a matrix-flushing method. The cumulus cells were cultured with melatonin 10 µM for 6 hours and for real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for evaluation of HAS2 and PGR expression levels. The fertilization rate was evaluated through IVF. All the data were analyzed using a t test. Results The results of this study showed that HAS2 and PGR expressions in the cumulus cells of the mice receiving melatonin increased in comparison to the control groups. Also, IVF results revealed an enhancement in fertilization rate in the experimental groups compared to the control groups. ConclusionTo improve the oocyte quality and provide new approaches for infertility treatment, administration of melatonin as an antioxidant, showed promising results. Thus, it is concluded that fertility outcomes can be improved by melatonin it enhances PGR .

TGF-β1 upregulates the expression of hyaluronan synthase 2 and hyaluronan synthesis in culture models of equine articular chondrocytes.

We investigated the effect of transforming growth factor beta 1 (TGF-β1) on equine hyaluronan synthase 2 (HAS2) gene expression and hyaluronan (HA) synthesis in culture models of articular chondrocytes. Equine chondrocytes were treated with TGF-β1 at different concentrations and times in monolayer cultures. In three-dimensional cultures, chondrocyte-seeded gelatin scaffolds were cultured in chondrogenic media containing 10 ng/mL of TGF-β1. The amounts of HA in conditioned media and in scaffolds were determined by enzyme-linked immunosorbent assays. HAS2 mRNA expression was analyzed by semi-quantitative reverse transcription polymerase chain reaction. The uronic acid content and DNA content of the scaffolds were measured by using colorimetric and Hoechst 33258 assays, respectively. Cell proliferation was evaluated by using the alamarBlue assay. Scanning electron microscopy (SEM), histology, and immunohistochemistry were used for microscopic analysis of the samples. The upregulation of HAS2 mRNA levels by TGF-β1 stimulation was dose and time dependent. TGF-β1 was shown to enhance HA and uronic acid content in the scaffolds. Cell proliferation and DNA content were significantly lower in TGF-β1 treatments. SEM and histological results revealed the formation of a cartilaginous-like extracellular matrix in the TGF-β1-treated scaffolds. Together, our results suggest that TGF-β1 has a stimulatory effect on equine chondrocytes, enhancing HA synthesis and promoting cartilage matrix generation.

Targeted HAS2 Expression Lessens Airway Responsiveness in Chronic Murine Allergic Airway Disease.

Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.

Potential treatment for vocal fold scar with pirfenidone.

Pirfenidone (PFD) is a strong antifibrotic agent that has been clinically approved in Japan for idiopathic pulmonary fibrosis. We examined the antifibrotic effects of PFD on fibroblasts isolated from scarred vocal folds (VFs) of ferrets in vitro. Prospective animal experiments with controls. Scar fibroblasts (SFs) were isolated from scarred VFs that had been electrocauterized 2weeks before harvesting (N = 4). Normal fibroblasts (NFs) were isolated from intact VFs (N = 4). SFs and NFs were incubated in the presence of 10ng/mL transforming growth factor β1 (TGF-β1), with or without PFD. After the 48-hour incubation, mRNA expression levels of α smooth muscle actin (αSMA), TGF-β1, collagen type I, and hyaluronan synthase 2 (HAS2) were examined by real-time polymerase chain reaction. Immunohistochemistry with anti-αSMA anti-collagen type I and phosphorylated Smad (p-Smad)2/3 antibodies in SFs with or without PFD was performed. SFs and NFs were cultured in collagen gel with or without PFD for 48 hours, and the extent of gel contraction was examined quantitatively. PFD treatment significantly (P < .05) decreased mRNA expression of collagen type I, significantly increased mRNA expression of TGF-β1 and HAS2, and significantly suppressed collagen gel contraction. However, it did not have a significant effect on the expression of αSMA. The expression of p-Smad2/3 in the nucleus was faded with PFD, possibly demonstrating the suppression of translocation of p-Smad2/3 from cytoplasm to nucleus with PFD. This is the first report to demonstrate the in vitro antifibrotic effects of PFD on fibroblasts isolated from scarred VFs of ferrets. NA. Laryngoscope, 128:E171-E177, 2018.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.