HAS2 Expression Research Articles

Sa2058 Hippo-YAP Signaling Pathway Modulates Stellate Cell Activation in Pancreatic Cancer and Pancreatitis

G A A b st ra ct s for sonic hedgehog (PTCH and SMOO), GPCRs (AGTRI) and TNFaR I and II. Pancreatic mesenchymal cells were also found, using Luminex magnetic bead-based immunodetection, to be a significant source of proinflammatory cytokines IL-6 and IL-8, secreting high IL-6 levels at all times subsequent to early passages. TNF-a expression was detected only during early passage when cells exhibited an acinar-like phenotype. Interestingly, ethanol (50 mM) provided in serum free medium induced a hormetic effect, modestly stimulating cell viability associated with significant (~2-fold) elevations in HAS1, HAS2 and UGDH mRNA levels. Insulin alone (0.5-10 nM) dose-dependently suppressed HAS2 expression, but at 1 nM, modestly induced HAS1 in the presence or absence of ethanol. Ethanol-induced HAS2 gene induction was abolished in the presence of 1 nM insulin. These effects suggested some shifting between relative levels of HAS genes in response to changes in metabolic conditions. In addition, we tested the effect of conditioned medium from PDAC cell lines on HAS gene expression. The human cancer cell lines BXPC3 and MiaPaCa-2 were found to secrete high levels of IL-8, but only very low levels of IL-6 and TNF-a. Interestingly, BXPC3 and MiaPaCa2 conditioned media stimulated HAS1 and, more potently, HAS2 expression in pancreatic mesenchymal cells. We conclude that, in the tumor microenvironment, autocrine and paracrine signaling contribute to a hyaluronan synthetic phenotype that may promote tumor progression.

Hyaluronan synthase 1: a mysterious enzyme with unexpected functions.

Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluronan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which have higher enzymatic activity. Furthermore, in most cell types studied so far, the expression of its gene is low and the enzyme requires high concentrations of sugar precursors for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states associated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. In addition, both full length and splice variants of HAS1 are expressed in malignancies like bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interestingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44–HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer.

Gene expression in salivary glands: effects of diet and mouse chromosome 17 locus regulating macronutrient intake.

Dcpp2, Prrt1, and Has1 are plausible candidate genes for the Mnic1 (macronutrient intake-carbohydrate) locus on mouse chromosome 17, based on their map positions and sequence variants, documented expression in salivary glands, and the important role of saliva in oral food processing and taste. We investigated the effects of genotype and diet on gene expression in salivary glands (parotid, submandibular, sublingual) of carbohydrate-preferring, C57BL6J.CAST/EiJ-17.1 subcongenic mice compared to fat-preferring wild-type C57BL/6J. To achieve accurate normalization of real-time quantitative RT-PCR data, we evaluated multiple reference genes to identify the most stably expressed control genes in salivary gland tissues, and then used geometric averaging to produce a reliable normalization factor. Gene expression was measured in mice fed different diets: (1) rodent chow, (2) macronutrient selection diets, (3) high-fat diet, and (4) low-fat diet. In addition, we measured salivary hyaluronan concentrations. All three genes showed strain differences in expression, in at least one major salivary gland, and diet effects were observed in two glands. Dcpp2 expression was limited primarily to sublingual gland, and strongly decreased in B6.CAST-17.1 subcongenic mice compared to wild-type B6, regardless of diet. In contrast, both genotype and diet affected Prrt1 and Has1 expression, in a gland-specific manner, for example, Prrt1 expression in the parotid gland alone was strongly reduced in both mouse strains when fed macronutrient selection diet compared to chow. Notably, we discovered an association between diet composition and salivary hyaluronan content. These results demonstrate robust effects of genetic background and diet composition on candidate gene expression in mouse salivary glands.

Histochemical structure and immunolocalisation of the hyaluronan system in the dromedary oviduct.

We investigated the local modulation of some histochemical properties of oviducts of the dromedary (Camelus dromedarius), focusing on the immnolocalisation of hyaluronic acid (HA) synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL1) and the HA receptor CD44 in the ampulla and isthmus. Abundant acidic mucopolysaccharides (glycosaminoglycans) were detected by Alcian blue staining along the luminal surface of both ciliated and non-ciliated epithelial cells (LE). Staining for HAS2 was higher in the primary epithelial folds of the ampulla compared with the isthmus, especially in secretory cells, adluminal epithelial surface and supranuclear cell domain. HAS3 staining was stronger in the LE of the isthmus than ampulla. HYAL2 was detected in the LE in the ampulla and isthmus and was more intense in the adluminal projections of secretory cells. HYAL1 was weakly detected in the LE with no difference between the ampulla and isthmus. Strong CD44 immunostaining was present in the LE of the ampulla and isthmus. CD44 staining was higher in secretory cells than in ciliated epithelial cells and was higher in the supranuclear region than the basal region of the cytoplasm. In conclusion, we provide evidence that HA synthesis and turnover occur in the camel oviduct. Differences in HAS2 and HAS3 expression suggest regional differences in the molecular size of HA secreted in oviductal fluid that may influence oviduct-gamete interaction in the camel.

Antitumor effects of hyaluronan inhibition in desmoid tumors.

Desmoid tumors (DTs) are rare, mesenchymal tumors that exhibit features of an abundant wound healing process. Previously, we showed that mesenchymal stem cells (MSCs) are constituents of DTs and may contribute to desmoid tumorigenesis via activities associated with wound healing. Hyaluronan (HA) is a long-charged chain of repeating glucuronate and N-acetylglucosamine disaccharides that is synthesized by HA synthases (HAS) and degraded by hyaluronidases (HYAL). HA is secreted into the extracellular matrix by injured stroma and is important for normal tissue repair and neoplastic progression. Here, we investigated the presence of HA in DTs and the antitumor effects of the HA inhibitor, 4-methylumbelliferone (4-MU), on DT-derived mesenchymal cells. By immunohistochemistry and enzyme-linked immunosorbent assay, we found abundant expression of HA in 29/30 DTs as well as >5-fold increased HA levels in DT-derived cell lines relative to controls. Immunohistochemistry also demonstrated high expression of HAS2 in DTs, and quantitative PCR analysis showed increased HAS2 upregulation in frozen DTs and DT-derived cells. 4-MU treatment of DT-derived cells significantly decreased proliferation as well as HA and HAS2 levels. Fluorescent immunohistochemistry showed that MSCs in DTs coexpressed HA, HAS2, HYAL2, as well as the major HA receptor CD44 and HA coreceptor TLR4. Taken together, our results suggest that paracrine regulation of HA signaling in DTs may contribute to MSC recruitment and tumor proliferation. Future studies investigating the role of HA in tumor-stroma crosstalk and inhibition of HA-MSC interactions as a novel therapeutic target in DTs and other solid tumors are warranted.

Hyaluronan Synthase 3 Null Mice Exhibit Decreased Intestinal Inflammation and Tissue Damage in the DSS-Induced Colitis Model.

Hyaluronan (HA) overproduction is a hallmark of multiple inflammatory diseases, including inflammatory bowel disease (IBD). Hyaluronan can act as a leukocyte recruitment molecule and in the most common mouse model of intestinal inflammation, the chemically induced dextran sodium sulfate (DSS) experimental colitis model, we previously determined that changes in colon distribution of HA occur before inflammation. Therefore, we hypothesized that, during a pathologic challenge, HA promotes inflammation. In this study, we tested the progression of inflammation in mice null for the hyaluronan synthase genes (HAS1, HAS3, or both HAS1 and HAS3) in the DSS-colitis model. Our data demonstrate that both the HAS1/HAS3 double and the HAS3 null mice are protected from colitis, compared to wild-type and HAS1 null mice, as determined by measurement of weight loss, disease activity, serum IL-6 levels, histologic scoring, and immunohistochemistry. Most notable is the dramatic increase in submucosal microvasculature, hyaluronan deposition, and leukocyte infiltration in the inflamed colon tissue of wild-type and HAS1 null mice. Our data suggest, HAS3 plays a crucial role in driving gut inflammation. Developing a temporary targeted therapeutic intervention of HAS3 expression or function in the microcirculation may emerge as a desirable strategy toward tempering colitis in patients undergoing flares of IBD.

Homeostasis of Hyaluronic Acid in Normal and Scarred Vocal Folds

Vocal fold scarring is one of the most challenging laryngeal disorders to treat. Hyaluronic acid (HA) is the main component of lamina propria, and it plays an important role in proper vocal fold vibration and is also thought to be important in fetal wound healing without scarring. Although several animal models of vocal fold scarring have been reported, little is known about the way in which HA is maintained in vocal folds. The purpose of this study was to clarify the homeostasis of HA by examining the expression of hyaluronan synthase (Has) and hyaluronidase (Hyal), which produce and digest HA, respectively. Experimental prospective animal study. Vocal fold stripping was performed on 38 Sprague-Dawley rats. Vocal fold tissue was collected at five time points (3days-2months). Expression of HA was examined by immunohistochemistry, and messenger RNA (mRNA) expression of Has and Hyal was examined by real-time polymerase chain reaction and in-situ hybridization. In scarred vocal folds, expression of Has1 and Has2 increased at day 3 together with expression of HA and returned to normal at 2weeks. At 2months, Has3 and Hyal3 mRNA showed higher expressions than normal. Expression patterns of Has and Hyal genes differed between normal, acute-scarred, and chronic-scarred vocal folds, indicating the distinct roles of each enzyme in maintaining HA. Continuous upregulation of Has genes in the acute phase may be necessary to achieve scarless healing of vocal folds.

121 EXPRESSION OF HYALURONAN SYSTEM COMPONENTS IN THE EARLY PREGNANT OVINE ENDOMETRIUM

Hyaluronan (HA; hyaluronic acid), a member of glycosaminoglycans (GAG) family, is the main polysaccharide and is expressed in almost all tissues including those of the reproductive tract. Three different hyaluronic acid-synthase (HAS) enzymes, HAS1, HAS2, and HAS3, synthesise HA. The action of HA depends on its molecular size and its cell surface receptor (CD44). Hyaluronidases (HYAL) are a group of enzymes, HYAL-1 and HYAL-2, that degrade HA. Roles of the HA system in reproductive events include oocyte maturation, fertilization, and implantation. It is also known that ovarian steroids, specifically progesterone, regulate the HA system in the endometrium. Moreover, HA-mediated cell signalling participates in embryonic development. The aim of this study was to evaluate expression of the HA system at the mRNA level in the early pregnant ovine endometrium at pre-implantation stage. Therefore, endometrial tissue samples were collected on Day 13 of the oestrous cycle (n = 10) and pregnancy (n = 14). Relative mRNA expression levels of HA system genes were quantified using quantitative RT-qPCR. Data were analysed using one-way ANOVA and l.s.d. test. All of the studied components of the HA system (HAS1, HAS2, HAS3, HYAL1, HYAL2, and CD44) were expressed in the ovine endometrium. Steady-state mRNA levels of HAS1, HYAL1, HYAL2, and CD44 were not significantly different between cyclic and early pregnant ovine endometrium on Day 13. However, expression of HAS2 and HAS3 appeared to be down-regulated in early pregnancy. Considering that both cyclic and pregnant endometrium on Day 13 is under the influence of progesterone, detected differential regulation of some components of HA system in the ovine endometrium may be directly related to the effects of the presence of an embryo. The role of the HA system in ovine endometrium at later stages of pregnancy warrants further investigation. Partially funded by TUBITAK-112R022 to ZK and SUBAP to AG.

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17β-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Up-regulation of proteoglycan 4 in temporomandibular osteoarthritic synovial cells by hyaluronic acid.

Hyaluronic acid (HA) injection is widely used in the treatment of temporomandibular joint (TMJ) osteoarthritis (OA). Proteoglycan 4 (PRG4) is another joint lubricant that protects surface of articular cartilage. But few studies had explored the role of HA in regulation of PRG4 expression in TMJ OA. In this study, the effects of HA on the expression of PRG4 in osteoarthritic TMJ synovial cells were investigated in hypoxia, which was similar to the TMJ physiologically. Synovial cells were isolated from the TMJ OA patients and were treated with or without HA under normoxia or hypoxia for indicated time periods. The proliferation of synovial cells was measured using Cell Counting Kit-8 (CCK-8). The gene expression of HAS2, VEGF, and PRG4 was detected by quantitative real-time PCR, and the secretion of PRG4 and VEGF was assayed by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to examine the protein expression of hypoxia-induced factor-1α (HIF-1α). Hyaluronic acid markedly increased the proliferation of osteoarthritic synovial cells in hypoxia. The expression of HAS2 and PRG4 mRNA of osteoarthritic synovial cells under hypoxia was enhanced by HA treatment. However, HA had no effect on reducing the VEGF and HIF-1α expression in synovial cells in hypoxia. Hyaluronic acid could promote the expression of HAS2 and PRG4, but could not modulate HIF-1α and VEGF expression of TMJ osteoarthritic synovial cells in hypoxia.

Spatially restricted hyaluronan production by Has2 drives epithelial tubulogenesis in vitro.

Generation of branched tubes from an epithelial bud is a fundamental process in development. We hypothesized that induction of hyaluronan synthase (Has) and production of hyaluronan (HA) drives tubulogenesis in response to morphogenetic cytokines. Treatment of J3B1A mammary cells with transforming growth factor-β1 or renal MDCK and mCCD-N21 cells with hepatocyte growth factor induced strong and specific expression of Has2. Immunostaining revealed that HA was preferentially produced at the tips of growing tubules. Inhibition of HA production, either by 4-methylumbelliferone (4-MU) or by Has2 mRNA silencing, abrogated tubule formation. HA production by J3B1A and mCCD-N21 cells was associated with sustained activation of ERK and S6 phosphorylation. However, silencing of either CD44 or RHAMM (receptor for HA-mediated motility), the major HA receptors, by RNA interference, did not alter tubulogenesis, suggesting that this process is not receptor-mediated.

4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression.

Background4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action.MethodsThe HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment.ConclusionsThese results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

Divergent Temporal Expression of Hyaluronan Metabolizing Enzymes and Receptors with Craniotomy vs. Controlled-Cortical Impact Injury in Rat Brain: A Pilot Study.

Traumatic brain injury (TBI) triggers many secondary changes in tissue biology, which ultimately determine the extent of injury and clinical outcome. Hyaluronan [hyaluronic acid (HA)] is a protective cementing gel present in the intercellular spaces whose degradation has been reported as a causative factor in tissue damage. Yet little is known about the expression and activities of genes involved in HA catabolism after TBI. Young adult male Sprague-Dawley rats were assigned to three groups: naïve control, craniotomy, and controlled-cortical impact-induced TBI (CCI-TBI). Four animals per group were sacrificed at 4 h, 1, 3, and 7 days post-CCI. The mRNA expression of hyaluronan synthases (HAS1-3), hyaluronidases (enzymes for HA degradation, HYAL 1–4, and PH20), and CD44 and RHAMM (membrane receptors for HA signaling and removal) were determined using real-time PCR. Compared to the naïve controls, expression of HAS1 and HAS2 mRNA, but not HAS3 mRNA increased significantly following craniotomy alone and following CCI with differential kinetics. Expression of HAS2 mRNA increased significantly in the ipsilateral brain at 1 and 3 days post-CCI. HYAL1 mRNA expression also increased significantly in the craniotomy group and in the contralateral CCI at 1 and 3 days post-CCI. CD44 mRNA expression increased significantly in the ipsilateral CCI at 4 h, 1, 3, and 7 days post-CCI (up to 25-fold increase). These data suggest a dynamic regulation and role for HA metabolism in secondary responses to TBI.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Collagen peptide and vitamin C additively attenuate age-related skin atrophy in Sod1-deficient mice.

Age-related skin thinning is correlated with a decrease in the content of collagen in the skin. Accumulating evidence suggests that collagen peptide (CP) and vitamin C (VC) transcriptionally upregulate type I collagen in vivo. However, the additive effects of CP and VC on age-related skin changes remain unclear. We herein demonstrate that CP and a VC derivative additively corrected age-related skin thinning via reduced oxidative damage in superoxide dismutase 1 (Sod1)-deficient mice. Co-treatment with these compounds significantly normalized the altered gene expression of Col1a1, Has2, and Ci1, a proton-coupled oligopeptide transporter, in Sod1(-/-) skin. The in vitro analyses further revealed that collagen oligopeptide, a digestive product of ingested CP, significantly promoted the bioactivity of the VC derivative with respect to the migration and proliferation of Sod1(-/-) fibroblasts. These findings suggest that combined treatment with CP and VC is effective in cases of age-related skin pathology.

In vitro model of hyaluronan synthase gene expression associated with lipopolysaccharide-induced inflammation in SW982 cell line.

The present study aimed to demonstrate the phenomena of hyaluronan synthesis in response to lipopolysaccharide-induced inflammation in SW982, a human synovial sarcoma cell line. The expression of IL-1ß, including Toll-like receptor 4 and IL-1ß-converting enzyme, was proved to be induced by a reverse transcription-polymerase chain reaction. The expression of HAS genes encoding enzyme hyaluronan synthase 2 and 3, including CD44 gene which encodes the cell surface receptor of hyaluronan were upregulated in association with the activation of inflammation, along with an increase in hyaluronan level in the culture medium. The highest expression of HAS2 and HAS3 was found at 9 h after treatment with lipopolysaccharide. However, HAS1 gene expression was not detectable neither with the non-treatment nor with the treatment with lipopolysaccharide. Dexamethasone at 30 nM significantly suppressed lipopolysaccharide-induced HAS genes expression, leading to the decline of the hyaluronan level in the culture medium. Our results demonstrated the effective tool for studying hyaluronan synthesis in association with inflammation in the SW982 cell line.

Growth promoting effect of hyaluronan synthesis promoting substances on Japanese eel leptocephali.

Hyaluronans (HAs) are glycosaminoglycans produced in the bodies of Anguilliform and Elopiform leptocephali, and play a role in metabolic energy. In mammals, HA synthesis-promoting substances (HASPS) up-regulate the expression of HA synthase (HAS) and increase the amount of HA in the body. In this study, Japanese eel leptocephali were fed a HASPS containing diet. We analyzed HAS1s and HAS2 expression, HA content, and their influence on growth. HASPS extracted from Grifola frondosa promoted HAS1s and HAS2 mRNA and HA content. Other than mammals, these results are first reported in vertebrate. Moreover, HASPS extracted from G. frondosa promoted leptocephalus growth. The relationship between growth and HA in the leptocephali is not yet clear. However, based on our results we hypothesize that HA is involved in the storage of energy, which is metabolized to sugars when needed for metabolic energy.

Asthmatic airway epithelial cells differentially regulate fibroblast expression of extracellular matrix components

Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression. We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase (HAS) 2, and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-β1 and TGF-β2 concentrations in media were measured by using ELISA. COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold, P < .02; 10.8-fold, P < .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P < .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-β2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P < .05), whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold, P < .006). HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling.

Expression of Hyaluronan Synthase and Collagen Type I mRNA by Hyaluronan Tetrasaccharides in Normal Human Dermal Fibroblasts

The major compounds in the dermis are hyaluronan and type I collagen, which decrease with aging, UV and various other factors. The loss of hyaluronan and collagen with aging is associated with increased dehydration and wrinkling of the skin. We aimed to investigate the influence of hyaluronan tetrasaccharide (HA4) on regulation of high molecular weight hyaluronan (HA) and collagen synthesis in normal human dermal fibroblasts (NHDFs). Expression of hyaluronan synthase (HAS) 1–3 and collagen (COL) 1A1 mRNA were evaluated by quantitative real-time PCR. HAS1 mRNA expression was found to be increased in NHDFs treated with HA and HA4. In addition, it was observed that NHDFs co-cultured with normal human epidermal keratinocytes (NHEKs) showed up-regulation of HAS1 mRNA expression, as compared with NHDFs single cell culture treated with HA4. Treatment of NHDFs with HA4 + derivatized vitamin C (VC-PMg) significantly increased COL1A1 mRNA expression. In this study, we confirmed that HA4 affected HAS1 and COL1A1 mRNA expression; thus, HA4 application may show various action in skin.

MP29-14 TARGETING HYALURONIC ACID FAMILY BIOMARKERS FOR TREATMENT OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-14 TARGETING HYALURONIC ACID FAMILY BIOMARKERS FOR TREATMENT OF RENAL CELL CARCINOMA Nicolas Ortiz, Michael Garcia-Roig, Travis Yates, Murugesan Manoharan, and Vinata Lokeshwar Nicolas OrtizNicolas Ortiz More articles by this author , Michael Garcia-RoigMichael Garcia-Roig More articles by this author , Travis YatesTravis Yates More articles by this author , Murugesan ManoharanMurugesan Manoharan More articles by this author , and Vinata LokeshwarVinata Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.756AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Up to 25% of patients with renal cell carcinoma (RCC) have metastasis at initial diagnosis. Current targeted therapies (e.g., Sorafenib) are not curative and the 5-year survival of these patients is <10%. Molecular determinants of RCC growth and metastasis may be accurate prognostic markers for metastasis and could be targeted for therapy. We evaluated the expression of 3 genes (and their splice variants) as prognostic indicators. We also tested the therapeutic efficacy of the combination of hymecromone (HC), a non-toxic, orally bioavailable hyaluronic acid (HA) synthesis inhibitor and Sorafenib (SF). METHODS Tumor specimens were collected from RCC patients undergoing nephrectomy (n=81; T1, 40; T2+, 41; Fuhrman Grade 1, 5; Grade 2, 32; Grade 3, 25; Grade 4, 19). Nine patients developed metastasis within 15 months. Tissue-RNA was subjected to Q-PCR for HA family of markers (i.e. HAS3, CD44, RHAMM). mRNA levels were correlated with metastasis by logistic regression. Effect of HC, SF and HC+SF on cell proliferation, cell cycle and apoptosis was examined in RCC cells (Caki-1, 786-O, ACHN, 769-P) by cell counting, flow cytometry and Cell Death ELISA. Boyden chamber was used for motility assay. Effect on cell cycle, apoptosis, and HA-receptor levels was evaluated by immunoblotting and Q-PCR. RESULTS The mRNA levels of HAS3, CD44 and RHAMM were 3 to 7-fold elevated in RCC tissues from patients who developed metastasis when compared to those who did not develop metastasis during the follow up period. In univariate analysis age, grade, stage, CD44 and RHAMM levels significantly correlated with metastasis. In multivariate analysis, CD44 (< 0.0001), HAS3 (P=0.03) and RHAMM (P< 0.0001) significantly correlated with metastasis. Among the three markers, RHAMM had the highest accuracy (89.5%) in predicting metastasis. Combination of HC (15-30-μg/ml) and SF (1.6 to 3.2 μg/ml), at non-cytotoxic doses caused 60-90% inhibition in proliferation, motility and invasion after 48-72 h treatment. Only the combination induced apoptosis (3-4-fold). HC+SF combination down regulated CD44 and RHAMM mRNA levels by 2- and 68-fold, respectively and inhibited HA synthesis. In Caki-1xenograft, oral treatment with combination caused 100% inhibition of tumor growth. CONCLUSIONS HAS3, CD44 and RHAMM expression in RCC tissues is potentially an independent prognostic indicator for metastasis. HC+SF combination may be a potentially effective treatment for metastatic RCC. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e310 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Nicolas Ortiz More articles by this author Michael Garcia-Roig More articles by this author Travis Yates More articles by this author Murugesan Manoharan More articles by this author Vinata Lokeshwar More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...