Expression Of Hyal2 Research Articles

Piceatannol enhances hyaluronic acid synthesis through SIRT1-Mediated HAS2 upregulation in human dermal fibroblasts

Dermal fibroblasts play a crucial role in skin structure and function by producing hyaluronic acid. Piceatannol (PIC), a polyphenol abundant in passion fruit seeds, has been reported to activate sirtuin 1 (SIRT1). Clinical trials have demonstrated that PIC intake improves skin moisture and maintains skin elasticity, yet the underlying mechanism remains unclear. This study aimed to investigate the effects of PIC on hyaluronic acid biosynthesis and the involvement of SIRT1 in this process. Human dermal fibroblast Hs68 cells were stimulated with PIC, and the expression levels of HAS2 and HYAL2, key enzymes in hyaluronic acid biosynthesis, as well as SIRT1 expression, were assessed using quantitative real-time PCR. Additionally, the role of SIRT1 in the hyaluronic acid biosynthesis pathway during PIC stimulation was examined using a SIRT1 inhibitor. The results demonstrated that PIC increased HAS2 expression while decreasing HYAL2 expression in human dermal fibroblasts. Furthermore, PIC enhanced SIRT1 expression, and pre-treatment with a SIRT1 inhibitor mitigated PIC-induced upregulation of HAS2, suggesting that PIC promotes hyaluronic acid synthesis by inducing SIRT1. These findings suggest that PIC could serve as a beneficial food ingredient, enhancing skin structure and function by promoting hyaluronic acid biosynthesis via SIRT1 induction.

Abstract A169: Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC

Abstract Background: Multiple clinical studies have addressed the role of immunotherapies for solid cancers. While these studies have demonstrated promising results and improvements over existing therapies, less than half of patients with advanced disease show the clinical benefits of immunotherapy. Furthermore, once tumors start to produce metastases, it becomes one of the leading causes of cancer-related mortality. Hyal2-ADC is a novel conjugate of anti-Hyal2 monoclonal antibody and cytotoxic payload PNU. Hyaluronidase 2 (Hyal2), which is a membrane-bound enzyme expressed by both MDSCs and epithelial tumor cells, serves as a molecular target for the Hyal2-ADC. Importantly, our anti-Hyal2 antibodies recognize both human and mouse Hyal2. Methods: Expression of Hyal2 in human and mouse cancer tissues was assessed using immunohistochemistry and Western Blotting. The in vitro effects of Hyal2-ADC tumor cells and MDSCs were evaluated using MTT assay and immunofluorescence. A single-cell RNA sequencing was utilized for the transcriptomic analysis of Hyal2-expressing myeloid cells. The in vivo anti-tumor activity of Hyal2-ADC was evaluated using syngeneic mouse tumor models and human xenograft tumor models in immunodeficient mice. Mice with established bladder or prostate tumors were used in this study. Results: Administration of Hyal-ADC alone led to the inhibition of tumor growth in a dose-dependent manner. Importantly, the therapeutic effect of Hyal2-ADC was associated with remodeling the tumor microenvironment, reduction of immunosuppressive myeloid cells, and influx of immune T cells. A combination of Hyal2-ADC with anti-PD1 therapy resulted in complete tumor eradication in mice with syngeneic established tumors (initial volume <200 mm3) in 100% treated mice. The cured mice demonstrated long-term protection from tumor re-challenge. Furthermore, the combination therapy of mice with large, advanced established tumors (>200mm3) resulted in 80% tumor eradication. Conclusions: Hyal2-ADC is a first-in-class drug that selectively targets Hyal2-expressing cells. The modulation of the immunosuppressive tumor microenvironment and development of efficient immune memory observed in murine models suggests that Hyal2-ADC alone or in combination with anti-PD1 therapy could be utilized for the treatment of solid tumors associated with deregulated HA metabolism, including bladder, prostate, breast, brain, and other cancers. Mechanistically, the enzyme Hyal2 is involved in the degradation of tumor-associated hyaluronan (HA), one of the major components of the extracellular matrix in tumor stroma. Depletion of Hyal2-expressing cells leads to normalization of HA metabolism in the tumor microenvironment supporting generation of T cell-mediated anti-tumor immune response. Overall, this study introduces a novel concept to remodeling the tumor microenvironment, bypassing tumor-associated immune suppression, and overcoming resistance to cancer immunotherapy, thus representing a novel therapeutic strategy for patients. Citation Format: Sergei Kusmartsev, William Donelan, Wayne Brisbane, Brianna Nguyen, Meng-Lun Hsieh, Shiwu Li, Paul Dominguez-Gutierrez, Padraic O'Malley, Paul L Crispen. Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A169.

Complement protein C1q stimulates hyaluronic acid degradation via gC1qR/HABP1/p32 in malignant pleural mesothelioma.

Complement component C1q can act as a pro-tumorigenic factor in the tumor microenvironment (TME). The TME in malignant pleural mesothelioma (MPM) is rich in C1q and hyaluronic acid (HA), whose interaction enhances adhesion, migration and proliferation of malignant cells. HA-bound C1q is also capable of modulating HA synthesis. Thus, we investigated whether HA-C1q interaction would affect HA degradation, analyzing the main degradation enzymes, hyaluronidase (HYAL)1 and HYAL2, and a C1q receptor candidate. We first proceeded with the characterization of HYALs in MPM cells, especially HYAL2, since bioinformatics survival analysis revealed that higher HYAL2 mRNA levels have an unfavorable prognostic index in MPM patients. Interestingly, Real-Time quantitative PCR, flow cytometry and Western blot highlighted an upregulation of HYAL2 after seeding of primary MPM cells onto HA-bound C1q. In an attempt to unveil the receptors potentially involved in HA-C1q signaling, a striking co-localization between HYAL2 and globular C1q receptor/HABP1/p32 (gC1qR) was found by immunofluorescence, surface biotinylation and proximity ligation assays. RNA interference experiments revealed a potentially regulatory function exerted by gC1qR on HYAL2 expression, since C1QBP (gene for gC1qR) silencing unexpectedly caused HYAL2 downregulation. In addition, the functional blockage of gC1qR by a specific antibody hindered HA-C1q signaling and prevented HYAL2 upregulation. Thus, C1q-HA interplay is responsible for enhanced HYAL2 expression, suggesting an increased rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments in the MPM TME. Our data support the notion of an overall tumor-promoting property of C1q. Moreover, the overlapping localization and physical interaction between HYAL2 and gC1qR suggests a potential regulatory effect of gC1qR within a putative HA-C1q macromolecular complex.

Low-Molecular-Weight Hyaluronic Acid Contributes to Noise-Induced Cochlear Inflammation

Introduction: Our previous work indicated that the activation of the Toll-like receptor (TLR) 4 signaling pathway contributed to noise-induced cochlear inflammation. Previous studies have reported that low-molecular-weight hyaluronic acid (LMW-HA) accumulates during aseptic trauma and promotes inflammation by activating the TLR4 signaling pathway. We hypothesized that LMW-HA or enzymes synthesizing or degrading HA might be involved in noise-induced cochlear inflammation. Methods: The present study included two arms. The first arm was the noise exposure study, in which TLR4, proinflammatory cytokines, HA, hyaluronic acid synthases (HASs), and hyaluronidases (HYALs) in the cochlea as well as auditory brainstem response (ABR) thresholds were measured before and after noise exposure. The second arm was analysis of HA delivery-induced reactions, in which control solution, high-molecular-weight HA (HMW-HA), or LMW-HA was delivered into the cochlea by cochleostomy or intratympanic injection. Then, the ABR threshold and cochlear inflammation were measured. Results: After noise exposure, the expression of TLR4, proinflammatory cytokines, HAS1, and HAS3 in the cochlea significantly increased over the 3rd to 7th day post-noise exposure (PE3, PE7). The expression of HYAL2 and HYAL3 dramatically decreased immediately after noise exposure, gradually increased thereafter to levels significantly greater than the preexposure level on PE3, and then rapidly returned to the preexposure level on PE7. The expression of HA, HAS2, and HYAL1 in the cochlea remained unchanged after exposure. After cochleostomy or intratympanic injection, both the hearing threshold shifts and the expression of TLR4, TNF-α, and IL-1β in the cochleae of the LMW-HA group were obviously greater than those of the control group and HMW-HA group. The expression of proinflammatory cytokines in the LMW-HA and control groups on the 7th day (D7) after cochleostomy tended to increase compared to that on the 3rd day (D3), whereas levels in the HMW-HA group tended to decrease on D7 compared to D3. Conclusion: HAS1, HAS3, HYAL2, and HYAL3 in the cochlea are involved in acoustic trauma-induced cochlear inflammation through the potential proinflammatory function of LMW-HA.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer

PurposeHyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients’ survival. The impact of a siRNA depletion of HAS2 was investigated in vitro.MethodsUsing the Kaplan–Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized.ResultsHAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin.ConclusionIn conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

High Levels of PD-L1+ and Hyal2+ Myeloid-derived Suppressor Cells in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) patients frequently have increased number of immunosuppressive myeloid cells in circulation. High number of myeloid-derived suppressor cells (MDSCs) in the blood are associated with immune suppression as well as with cancer-related inflammation which drives the mobilization of myeloid cells to tumor tissue. Here, we show that peripheral blood from a previously untreated RCC patient has increased the number of monocytic CD33+CD11b+ MDSCs, which also co-expressed PD-L1 and membrane-bound enzyme hyaluronidase 2 (Hyal2). PD-L1 expression is associated with immune suppression, whereas expression of Hyal2 is associated with inflammation, because Hyal2+ myeloid cells can degrade the extracellular hyaluronan (HA), leading to the accumulation of pro-inflammatory HA fragments with low molecular weight. These findings implicate the potential involvement of monocytic MDSCs in both tumor-associated immune suppression and cancer-related inflammation. Analysis of organotypic tumor-tissue slice cultures prepared from cancer tissue of the same patient revealed the significant presence of PD-L1+ HLA-DR+ macrophage-like or dendritic cell-like antigen-presenting cells in tumor stroma. Interestingly, stroma-associated PD-L1+ cells frequently have intracellular hyaluronan. Collectively, data presented in this study suggest that the interplay between tumor-recruited myeloid cells and stromal HA may contribute to the inflammation and immune tolerance in kidney cancer.

Detection of DNA methylation of HYAL2 gene for differentiating malignant from benign thyroid tumors

To assess the value of DNA methylation level of HYAL2 gene as a molecular marker for differential diagnosis of malignant and benign thyroid tumors. DNA methylation of HYAL2 gene in tissue specimens of 190 patients with papillary thyroid cancer (PTC) and 190 age- and gender-matched patients with benign thyroid tumors was examined by mass spectrometry, and the protein expression of HYAL2 was detected immunohistochemically for another 55 pairs of patients. Logistic regression analysis was performed to calculate the odds ratio (OR) and evaluate the correlation of per 10% reduction in DNA methylation with PTC. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) was calculated to assess the predictive value of alterations in HYAL2 methylation. Hypomethylation of HYAL2_CpG_3 was significantly correlated with early-stage PTC (OR=1.51, P=0.001), even in stage I cancer (OR=1.42, P=0.007). Age-stratified analysis revealed a significantly stronger correlation between increased HYAL2_CpG_ 3 methylation and early-stage PTC in patients below 50 years than in those older than 50 years (OR: 1.89 vs 1.37, P < 0.05); ROC analysis also showed a larger AUC of 0.787 in younger patients. The results of immunohistochemistry showed that patients with PTC had significantly higher protein expressions of HYAL2 than patients with benign tumors. The alterations of DNA methylation level of HYAL2 gene is significantly correlated with early-stage PTC, suggesting the value of DNA methylation level as a potential biomarker for differentiation of malignant from benign thyroid tumors.

Alterations in plasma hyaluronic acid in patients with clinically stable COPD versus (non)smoking controls

Hyaluronic acid (HA) is a key component of the extracellular matrix. HA and its metabolism are suggested to be altered in the lungs of patients with chronic obstructive pulmonary disease (COPD). The present study explored systemic HA, and its metabolic regulators, in patients with clinically stable COPD and smoking and non-smoking controls. Furthermore, associations of HA with acute exacerbations (AECOPD), airway-related hospitalizations, systemic inflammation and cardiovascular risk were studied. In total, 192 patients with moderate to very severe COPD [aged 62.3 y (± SD 7.0)], 84 smoking controls [aged 61.8 y (± 5.7)], and 107 non-smoking controls [aged 60.1 y (± 7.0)] were included. Plasma HA was reduced in patients with COPD compared to non-smoking controls (p = 0.033), but was comparable after adjusting for age and sex. Expression of HAS-3 did not differ between groups, but was substantially less detectable in more patients with COPD than (non)smoking controls (p < 0.001). Expression of HYAL-2 was enhanced in patients with COPD versus smoking (p = 0.019) and non-smoking (p < 0.001) controls, also in the age- and sex- adjusted model (p < 0.001). Plasma HA was not associated with AECOPD, airway-related hospitalizations in the previous year, or systemic inflammation in COPD. Arterial pulse wave velocity explained some of the variance (< 10%) in plasma HA (p = 0.006). Overall, these results indicate that expression of HYAL-2, but not plasma HA nor HAS-3, is enhanced in patients with COPD compared to (non)smoking controls. Furthermore, HA was not associated with clinical outcomes, yet, cardiovascular risk might play a role in its systemic regulation in stable COPD.

Hyal2 Expression in Tumor-Associated Myeloid Cells Mediates Cancer-Related Inflammation in Bladder Cancer.

The increased presence of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumor tissue has been extensively reported. However, their role in the regulation of hyaluronan (HA) metabolism in the tumor microenvironment has not been established. Here we describe a novel function of tumor-associated myeloid cells related to the enhanced breakdown of extracellular HA in human bladder cancer tissue, leading to the accumulation of small HA fragments with molecular weight (MW) <20 kDa. Increased fragmentation of extracellular HA and accumulation of low molecular weight HA (LMW-HA) in tumor tissue was associated with elevated production of multiple inflammatory cytokines, chemokines, and angiogenic factors. The fragmentation of HA by myeloid cells was mediated by the membrane-bound enzyme hyaluronidase 2 (Hyal2). Increased numbers of Hyal2+CD11b+ myeloid cells were detected in the tumor tissue as well as in the peripheral blood of patients with bladder cancer. Coexpression of CD33 suggested that these cells belong to monocytic myeloid-derived suppressor cells. The HA-degrading function of Hyal2-expressing MDSCs could be enhanced by exposure to tumor-conditioned medium, and IL1β was identified as one of the factors involved in the stimulation of Hyal2 activity. CD44-mediated signaling played an important role in the regulation of HA-degrading activity of Hyal2-expressing myeloid cells, as the engagement of CD44 receptor with specific mAb triggered translocation of Hyal2 enzyme to the cellular surface and stimulated secretion of IL1β. Taken together, this work identifies Hyal2-expressing tumor-associated myeloid cells as key players in the accumulation of LMW-HA in the tumor microenvironment and cancer-related inflammation and angiogenesis. SIGNIFICANCE: This study identifies Hyal2-expressing tumor-associated myeloid cells of monocyte-macrophage lineage as contributors to hyaluronan degradation in bladder cancer tissue, leading to accumulation of inflammatory and proangiogenic low molecular weight hyaluronan fragments.

MP17-05 MYELOID CELLS IN BLADDER CANCER MICROENVIRONMENT DEGRADE HYALURONIC ACID INTO INFLAMMATORY LOW MOLECULAR WEIGHT HYALURON

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-05 MYELOID CELLS IN BLADDER CANCER MICROENVIRONMENT DEGRADE HYALURONIC ACID INTO INFLAMMATORY LOW MOLECULAR WEIGHT HYALURON Paul Dominguez-Gutierrez*, Elizabeth Kwenda, Willim Donelan, Padraic O'Malley, Paul Crispen, and Sergei Kusmartsev Paul Dominguez-Gutierrez*Paul Dominguez-Gutierrez* More articles by this author , Elizabeth KwendaElizabeth Kwenda More articles by this author , Willim DonelanWillim Donelan More articles by this author , Padraic O'MalleyPadraic O'Malley More articles by this author , Paul CrispenPaul Crispen More articles by this author , and Sergei KusmartsevSergei Kusmartsev More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hyaluronic acid (HA) is a multifunctional glycosaminoglycan that is involved in tissue regeneration, inflammation response, and angiogenesis. HA is degraded by hyaluronidases (HYAL) 1-3 that cleave HA into specific low molecular weights (LMW) HA (<20KDa) that act as ligands for CD44, RHAMM, and ICAM-1. Multiple cancers have increased levels of HA, suggesting abnormal HA metabolism. This study sought to elucidate the role and factors associated with increased HA accumulation within the bladder cancer tumor microenvironment. METHODS: IRB approval and patient informed consent were given before collection of normal and tumor bladder tissue during cystectomy. 300µm precision-cut tissue slice were cultured for 3-14 days. Polyacrylamide gel electrophoresis was used for analysis of HA size using. To test the effect of the tumor microenvironment on normal cells, naïve myeloid cells were isolated from mouse bone marrow and cultured in plates precoated with high molecular weight (HMW) HA (>200KDa) in the presence or absence of human tumor conditioned media and examined using fluorescent microscopy. Realtime PCR was used to measure HYAL 1-3 expression and WES capillary electrophoresis was used to validate protein levels of Hyal2. RESULTS: Cultured slices from 25 bladder cancer patients were characterized by LMW-HA (Fig 1A). Normal bladder tissue was characterized by long structured linear pericellular HA, low inflammatory infiltrates, and > 200KDa HMW-HA (Fig1B). LMW-HA levels were undetectable in normal bladder samples. Furthermore, tumor bladder tissue was infiltrated with Hyal2 expressing myeloid cells. Hyal-2 expression in myeloid cells was upregulated by exposure to tumor-conditioned medium. CONCLUSIONS: Experimental and clinical samples of bladder cancer demonstrate high infiltration of Hyal-2 expressing myeloid cells. Increased Hyal-2 expression promotes degradation of HMW-HA into highly fragmented LMW-HA. This LMW-HA triggers inflammation via increased production of cytokines, chemokines, and growth factors within the tumor microenvironment. Future studies should focus on Hyal-2 as a potential target for bladder cancer therapy and LMW-HA as a prognostic marker. Source of Funding: J&E King Florida Biomedical Research Program1923 Fund © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e227-e227 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Dominguez-Gutierrez* More articles by this author Elizabeth Kwenda More articles by this author Willim Donelan More articles by this author Padraic O'Malley More articles by this author Paul Crispen More articles by this author Sergei Kusmartsev More articles by this author Expand All Advertisement PDF downloadLoading ...

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

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

Characteristics of Hyaluronan Synthesis Inhibition by 4-Methylumbelliferone in Orbital Fibroblasts.

PurposeHyaluronan (HA) overproduction by orbital fibroblasts (OFs) is a major factor in the pathogenesis of Graves’ orbitopathy (GO). 4-methylumbelliferone (4-MU) is an inhibitor of HA synthesis in different cell types in vitro and has beneficial effects in animal models of autoimmune diseases.MethodsHA production and mRNA expression of HA synthases (HAS1, HAS2, and HAS3) and hyaluronidases (HYAL1 and HYAL2) were measured in the presence and absence of 4-MU in unstimulated and transforming growth factor–β–stimulated fibroblasts from GO orbital (n = 4), non-GO orbital (n = 4), and dermal origin (n = 4).ResultsThe 4-MU treatment (1 mM) for 24 hours resulted in an average 87% reduction (P < 0.001) of HA synthesis, decreased the expression of the dominant HAS isoform (HAS2) by 80% (P < 0.0001), and increased the HYAL2 expression by 2.5-fold (P < 0.001) in control OFs, GO OFs, and dermal fibroblasts (DFs) regardless of the origin of the cells. The proliferation rate of all studied cell lines was reduced to an average 16% by 4-MU (P < 0.0001) without any effects on cell viability. HA production stimulated by transforming growth factor–β was decreased by 4-MU via inhibition of stimulated HAS1 expression in addition to the observed effects of 4-MU in unstimulated cases. Characteristics of HA synthesis inhibition by 4-MU did not differ in OFs compared with DFs.Conclusions4-MU has been found to inhibit the HA synthesis and the proliferation rate in OFs in vitro, adding it to the list of putative therapeutic agents in a disease the cure of which is largely unresolved.

Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc

PurposeHyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease.MethodsGene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1β, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression.ResultsGene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor–donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls.ConclusionAlthough HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target.Graphic abstractThese slides can be retrieved under Electronic Supplementary Material.

The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract—the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV–ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells.

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.

P4672Differential expression of HYAL2 and CD44 splicing variants in acute coronary syndromes, stable angina patients and healthy controls: correlation between hyaluronan-pathway and plaque erosion

P4672Differential expression of HYAL2 and CD44 splicing variants in acute coronary syndromes, stable angina patients and healthy controls: correlation between hyaluronan-pathway and plaque erosion

Decreased hyaluronidase 1 expression is associated with early disease recurrence in human endometrial cancer

ObjectiveHyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. MethodsA total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial–mesenchymal transition marker, E-cadherin, and disease recurrence. ResultsReduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131–23.270, p=0.034). ConclusionsThis study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.

A fermented barley and soybean formula enhances skin hydration

Skin hydration is one of the primary aims of beauty and anti-aging treatments. Barley (Hordeum vulgare) and soybean (Glycine max) are major food crops, but can also be used as ingredients for the maintenance of skin health. We developed a natural product-based skin treatment using a barley and soybean formula (BS) incorporating yeast fermentation, and evaluated its skin hydration effects as a dietary supplement in a clinical study. Participants ingested a placebo- (n = 33) or BS- (3 g/day) containing drink (n = 32) for 8 weeks. A significant increase in hydration in the BS group as compared to the placebo group was observed on the faces of subjects after 4 and 8 weeks, and on the forearm after 4 weeks. Decreases in stratum corneum (SC) thickness were also observed on the face and forearm. BS enhanced hyaluronan (HA) and skin barrier function in vitro and reduced Hyal2 expression in human dermal fibroblasts (HDF). BS also recovered ultraviolet (UV) B-induced downregulation of HA in HaCaT cells. These results suggest that BS has promising potential for development as a health functional food to enhance skin health.

The role of platelet activation in the expression and function of platelet hyaluronidase‐2 (1046.7)

Background: The deposition of hyaluronan (HA), which is a major glycosaminoglycan component of the extracellular matrix, increases during the course of inflammation. The inflammatory HA matrix, which can promote leukocyte binding and recruitment, can be fragmented be the hyaluronidase enzymes, specifically HYAL1 and HYAL2. Our lab has recently shown that human platelets express HYAL2 and that they can degrade HA from the surfaces of intestinal cells into smaller fragments capable of promoting inflammatory cytokine production. Objective: Define the mechanism by which platelets degrade HA and assess the role of platelet activation. Methods: Platelet degradation of purified HA in solution, immobilized HA and cell surface HA was assessed by carbohydrate gel electrophoresis, hyaluronidase assay, and HA ELISA‐like assay, respectively. HYAL2 expression was assessed by immunoblotting and flow cytometry. Results: 1) Platelets degrade purified HA only under acidic pH; 2) Platelets degrade cell surface HA in a HYAL2 dependent manner; 3) Activation of platelets results in increased surface HYAL2 expression and activity; 4) Platelet activation causes an increase in surface expression of HA binding proteins. Significance: Platelets circulate in an activated state during inflammatory diseases. Increased HYAL2 on activated platelets may lead to higher levels of HA fragments that perpetuate inflammation.Grant Funding Source: Supported by the NHLBI

MP28-15 HYAL4: A NEW HYALURONIDASE ASSOCIATED WITH BLADDER CANCER DIAGNOSIS AND PROGNOSIS

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2014MP28-15 HYAL4: A NEW HYALURONIDASE ASSOCIATED WITH BLADDER CANCER DIAGNOSIS AND PROGNOSIS John Shields, Soum Lokeshwar, Kelly Hoye, Travis Yates, Murugesan Manoharan, and Vinata Lokeshwar John ShieldsJohn Shields More articles by this author , Soum LokeshwarSoum Lokeshwar More articles by this author , Kelly HoyeKelly Hoye 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.2014.02.664AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The Hyaluronidase (HAase) family of enzymes degrade hyaluronic acid (HA). In the human genome there are 6 HAase genes clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). While PH20 and HYAL-2 are testicular and lysosomal HAases, HYAL1 is the tumor-derived HAase. HYLA1 promotes tumor growth, invasion and angiogenesis and is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal cells or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. METHODS By real time RT-PCR we examined the mRNA levels of 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). The levels were normalized to β-actin. HYAL4 function was analyzed by stably overexpressing HYAL4 in normal urothelial and BCa cells, using cell proliferation, motitlity and invasion assays and immunoblotting and Q-PCR. RESULTS HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (P<0.001). HYAL3 and PH20 were expressed at 100-fold lower levels. HYAL2 expression was comparable to HYAL1 and HYAL4, but the difference in the expression between NBL and TBL was not significant (P>0.05). cDNA cloning revealed that HYALP1 encodes a non-functional truncated protein. HYAL1, HYAL4 and HYALP1 mRNA levels were also significantly elevated in BCa patients’ urine (P< 0.001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than to detect BCa. Overexpression of HYAL-4 in normal urothelial and BCa cells significantly promoted invasion and chemotactic motility (> 3-fold). This increase was due to up-regulation of metastasis promoters, including CD44, RHAMM) MMP-9 and β-catenin. CONCLUSIONS This is the first study that shows like HYAL1, HYAL4 is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e301-e302 Advertisement Copyright & Permissions© 2014MetricsAuthor Information John Shields More articles by this author Soum Lokeshwar More articles by this author Kelly Hoye 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 ...