Anionic cardiolipin stabilizes the transmembrane region of hyaluronan synthase and promotes catalysis-relevant dynamics.

Hyaluronic acid (HA) is a glycosaminoglycan essential for cellular processes and finding increasingly applications in medicine, pharmaceuticals, and cosmetics. While membrane-integrated Class I hyaluronan synthase (HAS) catalyzes HA synthesis in most organisms, the molecular mechanisms by which HAS-lipid interactions impact HAS catalysis remain unclear. This study employed coarse-grained molecular dynamics simulation combined with dimensionality reduction to uncover the interplay between lipids and Streptococcus equisimilis HAS (SeHAS). A minimum of 67 % cardiolipin is necessary for HA synthesis, as determined through simulations using gradient-composed membranes. The anionic cardiolipin stabilizes the cationic transmembrane regions of SeHAS and thereby maintains its conformation. Moreover, the highly dynamic cardiolipin is required to modulate the catalysis-relevant motions in HAS and thus facilitate HA synthesis. These findings provide molecular insights essential not only for understanding the physiological functions of HAS, but also for the development of cell factories and enzyme catalysts for HA production.

Hyaluronic acid as a tumor progression agent and a potential chemotherapeutic biomolecule against cancer: A review on its dual role.

Hyaluronic acid is a major constituent of the extracellular matrix of vertebrate tissue that provides mechanical support to cells and acts as a mediator in regulation of necessary biochemical process essential for maintenance of tissue homeostasis. The variation in quantity of hyaluronic acid content in tissues is often associated with different pathological conditions. It is associated with tumor aggression and progression as it plays crucial role in regulating different aspects of tumorigenesis and several defined hallmarks of cancer. It assists in tumor progression by undergoing extracellular remodeling to establish tumor microenvironment which restricts the delivery of cytotoxic drugs to neoplastic cells due to increase in interstitial pressure. Hyaluronic acid catabolic and anabolic genes and low-molecular weight hyaluronic acid play significant role in the establishing tumor microenvironment by assisting in cell proliferation, metastasis and invasion. On the other hand, it is also used as an effective drug-delivery platform in cancer therapies as its biocompatibility and biodegradability lower the toxicity of chemotherapeutic drugs and increase drug retention. High-molecular weight hyaluronic acid-bioconjugates specifically bind with hyaladherins, facilitating targeted drug delivery and also exert anti-inflammatory properties. This review also highlights the market and patent trends in the development of effective chemotherapeutic hyaluronic acid formulations and the current scenario regarding clinical trials.

Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan.

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme-1 min-1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.

Lovastatin impairs cellular proliferation and enhances hyaluronic acid production in fibroblast-like synoviocytes.

INTRODUCTION: Statins have demonstrated chondroprotective effects by reducing inflammation and mitigating extracellular matrix degradation. However, statins are also reported to be cytotoxic to several types of cells. Early-onset osteoarthritis (OA) is characterized by synovial inflammation, which adversely affects hyaluronan (HA) production in fibroblast-like synoviocytes (FLSs). Nevertheless, the precise effects of statins on the synovium remain unclear. METHODS: This study investigated the impact of lovastatin on human FLSs, and HA secretion-related genes, signaling pathways, and production were evaluated. RESULTS: The findings revealed that high doses of lovastatin (20 or 40 µM) decreased FLS viability and increased cell death. FLS proliferation ceased when cultured in a medium containing 5 or 10 µM lovastatin. mRNA expression analysis demonstrated that lovastatin (5 and 10 µM) upregulated the gene level of hyaluronan synthase 1 (HAS1), HAS2, and proteoglycan 4 (PRG4), but not HAS3. While the expression of multidrug resistance-associated protein 5 transporter gene remained unaffected, both inward-rectifying potassium channel and acid-sensing ion channel 3 were upregulated. Western blot further confirmed that lovastatin increased the production of HAS1 and PRG4, and activated the PKC-α, ERK1/2, and p38-MAPK signaling pathways. Additionally, lovastatin elevated intracellular cAMP levels and HA production in FLSs. CONCLUSION: Lovastatin impairs cellular proliferation but enhances HA production in human FLSs.

A role for decorin in improving motor deficits after traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability due to injury worldwide. Extracellular matrix (ECM) remodeling is known to significantly contribute to TBI pathophysiology. Glycosaminoglycans, which are long-chain, variably sulfated polysaccharides abundant within the ECM, have previously been shown to be substantially altered after TBI. In this study, we sought to delineate the dynamics of glycosaminoglycan alterations after TBI and discover the precise biologic processes responsible for observed glycosaminoglycan changes after injury. We performed state-of-the art mass spectrometry on brain tissues isolated from mice after TBI or craniotomy-alone. We observed dynamic changes in glycosaminoglycans at Day 1 and 7 post-TBI, with heparan sulfate, chondroitin sulfate, and hyaluronan remaining significantly increased after a week vis-à-vis craniotomy-alone tissues. We did not observe appreciable changes in circulating glycosaminoglycans in mice after experimental TBI compared to craniotomy-alone nor in patients with TBI and severe polytrauma compared to control patients with mild injuries, suggesting increases in injury site glycosaminoglycans are driven by local synthesis. We subsequently performed an unbiased whole genome transcriptomics analysis on mouse brain tissues 7 days post-TBI and discovered a significant induction of hyaluronan synthase 2, glypican-3, and decorin. The functional role of decorin after injury was further examined through multimodal behavioral testing comparing wild-type and Dcn-/- mice. We discovered that genetic ablation of Dcn led to an overall negative effect of TBI on function, exacerbating motor impairments after TBI. Collectively, our results provide a spatiotemporal characterization of post-TBI glycosaminoglycan alterations in the brain ECM and support an important adaptive role for decorin upregulation after TBI.

Attenuated Replication-Competent Herpes Simplex Virus Expressing an ECM-Modifying Transgene Hyaluronan Synthase 2 of Naked Mole Rat in Oncolytic Gene Therapy.

Herpes simplex virus (HSV) has proven successful in treating human cancer. Since the approval of talimogene laherparepvec (T-VEC) in 2015, HSV has been thoroughly researched to discover novel mechanisms to combat cancer and treat other diseases. Another HSV-based drug, beremagene geperpavec (B-VEC), received approval in 2023 to treat the rare genetic disease dystrophic epidermolysis bullosa, and was also the first clinically approved HSV vector carrying an extracellular matrix (ECM)-modifying transgene. The ECM is a network of macromolecules surrounding cells, which provides support and regulates cell growth and differentiation, the disruption of which is common in cancer. The naked mole rat (NMR) has a thick ECM and a unique mutation in the hyaluronan synthase 2 (HAS2) gene, which has been linked to the high cancer resistance of the species. To study the effect of this mutation in human cancer, we have developed an attenuated, replication-competent HSV vector expressing the NMR-HAS2 gene. The viral replication, transgene expression and cytotoxic effect of the novel vector was studied in glioma cells. Our results show that an attenuated, replication-competent HSV vector expressing a foreign ECM-modifying transgene, namely HAS2, provides an effective tool to study and combat cancer in humans.

Selective adhesion inhibition and hyaluronan envelope reduction of dermal tumor cells by cold plasma-activated medium.

The sensitivity to cold plasma is specific to tumor cells while leaving normal tissue cells unaffected. This is the desired challenge in cancer therapy. Therefore, the focus of this work was a comparative study concerning the plasma sensitivity of dermal tumor cells (A-431) versus non-tumorigenic dermal cells (HaCaT) regarding their adhesion capacity. We found a selective inhibiting effect of plasma-activated medium on the adhesion of tumor cells while hardly affecting normal cells. We attributed this to a lower basal gene expression for the adhesion-relevant components CD44, hyaluronan synthase 2 (HAS2), HAS3, and the hyaluronidases in A431. Noteworthy, after plasma exposure, we revealed a significantly higher expression and synthesis of the hyaluronan envelope, the HAS3 gene, and the transmembrane adhesion receptors in non-tumorigenic HaCaTs.

Super Enhancer Driven Hyaluronan Synthase 3 Promotes Malignant Progression of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck with high metastatic and invasive nature. Super enhancers (SEs) control the expression of cell identity genes and oncogenes during tumorigenesis. As a glycosaminoglycan in the tumor microenvironment, hyaluronan (HA) is associated with cancer development. High expression of hyaluronan synthase 3 (HAS3) resulted in HA deposition, which promoted the growth of cancer cell. However, its role in NPC development remains elusive. We demonstrated that the levels of HAS3 mRNA or protein were increased in NPC cell lines. Transcription of HAS3 is associated with SE. Disruption of SE by bromodomain containing 4 (BRD4) inhibitor JQ1 resulted in downregulation of HAS3 and inhibition of cell proliferation and invasiveness in NPC cells. Inhibition of HA synthesis by HAS inhibitor 4-MU suppressed cell growth and invasion of NPC cells, whereas HA treatment exerted opposite effects. Genetically silencing HAS3 in HK1 and FaDu NPC cells attenuated cell proliferation and mobility, while re-expression of HAS3 enhanced malignant potential of CNE1 and CNE2 NPC cells. Furthermore, loss of HAS3 impaired metastatic potential of HK1 cells in nude mice. Mechanistically, inhibition of HA synthesis by chemical inhibitor or silencing HAS3 led to reduction of the levels of phosphorylation of EGFR, AKT, and ERK proteins. In contrast, exogenous HA treatment or forced expression of HAS3 activated EGFR/AKT/ERK signaling cascade. This study suggested that HAS3 is driven by SE and overexpressed in NPC. High expression of HAS3 promotes the malignant features of NPC via activation of EGFR/AKT/ERK signaling pathway.

Mimicking Antioxidases and Hyaluronan Synthase: A Zwitterionic Nanozyme for Photothermal Therapy of Osteoarthritis.

Restoring joint homeostasis is crucial for relieving osteoarthritis (OA). Current strategies are limited to unilateral efforts in joint lubrication, inhibition of inflammation, free radicals scavenging, and cartilage regeneration. Herein, by modifying molybdenum disulfide (MoS2 ) with Mg2+ -doped polydopamine and coating with polysulfobetaines, a dual-bionic photothermal nanozyme (MPMP) is constructed to mimic antioxidases/hyaluronan synthase for OA therapy. Photothermally enhanced lubrication lowers the coefficient of friction (0.028) in the early stage of OA treatment. The antioxidases-mimicking properties of MPMP nanozyme contribute to eliminating reactive oxygen and nitrogen species (ROS/RNS) (over 90% of scavenging ratio for H2 O2 /·OH/O· 2 - /DPPH/ABTS+ ) and supplying O2 . With NIR irradiation, the MPMP nanozyme triggers thermogenesis (upregulating HSP70 expression) and Mg2+ release, which promotes the chondrogenesis in inflammatory conditions by deactivating NF-κB/IL-17 signaling pathways and enhancing MAPK signaling pathway. Benefiting from HSP70 and Mg2+ , MPMP-NIR shows HAS-mimicking activity to increase the intracellular (twofold) and extracellular (3.12-fold) HA production. Therefore, MPMP-NIR demonstrates superior spatiotemporally therapeutic effect on OA in mice model, in terms of osteophytes (83.41% of reduction), OARSI scores (88.57% of reduction), and ACAN expression (2.70-fold of increment). Hence, insights into dual-bionic nanozymes can be a promising strategy for OA therapy or other inflammation-related diseases.

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

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

MicroRNA-376b is involved in the pathogenesis of thyroid-associated ophthalmopathy by regulating HAS2.

PURPOSE: The aim of this study was to investigate the microRNA (miRNA) expression profile in peripheral blood mononuclear cells (PBMC) of thyroid-associated ophthalmopathy (TAO) patients and to explore the molecular mechanisms of MicroRNA-376b (miR-376b) in the pathogenesis of TAO. METHODS: PBMCs from TAO patients and healthy controls were analyzed by miRNA microarray to screen for the significantly differentially expressed miRNAs. The miR-376b expression in PBMCs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The downstream target of miR-376b was screened by online bioinformatics, and detected by qRT-PCR and Western blotting. RESULTS: Compared with normal controls, 26 miRNAs were significantly different in PBMCs of TAO patients (14 miRNAs were down-regulated and 12 miRNAs were up-regulated). Among them, miR-376b expression was significantly decreased in PBMCs from TAO patients compared to healthy controls. Spearman correlation analysis revealed that miR-376b expression in PBMCs was significantly negatively correlated with free triiodothyronine (FT3), and positively correlated with thyroid-stimulating hormone (TSH). MiR-376b expression was obviously reduced in 6T-CEM cells after triiodothyronine (T3) stimulation compared to controls. MiR-376b mimics significantly decreased hyaluronan synthase 2 (HAS2) protein expression and the mRNA expression of intercellular cell adhesion molecule-1 (ICAM1) and tumor necrosis factor-α (TNF-α) in 6T-CEM cells, whereas miR-376b inhibitors markedly elevated HAS2 protein expression and gene expression of ICAM1 and TNF-α. CONCLUSIONS: MiR-376b expression in PBMCs was significantly decreased in PBMCs from TAO patients compared with the healthy controls. MiR-376b, regulated by T3, could modulate the expression of HAS2 and inflammatory factors. We speculate that miR-376b may be involved in the pathogenesis of TAO patients by regulating the expression of HAS2 and inflammatory factors.

Downregulation of HAS­2 regulates the chondrocyte cytoskeleton and induces cartilage degeneration by activating the RhoA/ROCK signaling pathway.

Osteoarthritis (OA) is a progressive joint disorder, which is principally characterized by the degeneration and destruction of articular cartilage. The cytoskeleton is a vital structure that maintains the morphology and function of chondrocytes, and its destruction is a crucial risk factor leading to chondrocyte degeneration and OA. Hyaluronan synthase­2 (HAS­2) is a key enzyme in synthesizing hyaluronic acid (HA) in vivo. The synthesis of high molecular weight HA catalyzed by HAS­2 serves a vital role in joint movement and homeostasis; however, it is unclear what important role HAS­2 plays in maintaining chondrocyte cytoskeleton morphology and in cartilage degeneration. The present study downregulated the expression of HAS­2 by employing 4­methylumbelliferone (4­MU) and RNA interference. In vitro experiments, including reverse transcription­quantitative PCR, western blotting, laser scanning confocal microscopy and flow cytometry were subsequently performed. The results revealed that downregulation of HAS­2 could activate the RhoA/ROCK signaling pathway, cause morphological abnormalities, decrease expression of the chondrocyte cytoskeleton proteins and promote chondrocyte apoptosis. In vivo experiments, including immunohistochemistry and Mankin's scoring, were performed to verify the effect of HAS­2 on the chondrocyte cytoskeleton, and it was revealed that inhibition of HAS­2 could cause cartilage degeneration. In conclusion, the present results revealed that downregulation of HAS­2 could activate the RhoA/ROCK pathway, cause abnormal morphology and decrease chondrocyte cytoskeleton protein expression, leading to changes in the signal transduction and biomechanical properties of chondrocytes, promotion of chondrocyte apoptosis and the induction of cartilage degeneration. Moreover, the clinical application of 4­MU may cause cartilage degeneration. Therefore, targeting HAS­2 may provide a novel therapeutic strategy for delaying chondrocyte degeneration, and the early prevention and treatment of OA.

Loss of hyaluronan synthases impacts bone morphology, quality, and mechanical properties.

Hyaluronan, a glycosaminoglycan synthesized by three isoenzymes (Has1, Has2, Has3), is known to play a role in regulating bone turnover, remodeling, and mineralization, which in turn can affect bone quality and strength. The goal of this study is to characterize how the loss of Has1 or Has3 affects the morphology, matrix properties, and overall strength of murine bone. Femora were isolated from Has1-/-, Has3-/-, and wildtype (WT) C57Bl/6 J female mice and were analyzed using microcomputed-tomography, confocal Raman spectroscopy, three-point bending, and nanoindentation. Of the three genotypes tested, Has1-/- bones demonstrated significantly lower cross-sectional area (p = 0.0002), reduced hardness (p = 0.033), and lower mineral-to-matrix ratio (p < 0.0001). Has3-/- bones had significantly higher stiffness (p < 0.0001) and higher mineral-to-matrix ratio (p < 0.0001) but lower strength (p = 0.0014) and bone mineral density (p < 0.0001) than WT. Interestingly, loss of Has3 was also associated with significantly lower accumulation of advanced glycation end-products than WT (p = 0.0478). Taken together, these results demonstrate, for the first time, the impact of the loss of hyaluronan synthase isoforms on cortical bone structure, content, and biomechanics. Loss of Has1 impacted morphology, mineralization, and micron-level hardness, while loss of Has3 reduced bone mineral density and affected organic matrix composition, impacting whole bone mechanics. This is the first study to characterize the effect of loss of hyaluronan synthases on bone quality, suggesting an essential role hyaluronan plays during the development and regulation of bone.

The role of hyaluronan in endothelial glycocalyx and potential preventative lifestyle strategy with advancing age.

The endothelial glycocalyx (EG) is a gel-like structure that forms a layer in between the surface of the endothelium and lumen. EG was once thought to be merely a structural support for the endothelium. However, in recent years, the importance of EG as a first line of defense and a key regulator to endothelial integrity has been illuminated. With advanced age, EG deterioration becomes more noticeable and at least partially associated with endothelial dysfunction. Hyaluronan (HA), one of the critical components of the EG, has distinct properties and roles to the maintenance of EG and endothelial function. Therefore, given the intimate relationship between the EG and endothelium during the aging process, HA may serve as a promising therapeutic target to prevent endothelial dysfunction.

Prognostic value of midkine, syndecan-1, hyaluronan synthase-2, sestrin-1, laminin subunit alpha-4 and fibulin-3 for malignant pleural mesothelioma.

Introduction: The prognosis of malignant pleural mesothelioma (MPM) is poor, with a limited survival time. In this study, we aimed to examine expression levels of genes selected from relevant literature and to utilize in silico methods to determine genes whose expression could reflect the prognosis of patients with MPM by ex-vivo validation experiments. Material and methods: The study group consisted of 54 MPM patients treated with chemotherapy. Expression of 6 genes - midkine (MDK), syndecan-1 (SDC1), hyaluronan synthase-2 (HAS2), sestrin-1 (SESN1), laminin subunit alpha-4 (LAMA4), and fibulin-3 (FBLN3) - was examined by qPCR in tumor tissues. Sestrin-1 and LAMA4 were identified using an in house R-based script: Unsupervised Survival Analysis Tool. Midkine, SDC1, HAS2, and FBLN3 were selected from current literature. We used two housekeeping genes, i.e. glucose-6-phosphate dehydrogenase and TATA-box binding protein, as controls. Results: Of the patients, 43 (79.6%) had epithelioid mesothelioma. The median survival for all patients was 10 (±1.2 SE) months (95% CI: 7.7-12.3). In multivariate analyses, MDK (p = 0.007), HAS2 (p = 0.008) and SESN1 (p = 0.014) expression levels were related to survival time in the whole group. In epithelioid type MPM patients, MDK (p = 0.014), FBLN3 (p = 0.029), HAS2 (p = 0.014) and SESN1 (p = 0.045) expression was related to survival time in multivariate analyses. Conclusions: High HAS2 and SESN1 expressions and low MDK are potential biomarkers of good prognosis in MPM. High HAS2 and SESN1 expression and low MDK and FBLN3 can also be utilized as biomarkers of good prognosis for epithelioid MPM. Those results should be further investigated in sera, plasma, and pleural effusions.

Conditional expression of endorepellin in the tumor vasculature attenuates breast cancer growth, angiogenesis and hyaluronan deposition.

The tumor stroma of most solid malignancies is characterized by a pathological accumulation of pro-angiogenic and pro-tumorigenic hyaluronan driving tumorigenesis and metastatic potential. Of all three hyaluronan synthase isoforms, HAS2 is the primary enzyme that promotes the build-up of tumorigenic HA in breast cancer. Previously, we discovered that endorepellin, the angiostatic C-terminal fragment of perlecan, evokes a catabolic mechanism targeting endothelial HAS2 and hyaluronan via autophagic induction. To explore the translational implications of endorepellin in breast cancer, we created a double transgenic, inducible Tie2CreERT2;endorepellin(ER)Ki mouse line that expresses recombinant endorepellin specifically from the endothelium. We investigated the therapeutic effects of recombinant endorepellin overexpression in an orthotopic, syngeneic breast cancer allograft mouse model. First, adenoviral delivery of Cre evoking intratumor expression of endorepellin in ERKi mice suppressed breast cancer growth, peritumor hyaluronan and angiogenesis. Moreover, tamoxifen-induced expression of recombinant endorepellin specifically from the endothelium in Tie2CreERT2;ERKi mice markedly suppressed breast cancer allograft growth, hyaluronan deposition in the tumor proper and perivascular tissues, and tumor angiogenesis. These results provide insight into the tumor suppressing activity of endorepellin at the molecular level and implicate endorepellin as a promising cancer protein therapy that targets hyaluronan in the tumor microenvironment.

Identification of a Small Molecule Inhibitor of Hyaluronan Synthesis, DDIT, Targeting Breast Cancer Cells.

Breast cancer is a common cancer in women. Breast cancer cells synthesize large amounts of hyaluronan to assist their proliferation, survival, migration and invasion. Accumulation of hyaluronan and overexpression of its receptor CD44 and hyaluronidase TMEM2 in breast tumors correlate with tumor progression and reduced overall survival of patients. Currently, the only known small molecule inhibitor of hyaluronan synthesis is 4-methyl-umbelliferone (4-MU). Due to the importance of hyaluronan for breast cancer progression, our aim was to identify new, potent and chemically distinct inhibitors of its synthesis. Here, we report a new small molecule inhibitor of hyaluronan synthesis, the thymidine analog 5'-Deoxy-5'-(1,3-Diphenyl-2-Imidazolidinyl)-Thymidine (DDIT). This compound is more potent than 4-MU and displays significant anti-tumorigenic properties. Specifically, DDIT inhibits breast cancer cell proliferation, migration, invasion and cancer stem cell self-renewal by suppressing HAS-synthesized hyaluronan. DDIT appears as a promising lead compound for the development of inhibitors of hyaluronan synthesis with potential usefulness in breast cancer treatment.

4-Methylumebelliferone Enhances Radiosensitizing Effects of Radioresistant Oral Squamous Cell Carcinoma Cells via Hyaluronan Synthase 3 Suppression.

Radioresistant (RR) cells are poor prognostic factors for tumor recurrence and metastasis after radiotherapy. The hyaluronan (HA) synthesis inhibitor, 4-methylumbelliferone (4-MU), shows anti-tumor and anti-metastatic effects through suppressing HA synthase (HAS) expression in various cancer cells. We previously reported that the administration of 4-MU with X-ray irradiation enhanced radiosensitization. However, an effective sensitizer for radioresistant (RR) cells is yet to be established, and it is unknown whether 4-MU exerts radiosensitizing effects on RR cells. We investigated the radiosensitizing effects of 4-MU in RR cell models. This study revealed that 4-MU enhanced intracellular oxidative stress and suppressed the expression of cluster-of-differentiation (CD)-44 and cancer stem cell (CSC)-like phenotypes. Interestingly, eliminating extracellular HA using HA-degrading enzymes did not cause radiosensitization, whereas HAS3 knockdown using siRNA showed similar effects as 4-MU treatment. These results suggest that 4-MU treatment enhances radiosensitization of RR cells through enhancing oxidative stress and suppressing the CSC-like phenotype. Furthermore, the radiosensitizing mechanisms of 4-MU may involve HAS3 or intracellular HA synthesized by HAS3.

Hyaluronan synthase 3 is protective after cardiac ischemia-reperfusion by preserving the T cell response.

Dysregulated extracellular matrix (ECM) is a hallmark of adverse cardiac remodeling after myocardial infarction (MI). Previous work from our laboratory suggests that synthesis of the major ECM component hyaluronan (HA) may be beneficial for post-infarct healing. Here, we aimed to investigate the mechanisms of hyaluronan synthase 3 (HAS3) in cardiac healing after MI. Mice with genetic deletion of Has3 (Has3 KO) and wildtype mice (WT) underwent 45 min of ischemia with subsequent reperfusion (I/R), followed by monitoring of heart function and analysis of tissue remodeling for up to three weeks. Has3 KO mice exhibited impaired cardiac function as evidenced by a reduced ejection fraction. Accordingly, Has3 deficiency also resulted in an increased scar size. Cardiac fibroblast activation and CD68+ macrophage counts were similar between genotypes. However, we found a significant decrease in CD4 T cells in the hearts of Has3 KO mice seven days post-MI, in particular reduced numbers of CD4+CXCR3+ Th1 and CD4+CD25+Treg cells. Furthermore, Has3 deficient cardiac T cells were less activated and more apoptotic as shown by decreased CD69+ and increased annexin V+ cells, respectively. In vitro assays using activated splenic CD3 T cells demonstrated that Has3 deficiency resulted in reduced expression of the main HA receptor CD44 and diminished T cell proliferation. T cell transendothelial migration was similar between genotypes. Of note, analysis of peripheral blood from patients with ST-elevation myocardial infarction (STEMI) revealed that HAS3 is the predominant HAS isoenzyme also in human T cells. In conclusion, our data suggest that HAS3 is required for mounting a physiological T cell response after MI to support cardiac healing. Therefore, our study may serve as a foundation for the development of novel strategies targeting HA-matrix to preserve T cell function after MI.

Hyaluronic acid production and characterization by novel Bacillus subtilis harboring truncated Hyaluronan Synthase.

Hyaluronic Acid (HA) is a natural biopolymer that has important physiological and industrial applications due to its viscoelastic and hydrophilic characteristics. The responsible enzyme for HA production is Hyaluronan synthase (HAS). Although in vitro structure-function of intact HAS enzyme has been partly identified, there is no data on in vivo function of truncated HAS forms. In the current study, novel recombinant Bacillus subtilis strains harboring full length (RBSFA) and truncated forms of SeHAS (RBSTr4 and RBSTr3) were developed and HA production was studied in terms of titer, production rate and molecular weight (Mw). The maximum HA titer for RBSFA, RBSTr4 and RBSTr3 was 602 ± 16.6, 503 ± 19.4 and 728 ± 22.9 mg/L, respectively. Also, the HA production rate was 20.02, 15.90 and 24.42 mg/L.h-1, respectively. The findings revealed that RBSTr3 produced 121% and 137% more HA rather than RBSFA and RBSTr4, respectively. More interestingly, the HA Mw was about 60 kDa for all strains which is much smaller than those obtained in prior studies.