Hyaluronan Synthase Research Articles

Pan-cancer analysis reveals the potential of hyaluronate synthase as therapeutic targets in human tumors

Hyaluronic acid (HA) is a crucial component of the extracellular matrix, and its level of accumulation is related to the progression of various malignant tumors. In this study, a pan-cancer analysis of the three enzymes called hyaluronan synthases (HAS1, HAS2, and HAS3) that produce HA was performed. The study comprehensively describes the characteristics of HAS1, HAS2, and HAS3 in cancers using public databases and tools, to identify the potential biological pathways involved at the molecular, protein, cellular, and clinical sample levels. The analysis showed that dysregulation of the three genes often occurs in cancer, contributing to cancer progression, metastasis, and prognosis. Overexpression of HAS2 promotes secretion of HA in GBM and enhances cell proliferation and migration. The common and specific functions of HAS in certain diseases have important research implications for the treatment and prognosis of tumors.

Effects of Water-Ethanol Extracts from Four Sphagnum Species on Gene Expression of Selected Enzymes in Normal Human Dermal Fibroblasts and Their Antioxidant Properties.

Mosses (Bryophyta), particularly species of the genus Sphagnum, which have been used for centuries for the treatment of skin diseases and damage, are still not explored enough in terms of their use in cosmetics. The purpose of this study was to determine the antioxidant properties of water-ethanol extracts from four selected species of the genus Sphagnum (S. girgenshonii Russow, S. magellanicum Brid., S. palustre L., and S. squarrosum Crome) and their impact on the expression of genes encoding key enzymes for the functioning of the skin. In this study, the effects of Sphagnum extracts on the expression of genes encoding tyrosinase, collagenase, elastase, hyaluronidase and hyaluronic acid synthase in human dermal fibroblasts were determined for the first time in vitro. The extracts inhibited tyrosinase gene expression and showed antioxidant activity. The experiment showed an increase in the expression of some genes encoding collagenase (MMP1) or hyaluronidase (HYAL2, HYAL3 and HYAL4) and a decrease in the hyaluronan synthase (HAS1, HAS2 and HAS3) genes expression by the tested extracts. The obtained results suggest that using extracts from the tested Sphagnum species in anti-aging cosmetics does not seem beneficial. Further studies are needed to clarify their impact on the skin.

Effects of Hyaluronan on Breast Cancer Aggressiveness.

The expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in breast cancer cells is critical for determining tumor aggressiveness and targeting therapies. The presence of such receptors allows for the use of antagonists that effectively reduce breast cancer growth and dissemination. However, the absence of such receptors in triple-negative breast cancer (TNBC) reduces the possibility of targeted therapy, making these tumors very aggressive with a poor outcome. Cancers are not solely composed of tumor cells, but also include several types of infiltrating cells, such as fibroblasts, macrophages, and other immune cells that have critical functions in regulating cancer cell behaviors. In addition to these cells, the extracellular matrix (ECM) has become an important player in many aspects of breast cancer biology, including cell growth, motility, metabolism, and chemoresistance. Hyaluronan (HA) is a key ECM component that promotes cell proliferation and migration in several malignancies. Notably, HA accumulation in the tumor stroma is a negative prognostic factor in breast cancer. HA metabolism depends on the fine balance between HA synthesis by HA synthases and degradation yielded by hyaluronidases. All the different cell types present in the tumor can release HA in the ECM, and in this review, we will describe the role of HA and HA metabolism in different breast cancer subtypes.

The hyaluronan metabolism in the UV-irradiated human epidermis and the relevance of in vitro epidermal models.

Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

Aqueous extract of Cordyceps cicadae (Miq.) promotes hyaluronan synthesis in human skin fibroblasts: A potential moisturizing and anti-aging ingredient.

Cordyceps cicadae (Miq.) is an edible fungus with unique and valuable medicinal properties that is commonly used in traditional Chinese medicine, but its anti-aging effects on the skin fibroblast are not well studied. The aim of the present study was to analyze the active components of aqueous C. cicadae extract (CCE), determine the effects of CCE on hyaluronan synthesis in human skin fibroblasts, and explore the underlying mechanisms. The results of this study indicate that CCE was rich in polysaccharides, five alditols (mainly mannitol), eight nucleosides, protein, and polyphenols, which were present at concentrations of 62.7, 110, 8.26, 35.7, and 3.8 mg/g, respectively. The concentration of extract required to inhibit 50% of 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging capacities were 0.36 ± 0.03 and 4.54 ± 0.10 mg/mL, respectively, indicating that CCE exhibits excellent antioxidant activities. CCE showed no cytotoxicity to skin fibroblasts at concentrations ≤ 100 μg/mL, and promoted HA synthesis in fibroblasts. Treatment of fibroblast cells with 100 μg/mL CCE enhances the HA content to 1293 ± 142 ng/mL, which is significantly more than that in the non-treatment (NT) group (p = 0.0067). Further, RNA sequencing detected 1,192 differentially expressed genes (DEGs) in CCE-treated fibroblasts, among which 417 were upregulated and 775 were downregulated. Kyoto Encyclopedia of Genes (KEGG) and Genomes pathway (GO) analysis based on RNA sequencing revealed that CCE mainly affected cytokine-cytokine receptor interaction regulated by HA synthesis-related genes. CCE upregulated HA synthase 2 (HAS2), epidermal growth factor (EGF)-related genes, heparin-binding EGF-like growth factor, C-C motif chemokine ligand 2, interleukin 1 receptor-associated kinase 2, and other genes related to fibroblast differentiation and proliferation. CCE downregulated the gene of matrix metallopeptidase 12 (MMP12), which leads to cell matrix loss. RT-qPCR further verified CCE significantly upregulated HAS2 expression and significantly downregulated MMP12 expression, thus promoting hyaluronan synthesis. CCE shows potential as a moisturizer and anti-aging agent in functional foods and cosmetics.

Novel β1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a β-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant β1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-β-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, β1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by β1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial β1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel β-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.

Decreased expression of hyaluronan synthase and loss of hyaluronan-rich cells in the anterior tibial fascia of the rat model of chemotherapy-induced peripheral neuropathy.

Previous studies on chemotherapy-induced peripheral neuropathy (CIPN) have focused on neuronal damage. Although some studies have revealed that the fascia is an important sensory organ, currently, we do not know about chemotherapy drug-induced fascial dysfunction. This study aimed to explore the fascia as a nonneural cause of mechanical hypersensitivity in CIPN by investigating the expression of hyaluronic acid synthase (HAS) and histology of the fascia in an animal model of CIPN. Rats were intraperitoneally administered with vincristine (VCR). Mechanical hypersensitivities of the hind paw and the anterior tibial muscle were assessed. The expression of HAS mRNA in the fascia of the anterior tibial muscles was quantitated using reverse transcription polymerase chain reaction. Immunohistochemistry was also performed for HAS2, hyaluronic acid-binding protein, and S100A4 in the fascia. Vincristine administration significantly decreased mechanical withdrawal thresholds in the hind paw and the anterior tibial muscle after day 3. Quantitative polymerase chain reaction showed significant downregulation of HAS mRNAs in the fascia of VCR-treated rats. Immunohistochemical analysis showed that the number of cells with strong HAS2 immunoreactivity, classified as fasciacytes by morphology and colocalized marker S100A4, decreased significantly in the VCR group. Hyaluronic acid plays a critical role in somatic pain sensation. Damaged fascia could be a possible cause of musculoskeletal pain in patients with CIPN. This study suggests that fascia is a nonneural cause and novel therapeutic target for chemotherapy-induced "peripheral neuropathy."

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.

Extracellular vimentin is sufficient to promote cell attachment, spreading, and motility by a mechanism involving N-acetyl glucosamine-containing structures

Vimentin intermediate filaments form part of the cytoskeleton of mesenchymal cells, but under pathological conditions often associated with inflammation, vimentin filaments depolymerize as the result of phosphorylation or citrullination, and vimentin oligomers are secreted or released into the extracellular environment. In the extracellular space, vimentin can bind surfaces of cells and the extracellular matrix, and the interaction between extracellular vimentin and cells can trigger changes in cellular functions, such as activation of fibroblasts to a fibrotic phenotype. The mechanism by which extracellular vimentin binds external cell membranes and whether vimentin alone can act as an adhesive anchor for cells is largely uncharacterized. Here, we show that various cell types (normal and vimentin null fibroblasts, mesenchymal stem cells, A549 lung carcinoma cells) attach to and spread on polyacrylamide hydrogel substrates covalently linked to vimentin. Using traction force microscopy and spheroid expansion assays, we characterize how different cell types respond to extracellular vimentin. Cell attachment to and spreading on vimentin-coated surfaces is inhibited by hyaluronic acid (HA) degrading enzymes, HA synthase inhibitors, soluble heparin, or N-acetyl glucosamine (GlcNAc), treatments that have little or no effect on the same cell types binding to collagen-coated hydrogels. These studies highlight the effectiveness of substrate-bound vimentin as a ligand for cells and suggest that carbohydrate structures, including the glycocalyx and glycosylated cell surface proteins that contain N-acetyl glucosamine, form a novel class of adhesion receptors for extracellular vimentin that can either directly support cell adhesion to a substrate or fine-tune the glycocalyx adhesive properties.

Whitening and moisturizing enhancing effects of three-dimensional human adipose-derived mesenchymal stem cell-conditioned medium-containing cream.

High-functional cosmetic products combined with the concept of "treatment" cosmetics are being introduced to the market. Cosmetic products containing a skin-derived microbiome, a three-dimensional (3D) stem cell culture medium, and low-molecular-weight collagen are being introduced, and these products are leading the cosmeceutical market. We aimed to confirm the potential of a 3D stem cell culture medium-containing cream as a skin-whitening and moisturizing product. To determine the enhancing effects of a cream containing 3D adipose tissue-derived mesenchymal stem cell-conditioned media (3D ADMSC-CM) on whitening and moisturization. The inhibitory activities of tyrosinase (TYR) and melanin were confirmed using 3D ADMSC-CM. Furthermore, hyaluronic acid expression in 3D ADMSC-CM was verified. The clinical efficacy of the cream containing 3D ADMSC-CM was established by evaluating its antioxidant properties and effects on skin tone, radiance, freckles, and moisturization. The use of 3D ADMSC-CM suppressed the inhibitory effects of TYR and melanin by approximately 24% and 33%, respectively, and increased the expression of hyaluronic acid synthase. A significant difference was observed after 4 weeks of using 3D ADMSC-CM in the skin antioxidant evaluation. After 2 and 4 weeks of use, skin tone and radiance increased and skin freckles decreased significantly. Under extremely cold and dry weather conditions, the use of the cream increased skin moisturization. The 3D ADMSC-CM cream evaluated in an environment similar to the human body was found to enhance skin whitening and moisturization and can therefore be used in the skin care and cosmetic industries as a biocosmetic product.

Hyaluronan synthesis inhibition normalizes ethanol-enhanced hepatic stellate cell activation.

Chronic ethanol overconsumption promotes alcohol-associated liver disease (ALD), characterized by hepatocyte injury, inflammation, hepatic stellate cell (HSC) activation, and fibrosis. Hyaluronan (HA) concentration is greater in livers and blood from advanced ALD patients than patients with advanced non-ALD. In the liver, HSCs are the major HA producers. The relationship between ethanol, HA, and HSC activation is incompletely understood. Thus, here, we tested the hypothesis that ethanol enhances HSC activation in a HA-dependent manner. Liver tissue microarrays (TMAs) containing steatotic livers from donors with or without a history of alcohol consumption were used to measure HA and collagen content. Mice were fed a moderate (2%, v/v) ethanol-containing diet or pair-fed control diet for 2 days, after which they were given a single carbon tetrachloride (CCl4 ) injection. To inhibit HA synthesis, we provided 4-methylumbelliferone (4MU) daily. We used LX2 cells, a human HSC cell line, to determine the impact ethanol had on LPS responses, with or without concurrent 4MU exposure. CCl4 induced liver injury, but it did not differ between ethanol or control diet fed mice with or without 4MU treatment. Ethanol feeding enhanced CCl4 -induced hepatic HA content, which was paralleled by HA synthase (Has)2 transcript abundance; 4MU treatment normalized both. Consistently, HSC activation, assessed by measuring αSMA mRNA and protein, was induced by CCl4 exposure, enhanced by ethanol feeding, and normalized by 4MU. Hepatic transcripts, but not protein, for Ccl2 were enhanced by ethanol feeding and normalized by 4MU exposure. Finally, ethanol-exposed LX2 cells made more LPS-stimulated CCL2 mRNA and protein than cells not exposed to ethanol; 4MU prevented this. These data show that ethanol augments HSC activation through HA synthesis and enhances hepatic profibrogenic features. Therefore, targeting HSC HA production could potentially attenuate liver disease in ALD patients.

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 35.57µg/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.

The Potential of Artemisinins as Novel Treatment for Thyroid Eye Disease by Inhibiting Adipogenesis in Orbital Fibroblasts.

Thyroid eye disease (TED) causes cosmetic defect and even threatens eyesight due to tissue remodeling in which orbital fibroblast (OF) plays a central role mainly by differentiating into adipocytes. Repurposing old drugs to novel applications is of particular interest. Here, we aimed to evaluate the effects of the antimalarials artemisinin (ARS) and the derivatives on the OFs isolated from patients with TED and their counterparts. OFs isolated from patients with TED or their counterparts were cultured and passaged in proliferation medium (PM) and stimulated by differentiation medium (DM) for adipogenesis. OFs were treated with or without ARS, dihydroartemisinin (DHA), and artesunate (ART) at different concentrations, before being examined in vitro. CCK-8 were used to assess cellular viability. Cell proliferation was determined by EdU incorporation and flow cytometry. Lipid accumulation within the cells was evaluated by Oil Red O staining. Hyaluronan production was determined by ELISA. RNAseq, qPCR, and Western blot analysis were performed to illustrate the underlying mechanisms. ARSs dose-dependently interfered with lipid accumulation of TED-OFs, rather than non-TED-OFs. Meanwhile, the expression of key adipogenic markers, such as PLIN1, PPARG, FABP4, and CEBPA, was suppressed. During adipogenesis as being cultivated in DM, instead of PM, ARSs also inhibited cell cycle, hyaluronan production and the expression of hyaluronan synthase 2 (HAS2) in a concentration-dependent manner. Mechanically, the favorable effects were potentially mediated by the repression of IGF1R-PI3K-AKT signaling by dampening IGF1R expression. Collectedly, our data evidenced that the conventional antimalarials ARSs were potentially therapeutic for TED.

#5664 PERITONEAL DIFFUSE PODOPLANIN EXPRESSION IS ASSOCIATED WITH SEVERE ARTERIOLOPATHY IN CHILDREN ON PERITONEAL DIALYSIS

Abstract Background and Aims Podoplanin is a transmembrane glycoprotein binding chemokines and modulating inflammatory reactions. In healthy peritoneum, it is expressed in mesothelial cells and in lymphatic endothelium, expression beyond these structures (diffuse podoplanin staining, DPS) has been observed in adult patients with encapsulating peritoneal sclerosis (EPS). Method Parietal peritoneal tissues from 71 children on PD fluids with low glucose degradation product (GDP) content underwent digital histomorphometry. DPS and pathomechanisms of peritoneal membrane transformation and vasculopathy were hierarchically clustered. A panel of 13 peritoneal cell markers was selected and tissue sections were stained with metal conjugated antibodies and acquired using imaging mass cytometry (IMC; Hyperion Imaging System®). After single-cell segmentation, spillover correction, and normalization, cell types were identified using manual gating to train a random forest classifier for final cell type identifications using published workflows. Single-cell analysis was used to characterize peritoneal cells with positive podoplanin signals. Results DPS was present in 22% of the PD patients, all devoid of any clinical, radiological or histological feature of EPS. DPS positive patients were younger, had higher dialytic glucose exposure (median 103 vs. 154 g/m²/day, p = 0.002), and lower peritoneal arteriolar lumen to vessel (L/V) ratio, i.e. exhibited more arteriolar lumen narrowing. PD duration [median 20 (14.5, 48) months] and peritonitis incidence were similar as in non-DPS patients. In multivariable analysis, glucose exposure, PD duration, epithelial-to-mesenchymal transformed (EMT) cells and lower patient body surface area (BSA) independently predicted the presence of DPS. DPS and lower mesothelial surface coverage predicted lower arteriolar L/V ratio. In subgroups matched for BSA, PD duration and dialytic glucose exposure, DPS patients had a lower L/V ratio, (0.44 ± 0.13 vs. 0.31 ± 0.18, p = 0.003) and higher submesothelial CD45 leucocyte, CD68 macrophage and EMT cell count. Submesothelial lipopolysaccharide and hyaluronic acid receptor CD44 were increased in DPS positive patients, hyaluronan synthase 1 and 2 were similar. Among DPS positive patients, DPS intensity grade was higher in those with history of bacterial or fungal peritonitis (7 out of 15). Two patients had three and four peritonitis episodes respectively, their DPS pattern did not differ from patients with history of only one peritonitis episode. Hierarchical clustering demonstrated highest similarity of DPS positive areas with CD68 positive areas. In IMC analysis of DPS positive areas, highest podoplanin signals were derived from M2 classified macrophages (CD68+CD163+) and fibroblastic cells [αSMA positive/non-endothelial/non-macrophage (αSMA+PROX1-CD31-CD68-CD163-)], signals derived from M1 classified macrophages (CD68+CD163-) were lowest. Conclusion Peritoneal DPS in pediatric patients on low-GDP PD is associated with lower BSA, higher dialytic glucose exposure, history of infectious peritonitis and peritoneal inflammatory and EMT cell invasion, and predicts obliterating peritoneal vasculopathy. We identified M2 macrophages as major source of extra-lymphatic peritoneal podoplanin accumulation, suggesting their role in severe peritoneal transformation.

Protective effect of Schizonepeta tenuifolia Briq. ethanolic extract against UVB-induced skin aging and photodamage in hairless mice.

The purpose of this study was to illuminate the mechanism by which Schizonepeta tenuifolia Briq. (ST) ethanolic extract prevents skin photoaging in HR-1 hairless mice (HR-1). The ST ethanolic extract alleviated wrinkle formation, epidermal skin thickness, and collagen degradation in skin tissues of ultraviolet B (UVB)-irradiated HR-1 mice. Expression of matrix metalloproteinases (a wrinkle-related marker) was reduced, and tissue inhibitor of metalloproteinase 1 expression was upregulated following application of ST ethanolic extract. Furthermore, skin dehydration and levels of hyaluronidase-1 and -2 (enzymes that break hyaluronic acid) were decreased. Moreover, protein expression of hyaluronan synthases (markers of skin hydration) and hyaluronic acid levels increased following ST ethanolic extract treatment in UVB-induced photoaging HR-1 mice. In addition, the phosphorylation of mitogen-activated protein kinases (MAPKs), including p38, extracellular signal-regulated kinase, and Jun N-terminal kinase was suppressed, and expression of nuclear factor-kappa was reduced. Treatment with ST ethanolic extract also reduced advanced glycation end product (AGE) accumulation and expression of the receptor for AGE (RAGE) in skin tissue. These results suggest that ST ethanolic extract moderates skin damage caused by UVB irradiation via regulating the expression of wrinkle- and hydration-related proteins, MAPKs, and RAGE.

The Endogenous Dual Retinoid Receptor Agonist Alitretinoin Exhibits Immunoregulatory Functions on Antigen-Presenting Cells.

Retinoids are a frequently used class of drugs in the treatment of inflammatory as well as malignant skin diseases. Retinoids have differential affinity for the retinoic acid receptor (RAR) and/or the retinoid X receptor (RXR). The endogenous dual RAR and RXR agonist alitretinoin (9-cis retinoic acid) demonstrated remarkable efficacy in the treatment of chronic hand eczema (CHE) patients; however, detailed information on the mechanisms of action remains elusive. Here, we used CHE as a model disease to unravel immunomodulatory pathways following retinoid receptor signaling. Transcriptome analyses of skin specimens from alitretinoin-responder CHE patients identified 231 significantly regulated genes. Bioinformatic analyses indicated keratinocytes as well as antigen presenting cells as cellular targets of alitretinoin. In keratinocytes, alitretinoin interfered with inflammation-associated barrier gene dysregulation as well as antimicrobial peptide induction while markedly inducing hyaluronan synthases without affecting hyaluronidase expression. In monocyte-derived dendritic cells, alitretinoin induced distinct morphological and phenotypic characteristics with low co-stimulatory molecule expression (CD80 and CD86), the increased secretion of IL-10 and the upregulation of the ecto-5'-nucleotidase CD73 mimicking immunomodulatory or tolerogenic dendritic cells. Indeed, alitretinoin-treated dendritic cells demonstrated a significantly reduced capacity to activate T cells in mixed leukocyte reactions. In a direct comparison, alitretinoin-mediated effects were significantly stronger than those observed for the RAR agonist acitretin. Moreover, longitudinal monitoring of alitretinoin-responder CHE patients could confirm in vitro findings. Taken together, we demonstrate that the dual RAR and RXR agonist alitretinoin targets epidermal dysregulation and demonstrates strong immunomodulatory effects on antigen presenting cell functions.

Modulation of lncRNA links endothelial glycocalyx to vascular dysfunction of tyrosine kinase inhibitor.

Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi-receptor tyrosine kinase inhibitor, has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored. We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human-induced pluripotent stem cell-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to down-regulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirmed that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and up-regulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in Has2os and degradation of the endothelial glycocalyx. Finally, we identified that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx. Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.

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

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. 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. 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. 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 HAS‑2 regulates the chondrocyte cytoskeleton and induces cartilage degeneration byactivating 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 synthase‑2 (HAS‑2) is a key enzyme in synthesizing hyaluronic acid (HA) invivo. The synthesis of high molecular weight HA catalyzed by HAS‑2 serves a vital role in joint movement and homeostasis; however, it is unclear what important role HAS‑2 plays in maintaining chondrocyte cytoskeleton morphology and in cartilage degeneration. The present study downregulated the expression of HAS‑2 by employing 4‑methylumbelliferone (4‑MU) and RNA interference. Invitro experiments, including reverse transcription‑quantitative PCR, western blotting, laser scanning confocal microscopy and flow cytometry were subsequently performed. The results revealed that downregulation of HAS‑2 could activate the RhoA/ROCK signaling pathway, cause morphological abnormalities, decrease expression of the chondrocyte cytoskeleton proteins and promote chondrocyte apoptosis. Invivo experiments, including immunohistochemistry and Mankin's scoring, were performed to verify the effect of HAS‑2 on the chondrocyte cytoskeleton, and it was revealed that inhibition of HAS‑2 could cause cartilage degeneration. In conclusion, the present results revealed that downregulation of HAS‑2 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 4‑MU may cause cartilage degeneration. Therefore, targeting HAS‑2 may provide a novel therapeutic strategy for delaying chondrocyte degeneration, and the early prevention and treatment of OA.

Heterologous Biosynthesis of Hyaluronic Acid Using a New Hyaluronic Acid Synthase Derived from the Probiotic Streptococcus thermophilus

Hyaluronic acid (HA) is a natural linear polysaccharide extensively used in many fields, including the food, medicine, and cosmetics industries. Currently, species that produce HA synthetase (HAS) from microbial sources are relatively small and mainly pathogenic, such as Streptococcus pyogenes and Pasteurella multicide. Moreover, there is limited research on the safe microbial sources of HAS. Thus, we characterized SthasA, a HAS derived from the probiotic Streptococcus thermophilus, and used it for the de novo synthesis of HA in a chassis strain of Bacillus amyloliquefaciens. Metabolic engineering of the precursor supply modules suggested that hasB (encoding UDPG dehydrogenase), which was derived from Corynebacterium glutamicum ATCC 13032, effectively promoted the accumulation of HA products. Furthermore, by combining the expression of the global regulatory factor CcpA, HA yield from the recombinant strain reached 3.20 g/L. Finally, we obtained a yield of 5.57 g/L HA with a molecular weight of 1.7 × 106 Da using various process optimization strategies in a 5 L bioreactor. This study enriches our understanding of obtaining HAS from non-pathogenic bacteria and provides a safe and effective process for producing HA, which has the potential to promote the industrial applications of HA further.