Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Overview of Enzyme Engineering Towards the Production of Hyaluronic Acid with Tailored Molecular Weight

: Hyaluronic acid or hyaluronan (HA) is a natural biopolymer composed of D-glucuronic acid and N-acetylglucosamine units, distributed as a non-sulfated and anionic glycosaminoglycan in important tissues of the body, and is commercially and biologically important. Its biological properties are determined by the molecular weight and dispersity which are suitable for particular medical and cosmetic applications. The synthesis of well-defined and monodisperse HA is still a significant obstacle and an impressive research field for advanced medical applications. High polydispersity by bacterial fermentation, the lack of knowledge of the mechanism required to start and continue the synthesis process, increased cost of raw materials to produce HA, clarification and explanation of factors limiting synthesis in bacterial systems are among the important challenges of hyaluronic acid synthesis. Hyaluronan synthase plays a critical role in HA molecular mass by producing a wide range of HA involved in various biological processes. Hyaluronan biosynthesis has been considered extensively; however, the control of its size and weight during the synthesis process is poorly investigated. This review focuses on these uncharted biochemical details to obtain the uniform chain lengths of Hyaluronan by protein engineering and regulating the function of Hyaluronan synthase.

Hyaluronan and Derivatives: An In Vitro Multilevel Assessment of Their Potential in Viscosupplementation

In this research work, viscosupplements based on linear, derivatized, crosslinked and complexed HA forms were extensively examined, providing data on the hydrodynamic parameters for the water-soluble-HA-fraction, rheology, sensitivity to enzymatic hydrolysis and capacity to modulate specific biomarkers’ expression in human pathological chondrocytes and synoviocytes. Soluble HA ranged from 0 to 32 mg/mL and from 150 to 1330 kDa MW. The rheological behavior spanned from purely elastic to viscoelastic, suggesting the diversity of the categories that are suitable for restoring specific/different features of the healthy synovial fluid. The rheological parameters were reduced in a diverse manner upon dilution and hyaluronidases action, indicating different durations of the viscosupplementation effect. Bioactivity was found for all the samples, increasing the expression of different matrix markers (e.g., hyaluronan-synthase); however, the hybrid cooperative complexes performed better in most of the experiments. Hybrid cooperative complexes improved COLII mRNA expression (~12-fold increase vs. CTR), proved the most effective at preserving cell phenotype. In addition, in these models, the HA samples reduced inflammation. IL-6 was down-regulated vs. CTR by linear and chemically modified HA, and especially by hybrid complexes. The results represent the first comprehensive panel of data directly comparing the diverse HA forms for intra-articular injections and provide valuable information for tailoring products’ clinical use as well as for designing new, highly performing HA-formulations that can address specific needs.

Endothelial Hyaluronan Synthase 3 Augments Postischemic Arteriogenesis Through CD44/eNOS Signaling

Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 different HAS1, -2, and -3 (hyaluronan synthase isoenzymes) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3 -deficient ( Has3 -KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3 -KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3 -KO thigh muscles. Endothelial-specific Has3 -KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3 -deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.

Differences in human placental mesenchymal stromal cells may impair vascular function in FGR

Placentae from pregnancies with fetal growth restriction (FGR) exhibit poor oxygen and nutrient exchange, in part due to impaired placental vascular development. Placental mesenchymal stromal cells (pMSCs) reside in a perivascular niche, where they may influence blood vessel formation/ function. However, the role of pMSCs in vascular dysfunction in FGR is unclear. To elucidate the mechanisms by which pMSCs may impact placental vascularisation we compared the transcriptomes of human pMSCs isolated from FGR (<5th centile) (n=7) and gestation-matched control placentae (n=9) using Affymetrix microarrays. At the transcriptome level there were no statistically significant differences between normal and FGR pMSCs, however several genes linked to vascular function exhibited notable fold changes, and thus the dataset was used as a hypothesis-generating tool for possible dysfunction in FGR. Genes/proteins of interest were followed up by real-time PCR and by immunohistochemistry. Gene expression of ADAMTS1 and FBLN2 (fibulin-2) were significantly upregulated, whilst HAS2 (hyaluronan synthase-2) was significantly downregulated, in pMSCs from FGR placentae (n=8) relative to controls (n=7, p<0.05 for all). At the protein level, significant differences in the level of fibulin-2 and hyaluronan synthase-2, but not ADAMTS1 were confirmed between pMSCs from FGR and control pregnancies by western blot. All three proteins demonstrated perivascular expression in third-trimester placentae. Fibulin-2 maintains vessel elasticity, and its increased expression in FGR pMSCs could help explain the increased distensibility of FGR blood vessels. ADAMTS1 and hyaluronan synthase-2 regulate angiogenesis, and their differential expression by FGR pMSCs may contribute to the impaired angiogenesis in these placentae.

PROGNOSTIC VALUES OF MIDKINE, SYNDECAN-1, HYALURONAN SYNTHASE-2, SESTRIN-1, LAMININ SUBUNIT ALPHA-4 AND FIBULIN-3 FOR MALIGNANT PLEURAL MESOTHELIOMA

IntroductionThe 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 the patients with MPM by ex-vivo validation experimentsMaterial and methodsThe study group consisted of 54 MPM patients treated by 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) were examined by qPCR in tumor tissues. SESN1 and LAMA4 were identified using an in house R-based script “Unsupervised Survival Analysis Tool." MDK, SDC1, HAS2, and FBLN3 were selected from current literature. We used two housekeeping genes; glucose-6-phosphate dehydrogenase and TATA-box binding protein as controls.ResultsOf the patients, 43 (79.6%) had epithelioid mesothelioma. The median survival for all patients was 10 (±1.2 SE) months (CI 95%; 7.7-12.3). In multivariate analyses, MDK (p=0.007), HAS2 (p=0.008) and SESN1 (p=0.014) expression were related with survival time in 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 with survival time by multivariate analyses.ConclusionsHigh 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

Zingiber mioga Extract Improves Moisturization and Depigmentation of Skin and Reduces Wrinkle Formation in UVB-Irradiated HRM-2 Hairless Mice

Here, we investigated the effects of Zingiber mioga extracts (FSH-ZM) on the moisturization and depigmentation of skin as well as wrinkle formation in UVB-irradiated HRM-2 hairless mice. The mice were divided into six groups as follows: normal control (NC), UVB-irradiated control (C), positive control 1 (PC1, L-ascorbic acid 200 mg/kg b.w.), positive control 2 (PC2, Arbutin 200 mg/kg b.w.), Z100 (FSH-ZM 100 mg/kg b.w.), and Z200 (FSH-ZM 200 mg/kg b.w.). The experiment spanned a period of 6 weeks. We found that FSH-ZM led to an increase in the expression of hyaluronan synthase 2, fibrillin-1, and elastin mRNAs, and showed improved skin hydration in HRM-2 hairless mice compared to that in the UVB-irradiated control group. Furthermore, FSH-ZM also inhibited the expression of inflammatory cytokines and wrinkle forming factors generated by UVB and reduced the formation of wrinkles in the test group relative to that in the control group by increasing collagen synthesis. Moreover, we found that FSH-ZM decreased the expression of melanogenesis factors, which improved depigmentation in UVB-irradiated hairless mice. These results suggest that Zingiber mioga can potentially be utilized to develop products aimed at improving skin moisturization and depigmentation and reducing wrinkle formation.