HAS2 Expression Research Articles

Bone quality relies on hyaluronan synthesis – Insights from mice with complete knockout of hyaluronan synthase expression

Bone consists of a complex mineralised matrix that is maintained by a controlled equilibrium of synthesis and resorption by different cell types. Hyaluronan (HA) is an important glycosaminoglycan in many tissues including bone.Previously, the importance of HA synthesis for bone development during embryogenesis has been shown. We therefore investigated whether HA synthesis is involved in adult bone turnover and whether abrogation of HA synthesis in adult mice would alter bone quality.To achieve complete abrogation of HA synthesis in adult mice, we generated a novel Has-total knockout (Has-tKO) mouse model in which a constitutive knockout of Has1 and Has3 was combined with an inducible, Ubc-Cre-driven Has2 knockout.By comparing bone tissue from wild-type, Has1,3 double knockout and Has-tKO mice, we demonstrate that Has2-derived HA mainly contributes to the HA content in bone. Furthermore, Has-tKO mice show a significant decrease of bone integrity in trabecular and cortical bone, as shown by µ-CT analysis. These effects are detectable as early as five weeks after induced Has2 deletion, irrespective of sex and progress with age.Mesenchymal stem cells (MSC) during osteogenic differentiation in vitro showed that Has2 expression is increased while Has3 expression is decreased during differentiation. Furthermore, the complete abrogation of HA synthesis results in significantly reduced osteogenic differentiation as indicated by reduced marker gene expression (Runx-2, Tnalp, Osterix) as well as alizarin red staining. RNAseq analysis revealed that MSC from Has-tKO are characterised by decreased expression of genes annotated for bone and organ development, whereas expression of genes associated with chemokine related interactions and cytokine signalling is increased.Taken together, we present a novel mouse model with complete deletion of HA synthases in adult mice which has the potential to study HA function in different organs and during age-related HA reduction. With respect to bone, HA synthesis is important for maintaining bone integrity, presumably based on the strong effect of HA on osteogenic differentiation.

A point-of-research decision in synovial tissue engineering: Mesenchymal stromal cells, tissue derived fibroblast or CTGF-mediated mesenchymal-to-fibroblast transition

Rheumatoid arthritis (RA) and osteoarthritis (OA) are prevalent inflammatory joint diseases characterized by synovitis, cartilage, and bone destruction. Fibroblast-like synoviocytes (FLSs) of the synovial membrane are a decisive factor in arthritis, making them a target for future therapies. Developing novel strategies targeting FLSs requires advanced in vitro joint models that accurately replicate non-diseased joint tissue. This study aims to identify a cell source reflecting physiological synovial fibroblasts. Therefore, we newly compared the phenotype and metabolism of “healthy” knee-derived FLSs from patients with ligament injuries (trauma-FLSs) to mesenchymal stromal cells (MSCs), their native precursors. We differentiated MSCs into fibroblasts using connective tissue growth factor (CTGF) and compared selected protein and gene expression patterns to those obtained from trauma-FLSs and OA-FLSs. Based on these findings, we explored the potential of an MSC-derived synovial tissue model to simulate a chronic inflammatory response akin to that seen in arthritis. We have identified MSCs as a suitable cell source for synovial tissue engineering because, despite metabolic differences, they closely resemble human trauma-derived FLSs. CTGF-mediated differentiation of MSCs increased HAS2 expression, essential for hyaluronan synthesis. It showed protein expression patterns akin to OA-FLSs, including markers of ECM components and fibrosis, and enzymes leading to a shift in metabolism towards increased fatty acid oxidation. In general, cytokine stimulation of MSCs in a synovial tissue model induced pro-inflammatory and pro-angiogenic gene expression, hyperproliferation, and increased glucose consumption, reflecting cellular response in human arthritis. We conclude that MSCs can serve as a proxy to study physiological synovial processes and inflammatory responses. In addition, CTGF-mediated mesenchymal-to-fibroblast transition resembles OA-FLSs. Thus, we emphasize MSCs as a valuable cell source for tools in preclinical drug screening and their application in tissue engineering.

Evaluating the Dermatological Benefits of Snowberry (Symphoricarpos albus): A Comparative Analysis of Extracts and Fermented Products from Different Plant Parts.

Skin ageing is influenced by both intrinsic and extrinsic factors, with excessive ultraviolet (UV) exposure being a significant contributor. Such exposure can lead to moisture loss, sagging, increased wrinkling, and decreased skin elasticity. Prolonged UV exposure negatively impacts the extracellular matrix by reducing collagen, hyaluronic acid, and aquaporin 3 (AQP-3) levels. Fermentation, which involves microorganisms, can produce and transform beneficial substances for human health. Natural product fermentation using lactic acid bacteria have demonstrated antioxidant, anti-inflammatory, antibacterial, whitening, and anti-wrinkle properties. Snowberry, traditionally used as an antiemetic, purgative, and anti-inflammatory agent, is now also used as an immune stimulant and for treating digestive disorders and colds. However, research on the skin benefits of Fermented Snowberry Extracts remains limited. Thus, we aimed to evaluate the skin benefits of snowberry by investigating its moisturising and anti-wrinkle effects, comparing extracts from different parts of the snowberry plant with those subjected to fermentation using Lactobacillus plantarum. Chlorophyll-free extracts were prepared from various parts of the snowberry plant, and ferments were created using Lactobacillus plantarum. The extracts and ferments were analysed using high-performance liquid chromatography (HPLC) to determine and compare their chemical compositions. Moisturising and anti-ageing tests were conducted to assess the efficacy of the extracts and ferments on the skin. The gallic acid content remained unchanged across all parts of the snowberry before and after fermentation. However, Fermented Snowberry Leaf Extracts exhibited a slight decrease in chlorogenic acid content but a significant increase in ferulic acid content. The Fermented Snowberry Fruit Extract demonstrated increased chlorogenic acid and a notable rise in ferulic acid compared to its non-fermented counterpart. Skin efficacy tests revealed that Fermented Snowberry Leaf and Fruit Extracts enhanced the expression of AQP-3, HAS-3, and COL1A1. These extracts exhibited distinct phenolic component profiles, indicating potential skin benefits such as improved moisture retention and protection against ageing. These findings suggest that Fermented Snowberry Extracts could be developed into effective skincare products, providing a natural alternative for enhancing skin hydration and reducing signs of ageing.

Abstract P454: Screening of risk genes for carotid vulnerable plaque based on transcriptomics and experimental verification

Purpose and background: This study aims to conduct an in-depth analysis of human carotid atherosclerotic plaques using transcriptomic data to elucidate the relationship between hypoxia-related genes and the stability of carotid artery plaques and explore their connection with immune infiltration. Methods: Four GEO datasets (GSE21545, GSE24495, GSE159677, and GSE189300) were used. Initially, gene expression data from carotid artery plaque samples in GSE21545 and GSE24495 were analyzed. Hypoxia-related gene sets were obtained from MSigDB, and the "HALLMARK_HYPOXIA" gene set was explicitly selected. GSEA software was used for ultimately selecting CDKN1C and HAS1 as characteristic genes. Subsequently, sample clustering analysis was conducted to determin the optimal number of clusters. Gene set enrichment and differential analysis were performed. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting experiments were conducted for preliminary validation of CDKN1C and HAS1 expression levels in ApoE knockout mice at 16 and 6 weeks. Subsequently, single-cell data from GSE159677 were processed to determine the differential localization of CDKN1C in carotid atherosclerotic plaques, with functional analysis conducted using GSE189300. Results: CDKN1C and HAS1 genes were found to exhibit differential expression in plaques and were correlated with hypoxia and immune scores. Clustering analysis revealed two main clusters, with patients in cluster A showing a higher risk of adverse events and a stronger association with immunity. Genes with a positive effect on atherosclerosis were highly expressed in poorer prognosis cluster A. Validation dataset GSE24495 also confirmed the grouping ability of CDKN1C and HAS1 genes, showing enrichment in atherosclerosis-related pathways. RT-qPCR and protein immunoblotting experiments demonstrated no significant differences in HAS1 expression levels, while CDKN1C showed lower protein expression in 16-week ApoE knockout mice compared to 6 weeks. Single-cell data from GSE159677 localized differential expression of CDKN1C in plaque cells. Finally, immunofluorescence validation confirmed the coexistence of CDKN1C and vascular smooth muscle cells. Conclusion: This study comprehensively analyzed transcriptomics data from carotid atherosclerotic plaques, revealing the significant roles of CDKN1C and HAS1 genes. Immunofluorescence validation further supported the crucial role of these genes in carotid atherosclerosis.

Calycosin inhibits the proliferation and metastasis of renal cell carcinoma through the MAZ/HAS2 signaling pathway.

Calycosin (Caly), a flavonoid compound, demonstrates a variety of beneficial properties. However, the specific mechanisms behind Caly's anticancer effects remain largely unexplored. Network pharmacology was used to explore the potential targets of Caly in renal cancer. Additionally, RNA-seq sequencing was used to detect changes in genes in renal cancer cells after Caly treatment. Validation was carried out through quantitative reverse transcription-PCR and Western blot analysis. The luciferase reporter assay was applied to pinpoint the interaction site between MAZ and HAS2. Furthermore, the immunoprecipitation assay was utilized to examine the ubiquitination and degradation of MAZ. In vivo experiments using cell line-derived xenograft mouse models were performed to assess Calycosin's impact on cancer growth. Network pharmacology research suggests Caly plays a role in promoting apoptosis and inhibiting cell adhesion in renal cancer. In vitro, Caly has been observed to suppress proliferation, colony formation, and metastasis of renal cancer cells while also triggering apoptosis. Additionally, it appears to diminish hyaluronic acid synthesis by downregulating HAS2 expression. MAZ is identified as a transcriptional regulator of HAS2 expression. Calycosin further facilitates the degradation of MAZ via the ubiquitin-proteasome pathway. Notably, Caly demonstrates efficacy in reducing the growth of renal cell carcinoma xenograft tumors in vivo. Our findings indicate that Caly suppresses the proliferation, metastasis, and progression of renal cell carcinoma through its action on the MAZ/HAS2 signaling pathway. Thus, Caly represents a promising therapeutic candidate for the treatment of renal cell carcinoma.

Abstract Tu088: Fibroblast Activation, Collagen Secretion, and Migration Occurs Independently of Has2 Expression

Background: Hyaluronan (HA) accumulates after tissue injury and may regulate the wound-healing process. We found that HA is increased in and around the scar following myocardial infarction (MI). Fibroblasts are the main producers of the collagen that comprises the scar, and we have shown that they are responsible for HA accumulation. Given that these two extraordinarily high-volume synthetic processes occur in the same place and simultaneously, we queried whether alterations in Has2 -dependent HA synthesis impacted collagen secretion and other fibroblast functions. Goal: To determine the impact of HA synthesis on fibroblast function. Methods and Results: We isolated cardiac fibroblasts from Has2 fl/fl mice and treated with Cre adenovirus to delete Has2 expression or Has2 adenovirus to overexpress Has2. We assessed cell number and found no differences when Has2 was deleted or overexpressed in both naïve fibroblasts (n=6); activation with TGF-b did not change this response . We used a scratch assay to assess migration and found no change between treatments in naïve fibroblasts (n=6). Hydroxyproline assessment of deposited collagen and immunoblotting for soluble collagen secretion in the media indicated no differences between treatments in naïve fibroblasts (n=3). Conclusion: Genetic manipulation of HA production did not impact proliferation, migration, or collagen secretion in fibroblasts. Considering the enormous metabolic requirement of fibroblasts to produce HA and collagen, these results suggest that the regulation of these processes may be distinct or that the substrate is not limiting in our experimental conditions. In the intact heart, substrate could become limiting and a relationship between these two biosynthetic processes may emerge

Targeting hyaluronan metabolism-related molecules associated with resistant tumor-initiating cells potentiates chemotherapy efficacy in lung cancer

The success of chemotherapy regimens in patients with non-small cell lung cancer (NSCLC) could be restricted at least in part by cancer stem cells (CSC) niches within the tumor microenvironment (TME). CSC express CD133, CD44, CD47, and SOX2, among other markers and factors. Analysis of public data revealed that high expression of hyaluronan (HA), the main glycosaminoglycan of TME, correlated positively with CSC phenotype and decreased disease-free interval in NSCLC patients. We aimed to cross-validate these findings on human and murine lung cancer cells and observed that CD133 + CSC differentially expressed higher levels of HA, HAS3, ABCC5, SOX2, and CD47 (p < 0.01). We modulated HA expression with 4-methylumbelliferone (4Mu) and detected an increase in sensitivity to paclitaxel (Pa). We evaluated the effect of 4Mu + chemotherapy on survival, HA metabolism, and CSC profile. The combination of 4Mu with Pa reduced the clonogenic and tumor-forming ability of CSC. Pa-induced HAS3, ABCC5, SOX2, and CD47 expression was mitigated by 4Mu. Pa + 4Mu combination significantly reduced in vivo tumor growth, enhancing animal survival and restoring the CSC profile in the TME to basal levels. Our results suggest that HA is involved in lung CSC phenotype and chemosensitivity, and its modulation by 4Mu improves treatment efficacy to inhibit tumor progression.

Risk of anthracycline-induced cardiac dysfunction in adolescent and young adult (AYA) cancer survivors: role of genetic susceptibility loci.

There is a known genetic susceptibility to anthracycline-induced cardiac dysfunction in childhood cancer survivors, but this has not been adequately shown in adolescent and young adult (AYA) patients. Our aim was to determine if the previously identified variants associated with cardiac dysfunction in childhood cancer patients affect AYA cancer patients similarly. Forty-five variants were selected for analysis in 253 AYAs previously treated with anthracyclines. We identified four variants that were associated with cardiac dysfunction: SLC10A2:rs7319981 (p = 0.017), SLC22A17:rs4982753 (p = 0.019), HAS3:rs2232228 (p = 0.023), and RARG:rs2229774 (p = 0.050). HAS3:rs2232228 and SLC10A2:rs7319981 displayed significant effects in our AYA cancer survivor population that were in the opposite direction than that reported in childhood cancer survivors. Genetic variants in the host genes were further analyzed for additional associations with cardiotoxicity in AYA cancer survivors. The host genes were then evaluated in a panel of induced pluripotent stem cell-derived cardiomyocytes to assess changes in levels of expression when treated with doxorubicin. Significant upregulation of HAS3 and SLC22A17 expression was observed (p < 0.05), with non-significant anthracycline-responsivity observed for RARG. Our study demonstrates that there is a genetic influence on cardiac dysfunction in AYA cancer patients, but there may be a difference in the role of genetics between childhood and AYA cancer survivors.

γ-Mangosteen, an autophagy enhancer, prevents skin-aging via activating KEAP1/NRF2 signaling and downregulating MAPKs/AP-1/NF-κB-mediated MMPs

BackgroundMangosteens, a naturally occurring xanthones, found abundantly in mangosteen fruits. The anti-skin aging potential of γ-mangosteen (GM) remains unexplored; therefore, we investigated the UVB-induced anti-skin aging of GM via activation of autophagy. HypothesisWe hypothesized that GM exerts antioxidant and anti-aging capabilities both in vitro and in vivo through activation of autophagy as well as control of KEAP1/NRF2 signaling and MAPKs/AP-1/NF-κB-mediated MMPs pathways. MethodsThe anti-skin aging effects of GM were studied using HDF cells and a mice model. Various assays, such as DPPH, ABTS, CUPRAC, FRAP, and ROS generation, assessed antioxidant activities. Kits measured antioxidant enzymes, SA-β-gal staining, collagen, MDA content, si-RNA experiments, and promoter assays. Western blotting evaluated protein levels of c-Jun, c-Fos, p-IκBα/β, p-NF-κB, MAPK, MMPs, collagenase, elastin, KEAP1, NRF2, HO-1, and autophagy-related proteins. ResultsGM exhibited strong antioxidant, collagenase and elastase enzyme inhibition activity surpassing α- and β-mangosteen. GM competitively inhibited elastase with a Ki value of 29.04 µM. GM orchestrated the KEAP1-NRF2 pathway, enhancing HO-1 expression, and suppressed UVB-induced ROS in HDF cells. NRF2 knockdown compromised GM's antioxidant efficacy, leading to uncontrolled ROS post-UVB. GM bolstered endogenous antioxidants, curbing lipid peroxidation in UVB-exposed HDF cells and BALB/c mice. GM effectively halted UVB-induced cell senescence, and reduced MMP-1/-9, while elevated TIMP-1 levels, augmented COL1A1, ELN, and HAS-2 expression in vitro and in vivo. Additionally, it suppressed UVB-induced MAPKs, AP-1, NF-κB phosphorylation. Pharmacological inhibitors synergistically enhanced GM's anti-skin aging potential. Moreover, GM inhibited UVB-induced mTOR activation, upregulated LC3-II, Atg5, Beclin 1, and reduced p62 in both UVB induced HDF cells and BALB/c mice, while blocking of autophagy successfully halt the GM effects against the UVB-induced increase of cell senescence, degradation of collagen through upregulation of MMP-1, underscoring GM's substantial anti-skin aging impact via autophagy induction in vitro and in vivo. ConclusionTogether, GM has potent antioxidant and anti-skin aging ingredients that can be used to formulate skin care products for both the nutraceutical and cosmeceutical industries.

Epidermal keratinocytes regulate hyaluronan metabolism via extracellularly secreted hyaluronidase 1 and hyaluronan synthase 3

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by HYAL1 knockdown, but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, HAS3 knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated to increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production.

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.

#2339 All-trans retinoic acid enhances the efficacy of mesenchymal stem cells in acute kidney injury

Abstract Background and Aims Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy for acute kidney injury (AKI), but still suffer from the drawbacks of low survival rate, low homing rate, and uncertain differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy on AKI. Method We established a renal ischemia/reperfusion (I/R) injury model induced by clamping of left renal artery for 30 minutes and contralateral nephrectomy. C57BL/6 mice were randomly divided into 4 groups: sham group, IRI group, MSCs group, and ATRA-MSCs group. MSCs or ATRA-pretreated MSCs were injected via tail vein. GFR was measured by transcutaneous monitoring the clearance of FITC-sinistrin. These mice were sacrificed 3 days after surgery, and the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured. The renal pathological changes were examined by Periodic Acid-Schiff staining (PAS). The in vitro hypoxia/reoxygenation (H/R) model was established using human proximal tubular epithelial cells (HK-2 cells). Co-culturing HK-2 cells with MSCs or ATRA-pretreated MSCs. Western blot, quantitative real time-PCR (qRT-PCR), and immunohistochemistry were used to detect mRNA or protein expression. We performed RNA sequencing (RNA-seq) analysis in ATRA-pretreated MSCs. The content of hyaluronic acid (HA) in the supernatant of ATRA-pretreated MSCs culture was detected by ELISA. Hyaluronan synthase mRNA expression level in ATRA-pretreated MSCs assessed by qRT-PCR analysis. The HAS2 promoter-binding transcription factors were forecasted by the JASPAR Transcription Factor Binding Site database. Western blot was used to detect whether P13K/AKT pathway was activated by ATRA-pretreated MSCs. Whether this nephroprotective effect could be reversed were explored by silencing HAS2 or CD44 antibodies. Results The AKI mice showed a significant decrease in GFR and even complete loss of renal function. Administration of MSCs significantly increased GFR, and MSCs pretreated with ATRA further increased GFR. Mice injected with ATRA-pretreated MSCs had significantly lower levels of SCr, BUN and ATN scores compared with untreated MSCs. Western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemistry showed that ATRA pretreated MSCs were more effective in reducing AKI inflammation and apoptosis and promoting proliferative repair than untreated MSCs. GO enrichment analysis demonstrated that the hyaluronan biosynthetic process may be a significantly expressed. HAS2 expression and HA content of MSCS were significantly increased after the addition of ATRA. Analysis for potential transcription factor binding sites revealed that the HAS2 promoter region has multiple potential binding sites for the retinoic acid receptor. Based on the predictions from the JASPAR database, the RXR-RAR heterodimer had the highest prediction score. We then performed ChIP analysis to experimentally verify the binding of RXR-RAR heterodimer transcription factor to HAS2 promoters. We further found that ATRA-pretreated MSCs activated the PI3K/AKT signaling pathway in AKI. As expected, we found that ATRA-pretreated MSCs anti-inflammatory, anti-apoptosis, and pro-proliferation effects were substantially reversed by silencing HAS2 or blocking CD44. Conclusion ATRA promotes HA secretion from MSCs and activates the PI3K/AKT pathway, which in turn enhances the therapeutic efficacy of MSCs on AKI.

Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 μM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

Upregulation of HAS2 promotes glioma cell proliferation and chemoresistance via c-myc

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant human brain tumor. Although comprehensive therapies, including chemotherapy and radiotherapy following surgery, have shown promise in prolonging survival, the prognosis for GBM patients remains poor, with an overall survival rate of only 14.6 months. Chemoresistance is a major obstacle to successful treatment and contributes to relapse and poor survival rates in glioma patients. Therefore, there is an urgent need for novel strategies to overcome chemoresistance and improve treatment outcomes for human glioma patients. Recent studies have shown that the tumor microenvironment plays a key role in chemoresistance. Our study demonstrates that upregulation of HAS2 and subsequent hyaluronan secretion promotes glioma cell proliferation, invasion, and chemoresistance in vitro and in vivo through the c-myc pathway. Targeting HAS2 sensitizes glioma cells to chemotherapeutic agents. Additionally, we found that hypoxia-inducible factor HIF1α regulates HAS2 expression. Together, our findings provide insights into the dysregulation of HAS2 and its role in chemoresistance and suggest potential therapeutic strategies for GBM.

Corn Silk Polysaccharide Reduces the Risk of Kidney Stone Formation by Reducing Oxidative Stress and Inhibiting COM Crystal Adhesion and Aggregation.

HK-2 cells were damaged by 100 nm COM crystals to build an injury model. The cells were protected by CSPs with -COOH contents of 3.92% (CSP0) and 16.38% (CCSP3), respectively. The changes in the biochemical indexes of HK-2 cells and the difference in adhesion amount and aggregation degree of nano-COM on the cell surface before and after CSP protection were detected. CSP0 and CCSP3 protection can obviously inhibit HK-2 cell damage caused by nano-COM crystals, restore cytoskeleton morphology, reduce intracellular ROS level, inhibit phosphoserine eversion, restore the polarity of the mitochondrial membrane potential, normalize the cell cycle process, and reduce the expression of adhesion molecules, OPN, Annexin A1, HSP90, HAS3, and CD44 on the cell surface. Finally, the adhesion and aggregation of nano-COM crystals on the cell surface were effectively inhibited. The carboxymethylated CSP3 exhibited a higher protective effect on cells than the original CSP0, and cell viability was further improved with the increase in polysaccharide concentration. CSPs can protect HK-2 cells from calcium oxalate crystal damage and effectively reduce the adhesion and aggregation of nano-COM crystals on the cell surface, which is conducive to inhibiting the formation of calcium oxalate kidney stones.

Abstract 3283: Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance

Abstract Introduction: Despite numerous available treatments, metastatic renal cell carcinoma (mRCC) remains highly lethal, with over 90% of patients succumbing within 5 years. Sorafenib (SF), a second-line treatment for mRCC, exhibits modest efficacy, and the underlying molecular basis for its failure is elusive. Hyaluronic acid (HA), implicated in promoting cell proliferation, invasion, and motility through CD44 and RHAMM receptors, represents a potential player in SF resistance. Hymecromone (HC), a non-toxic dietary supplement, is known to inhibit HA synthesis and downregulate CD44 and RHAMM mRNA levels. This study investigates the elusive mechanisms of SF resistance and evaluates the effectiveness of the HC+SF combination as a targeted therapy for advanced RCC, providing insights into a novel and potentially impactful treatment approach. Methods: HA levels were measured in normal and tumor kidney specimens using ELISA-like assay and IHC. Similarly, HAS3 and UGT1A9 (A9) levels and SF glucuronidation were measured by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination was examined in A9 and HAS3 knockdown and overexpression cell lines (transfectants) and primary RCC spheroids by proliferation, apoptosis, motility, and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in subcutaneous and orthotopic xenograft mouse models, with bioluminescence imaging. Results: SF failure in mRCC was linked due to its glucuronidation and inactivation by A9. Tumor microsomes from patients who developed mRCC glucuronidated SF 5-fold higher than from non-mRCC patients. A9 levels were elevated (8-10 folds) in mRCC patients’ tumor along with HA and HAS3 (5-10 folds) as compared to non-mRCC patients and normal kidney tissues. HC treatment downregulated A9 promoter activity and inhibited SF-glucuronidation in RCC cells. SF (5uM) and HC (20, 40ug/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (&amp;gt;90%), motility/invasion (&amp;gt;80%), and induced apoptosis (5-fold) and downregulated HAS3 expression (&amp;gt;80%), and HA synthesis in RCC cells. While A9 and HAS3 transfectants both were resistant to SF+HC treatment, A9 and HAS3 - KO transfectants were sensitive to SF. Endothelial cells co-cultured with either A9 or HAS3 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in A9 or HAS3 transfectants. HC (100 or 200mg/kg) and SF (30mg/kg) oral combination abrogated Caki-1 tumor growth (&amp;gt;80%), without serum/tissue toxicity. HC+SF treatment decreased A9, HA and HAS3 levels, Ki67 index and microvessel density. Both A9 and HAS3 overexpressing tumors were resistant to treatment. Conclusion: This study demonstrates the mechanism of SF failure in mRCC and proposes a safe, oral adjunct combination for effective treatment. Citation Format: Anuj Kumar Sharma, Karina Aguilar, Chandramukhi S. Panda, Vinata B. Lokeshwar. Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3283.

Unravelling the role of HAS2, GREM1, and PTGS2 gene expression in cumulus cells: implications for human oocyte development competency - a systematic review and integrated bioinformatic analysis.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities - DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Long Term Effect of Echinochrome A on the Extracellular Matrix of Vocal Folds Lamina Propria in Ovariectomized Rats

Background and Objectives Voice problems after menopause are caused by extracellular matrix (ECM) changes in the vocal fold lamina propria. Echinochrome A (ECH), which is known to a strong anti-oxidant, was injected into ovariectomized rats (OVX) to evaluate the effect on the ECM of the vocal fold lamina propria.Materials and Method Sprague-Dawley female rats were randomly divided into three groups as follows: group I (sham-operated rats, SHAM), group II (OVX), group III (ovariectomized rats injected with ECH, OVX+ECH). Rats were sacrificed 12 weeks after ovariectomy.Results ECH treatment had no effect on the levels of estradiol and expression of estrogen receptor β in vocal fold. The expression of Has2 and Has3 were decreased in the OVX and increased with ECH treatment. The expression of Col1a1, Col1a2, and Col3a1 were decreased in the OVX group, but significantly elevated in the OVX+ECH group. The expression of Eln was decreased in the OVX group, but the level of Eln increased in the OVX+ECH group. The expression of matrix metalloproteinases (MMP)-1, 8, and 9 was increased in the OVX group, but the expression of MMP-1 and 8 were decreased after ECH treatment. ECH treatment had no effect on 4-hydroxynonenal in vocal fold, superoxide dismutase, catalase, and glutathione peroxidase were decreased in the OVX and increased with ECH treatment.Conclusion It was confirmed that ECH injection for 12 weeks suppressed changes in collagen, elastin, and MMPs expression caused by menopause. These findings suggest that ECH can be used as a novel alternative therapy for postmenopausal voice changes.

Enhanced Anti‐Photoaging Effects of Adipose‐Derived Stem Cell (ADSC) Secretome via Liposomal and Iontophoretic Intradermal Delivery

AbstractThe growing societal concern regarding skin aging caused by ultraviolet (UV) exposure has spurred the quest for effective therapeutic solutions. Among these, secreted factors derived from adipose‐derived stem cells (ADSC) have emerged as promising candidates with anti‐photoaging properties. The secretome of ADSC stimulates resident fibroblasts, triggering antioxidant effects and promoting the synthesis of the extracellular matrix, thus counteracting the effects of photoaging. In this study, the potential of dried ADSC secretome (AD‐Sc), obtained through a compressed fluid antisolvent process, is assessed as an anti‐photoaging agent. In addition, liposome nanocarriers are used to encapsulate AD‐Sc (Sc_Lipo) to enhance its delivery efficiency. The findings demonstrate that both AD‐Sc and Sc_Lipo effectively reduce reactive oxygen species levels in UV‐damaged human fibroblasts, upregulate the expression of HAS1 and COL1, and inhibit MMP2. Moreover, it is discovered that reverse electrodialysis (RED)‐driven iontophoresis improves the intradermal delivery of Sc_Lipo, as validated in an ex vivo porcine skin. Finally, in an in vivo UV‐induced wrinkling mice model, the Lipo+RED group shows noticeable skin photoaging improvements. This study provides additional evidence that a combined intradermal delivery system for AD‐Sc may represent a viable therapeutic approach for mitigating and preventing photoaging in clinical settings.

The skin hydration and anti-inflammatory potential of zerumbone, a natural sesquiterpene of Zingiber zerumbet, enhanced Src/ERK-mediated HAS-2/AQP-3 and inhibited NFκB/AP-1 expression in UVB-irradiated human keratinocytes

We assayed skin hydration and anti-inflammatory efficacies of zerumbone (Zer, 2.5–10 μM), a natural sesquiterpene of Zingiber zerumbet, using non– or UVB (30 mJ/cm2)-irradiated keratinocytes (HaCaT). & Zer increased cell viability, upregulated hyaluronic acid, and inhibited ROS generation in UVB-irradiated HaCaT cells. Zer promoted antioxidant Nrf2 nuclear translocation resulting in HO-1 and γ-GCLC expression. Zer promotes skin hyaluronic acid by increasing protein and mRNA expression of HAS-2 and AQP-3 in non– or UVB-irradiated HaCaT cells. Furthermore, Zer increased Src and ERK phosphorylation. Src silencing or ERK inhibitor (PD98059) diminished Zer-mediated skin hydration, as evidenced by decreased HAS-2 and AQP-3 expression. Interestingly, UVB-induced Src/ERK inhibition was reversed by Zer or N-acetylcysteine. Additionally, Zer inhibited inflammatory iNOS, COX-2, and IL-1β expression through NFκB (p65) and AP-1 (c-Jun/c-Fos) pathway in UVB-irradiated HaCaT cells. HaCaT cells treated with Zer enhanced the growth factors PDGF-A, VEGF, and EGFR expressions. Zerumbone might be utilized in cosmetic formulations.