Accumulation Of Hyaluronic Acid Research Articles

Hyaluronic Acid Influences Amino Acid Metabolism via Differential L‐Type Amino Acid Transporter 1 Expression in the U87‐Malignant Glioma Cell Line

The glioblastoma (GBM) tumor microenvironment is heterogeneous, complex, and being increasingly understood as a significant contributor to tumor progression. In brain tumors, the extracellular matrix contains a large concentration of hyaluronic acid (HA) that makes it important to study its role in cancer progression. In particular, abnormal accumulation of HA is observed in gliomas and is often associated with poor prognosis. In addition, HA is a polymer and its molecular weight (MW) distribution may influence tumor cell activity. Herein, the influence of the MW of HA on tumor cell metabolism is evaluated. A 2D cell culture approach is used to expose the U87‐MG (medium glucose [MG]) cell line to different HA MWs (10, 60, and 500 kDa) and glucose concentrations (0, 5.5, and 25 mm). Notably, it is found that HA influences GBM amino acid metabolism via reduction in LAT1 transporter protein expression. Also an influence on mitochondrial respiration levels and a difference in the accumulation of some key products of cell metabolic activity (lactic acid, glutamic acid, and succinic acid) are reported. Overall, in these results, it is indicated that HA MW can influence GBM metabolic state, with implications for cell invasion and tumor progression.

Melatonin inhibits the stemness of head and neck squamous cell carcinoma by modulating HA synthesis via the FOSL1/HAS3 axis.

Hyaluronic acid (HA) is a glycosaminoglycan and the main component of the extracellular matrix (ECM), which has been reported to interact with its receptor CD44 to play critical roles in the self-renewal and maintenance of cancer stem cells (CSCs) of multiple malignancies. Melatonin is a neuroendocrine hormone with pleiotropic antitumor properties. However, whether melatonin could regulate HA accumulation in the ECM to modulate the stemness of head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we found that melatonin suppressed CSC-related markers, such as CD44, of HNSCC cells and decreased the tumor-initiating frequency of CSCs in vivo. In addition, melatonin modulated HA synthesis of HNSCC cells by downregulating the expression of hyaluronan synthase 3 (HAS3). Further study showed that the Fos-like 1 (FOSL1)/HAS3 axis mediated the inhibitory effects of melatonin on HA accumulation and stemness of HNSCC in a receptor-independent manner. Taken together, melatonin modulated HA synthesis through the FOSL1/HAS3 axis to inhibit the stemness of HNSCC cells, which elucidates the effect of melatonin on the ECM and provides a novel perspective on melatonin in HNSCC treatment.

Use of Minimal Amounts of Hyaluronidase in the Ultrasound-Guided Treatment of Acute Vascular Occlusion by Hyaluronic Acid: A Preliminary Report

Abstract Background Complications from hyaluronic acid (HA) fillers are increasingly frequent, given the very high number of treatments performed every day worldwide. They are routinely treated with high doses of hyaluronidase, which can cause significant local and general unwanted effects. Objectives The aim of our work is to demonstrate that when the origin of the complication is precisely identified and treated under ultrasound guidance, a few hyaluronidase units are enough to treat it effectively. Methods Five young female patients came to our observation for vascular lesions from injection of HA fillers performed in the immediately preceding days, in 4 cases, and a few weeks earlier in the fifth case. All lesions were accurately identified by ultrasound and treated with hyaluronidase. Results The 4 promptly treated patients fully recovered with a hyaluronidase dose of 87 ± 44 IU (range, 30-150 IU). The fifth patient, treated later, markedly improved regarding clinical picture and symptoms. No early or late side effects have been reported from this hyaluronidase dosage. Conclusions Our data confirm that if the lesion at the origin of the skin damage is precisely localized with ultrasound examination and treated under ultrasound guidance, a few units of hyaluronidase, injected directly into the HA accumulation, effectively resolve the skin damage. At the doses we used, which were much lower than those usually recommended, hyaluronidase proved to be not only effective but also free of any side effects. Level of Evidence: 5

In-silico analysis of TMEM2 as a pancreatic adenocarcinoma and cancer-associated fibroblast biomarker, and functional characterization of NSC777201, for targeted drug development.

Pancreatic adenocarcinoma (PAAD), known as one of the deadliest cancers, is characterized by a complex tumor microenvironment, primarily comprised of cancer-associated fibroblasts (CAFs) in the extracellular matrix. These CAFs significantly alter the matrix by interacting with hyaluronic acid (HA) and the enzyme hyaluronidase, which degrades HA - an essential process for cancer progression and spread. Despite the critical role of this interaction, the specific functions of CAFs and hyaluronidase in PAAD development are not fully understood. Our study investigates this interaction and assesses NSC777201, a new anti-cancer compound targeting hyaluronidase. This research utilized computational methods to analyze gene expression data from the Gene Expression Omnibus (GEO) database, specifically GSE172096, comparing gene expression profiles of cancer-associated and normal fibroblasts. We conducted in-house sequencing of pancreatic cancer cells treated with NSC777201 to identify differentially expressed genes (DEGs) and performed functional enrichment and pathway analysis. The identified DEGs were further validated using the TCGA-PAAD and Human Protein Atlas (HPA) databases for their diagnostic, prognostic, and survival implications, accompanied by Ingenuity Pathway Analysis (IPA) and molecular docking of NSC777201, in-vitro, and preclinical in-vivo validations. The result revealed 416 DEGs associated with CAFs and 570 DEGs related to NSC777201 treatment, with nine overlapping DEGs. A key finding was the transmembrane protein TMEM2, which strongly correlated with FAP, a CAF marker, and was associated with higher-risk groups in PAAD. NSC777201 treatment showed inhibition of TMEM2, validated by rescue assay, indicating the importance of targeting TMEM2. Further analyses, including IPA, demonstrated that NSC777201 regulates CAF cell senescence, enhancing its therapeutic potential. Both in-vitro and in-vivo studies confirmed the inhibitory effect of NSC777201 on TMEM2 expression, reinforcing its role in targeting PAAD. Therefore, TMEM2 has been identified as a theragnostic biomarker in PAAD, influenced by CAF activity and HA accumulation. NSC777201 exhibits significant potential in targeting and potentially reversing critical processes in PAAD progression, demonstrating its efficacy as a promising therapeutic agent.

Pretibial Myxedema in a Hypothyroid Patient with Grave’s Disease: A Case Report

Pretibial myxedema, a rare dermatological manifestation primarily associated with Graves' disease, presents as nonpitting edema, nodular, or plaque-like lesions on the lower legs due to accumulation of hyaluronic acid. We present the case of a 42-year-old woman with a history of Graves' disease who developed dark plaques and indurated skin lesions on her shins, extending to the dorsal feet. Diagnosed with Grave’s disease 20 years prior, she underwent subtotal thyroidectomy following antithyroid treatment. Pretibial myxedema developed one-year post-thyroidectomy and was managed with systemic glucocorticoids. She later received iodine-131 therapy, which led to hypothyroidism, and started to admit thyroid hormone replacement. The thyroid function test revealed normal TSH levels and increased TRAbs. Histopathology of skin lesions showed mucopolysaccharide deposition and increased collagen confirming myxedema. This case highlights the rare elephantiasis form of pretibial myxedema and underscores the importance of recognizing characteristic skin lesions in patients with hypothyroidism.

Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro–computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin β1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Development of In Situ Product Recovery (ISPR) System Using Amberlite IRA67 for Enhanced Biosynthesis of Hyaluronic Acid by Streptococcus zooepidemicus

High broth viscosity due to the accumulation of hyaluronic acid (HA) causes a limited yield of HA. It is a major problem of HA production using Streptococcus zooepidemicus. Extractive fermentation via in situ product recovery (ISPR) was utilized to enhance the HA production. Resins from Amberlite: IRA400 Cl; IRA900 Cl; IRA410 Cl; IRA402 Cl; and IRA67 were tested for the HA adsorption. IRA67 showed high adsorption capacity on HA. The study of the adsorption via a 2 L stirred tank bioreactor of S. zooepidemicus fermentation was investigated to elucidate the adsorption of HA onto IRA67 in dispersed and integrated internal column systems. The application of a dispersed IRA67 improved the HA production compared to the fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column (3.928 g/L) over that obtained with dispersed IRA67. The cultivation with an internal column shows the highest reduction of viscosity value after the addition of IRA67 resin: from 58.8 to 23.7 (mPa·s), suggesting the most effective ISPR of HA. The improved biosynthesis of HA indicated that an extractive fermentation by ISPR adsorption is effective and may streamline the HA purification.

FGF9 Promotes Expression of HAS2 in Palatal Elevation via the Wnt/β-Catenin/TCF7L2 Pathway.

Fgf9 mutation was found in cleft palate patients. Our previous study indicated that Fgf9 promotes timely elevation of palate by regulating hyaluronic acid (HA) accumulation at embryonic day 13.5 (E13.5). HA is synthesized by hyaluronic acid synthases (HAS) isoforms 1, 2, or 3. However, how FGF9 regulates HA in palatogenesis is still unclear. Using Ddx4-Cre mice, we generated the Fgf9-/- mouse model (with exon 2 deletion). Immunohistochemistry was used to detect the location and expression of HAS2 in WT and the Fgf9-/- palate at E13.5. We also predicted the association between Fgf9 and Has2 within the developing palate by performing a bioinformatics analysis. The expression of β-catenin, HAS2, and TCF7L2 were verified by Western blotting after knockout of Fgf9. Rescue experiments were performed by ELISA in vitro. Fgf9-/- mice exhibited 100% penetrance of the cleft palate. A knockout of Fgf9 confirmed that HAS2 and TCF7L2 expression was positively correlated with FGF9. TCF7L2 binds to the Has2 promoter, exhibiting the high specificity predicted by JASPAR. Additionally, increased HA expression by BML-284, TCF-dependent agonist, was blocked in Fgf9-/- palate because of the significant decline in TCF7L2 expression. FGF9 promotes HAS2 expression via Wnt/β-catenin/TCF7L2 pathway with TCF7L2 activating transcription of Has2 in the palate.

Hyaluronidase 1 deficiency decreases bone mineral density in mice

Mucopolysaccharidosis IX is a lysosomal storage disorder caused by a deficiency in HYAL1, an enzyme that degrades hyaluronic acid at acidic pH. This disease causes juvenile arthritis in humans and osteoarthritis in the Hyal1 knockout mouse model. Our past research revealed that HYAL1 is strikingly upregulated (~ 25x) upon differentiation of bone marrow monocytes into osteoclasts. To investigate whether HYAL1 is involved in the differentiation and/or resorption activity of osteoclasts, and in bone remodeling in general, we analyzed several bone parameters in Hyal1 −/− mice and studied the differentiation and activity of their osteoclasts and osteoblasts when differentiated in vitro. These experiments revealed that, upon aging, HYAL1 deficient mice exhibit reduced femur length and a ~ 15% decrease in bone mineral density compared to wild-type mice. We found elevated osteoclast numbers in the femurs of these mice as well as an increase of the bone resorbing activity of Hyal1 −/− osteoclasts. Moreover, we detected decreased mineralization by Hyal1 −/− osteoblasts. Taken together with the observed accumulation of hyaluronic acid in Hyal1 −/− bones, these results support the premise that the catabolism of hyaluronic acid by osteoclasts and osteoblasts is an intrinsic part of bone remodeling.

Synovial joint cavitation initiates with microcavities in interzone and is coupled to skeletal flexion and elongation in developing mouse embryo limbs.

ABSTRACTThe synovial cavity and its fluid are essential for joint function and lubrication, but their developmental biology remains largely obscure. Here, we analyzed E12.5 to E18.5 mouse embryo hindlimbs and discovered that cavitation initiates around E15.0 with emergence of multiple, discrete, µm-wide tissue discontinuities we term microcavities in interzone, evolving into a single joint-wide cavity within 12 h in knees and within 72-84 h in interphalangeal joints. The microcavities were circumscribed by cells as revealed by mTmG imaging and exhibited a carbohydrate and protein content based on infrared spectral imaging at micro and nanoscale. Accounting for differing cavitation kinetics, we found that the growing femur and tibia anlagen progressively flexed at the knee over time, with peak angulation around E15.5 exactly when the full knee cavity consolidated; however, interphalangeal joint geometry changed minimally over time. Indeed, cavitating knee interzone cells were elongated along the flexion angle axis and displayed oblong nuclei, but these traits were marginal in interphalangeal cells. Conditional Gdf5Cre-driven ablation of Has2 – responsible for production of the joint fluid component hyaluronic acid (HA) – delayed the cavitation process. Our data reveal that cavitation is a stepwise process, brought about by sequential action of microcavities, skeletal flexion and elongation, and HA accumulation. This article has an associated First Person interview with the first author of the paper.

The extracellular matrix maintained by hypodermal macrophages via IGF1 is a niche for Staphylococcus aureus infection

Abstract Macrophages (MΦ) play a vital role in tissue homeostasis, but their functions in skin hypodermis, a common site of Staphylococcus aureus (S. aureus) infection, remains largely unexplored. Using flow-cytometry, bulk RNA-seq and single-cell RNA-seq, two MΦ subsets with distinct transcriptomes were uncovered. Both MΦ subsets relied on colony stimulating factor 1 (CSF1) receptor signaling for their survival. Single-cell RNA-seq of non-immune cells identified fibroblasts (FB) as a major source of dermal and hypodermal CSF1. Analysis of dermal and hypodermal stromal cells transcriptomes revealed Tek (encoding Tie2) in hypodermal FB as a potential driver for skin layer-specific ablation of CSF1. Tek-cre x Csf1-floxed mice (Csf1ΔTek) displayed a striking post-natal ablation of hypodermal MΦ associated with an alteration of the collagen network and increased accumulation of hyaluronic acid (HA). Generation of a ligand-receptor map identified candidate genes involved in hypodermal MΦ-FB crosstalk. Among which, depletion of insulin-like growth factor 1 (IGF1) from MΦ in Csf1r-cre x Igf1-floxed (Igf1ΔCsf1r) recapitulated the alteration of the ECM observed in absence of hypodermal MΦ. Interestingly, upon injecting S. aureus into the hypodermis, Csf1ΔTek and Igf1ΔCsf1r mice displayed striking reduction of the cellulitis phenotype as compared to WT mice. Pre-injection of HA attenuated cellulitis in WT mice, suggesting that deposition of HA in the absence of MΦ or MΦ-derived IGF1 conferred host protection against S. aureus. Thus, while hypodermal MΦ critically maintained ECM homeostasis, this provided a niche for S. aureus infection, highlighting implications for novel therapeutic strategies in S. aureus soft-tissue infections.

HYPOTHYROIDISM IN THE PRACTICE OF A GENERAL PRACTITIONER

Thyroid pathology is widespread and is detected in almost half of the population of the Chuvash Republic. The thyroid gland plays an important role in the functioning of many organs and systems in the human body. Thyroid hormones are vital for humans; they regulate water-electrolyte, protein, fat, carbohydrate and energy metabolism, normal functioning of organs and systems, including the central nervous system. One of thyroid gland pathologies associated with a deficiency of its hormones is hypothyroidism. Primary, secondary and tertiary hypothyroidisms are distinguished. Clinical manifestations of hypothyroidism are nonspecific, mostly they depend on the patient's age, duration and severity of hormone deficiency. Hypothyroidism is suspected if patients have the following symptoms: hypothermia, myxedemas due to accumulation of hyaluronic acid and mucin in the tissues, bradyphrenia, myopathy, anemia. Determining the level of TSH and free T4 in the serum helps to confirm it, and a thyrotrophin-releasing hormone test is used to diagnose secondary and tertiary hypothyroidism. The present article discusses a clinical case of hypothyroidism onset and the difficulties in diagnosing this pathology.

Justification of treatment and possible outcomes of severe COVID-19

The role of hyaluronic acid in the pathogenesis of acute respiratory distress syndrome, including those associated with severe COVID-19, is known. Pro-inflammatory cytokines (interleukin-1, tumor necrosis factor) are strong inducers of hyaluronic acid synthase (HAS2) in CD31+ endothelial cells, EpCAM+ cells of the alveolar epithelium of the lungs and fibroblasts. Hyaluronic acid can absorb water in quantities significantly exceeding its own molecular weight. Reducing the presence or inhibiting of hyaluronic acid synthesis is of great importance for facilitating the breathing of COVID-19 patients. Hyaluronidase-based preparations can reduce the accumulation of hyaluronic acid and promote pulmonary alveoli cleansing. Respiratory viral infections, including pandemic strains of coronaviruses, especially in severe cases with acute respiratory distress syndrome, can be complicated by the development of pulmonary fibrosis. It has been shown that changes in X-ray Computed Tomography findings characteristic of fibrosis in the first year after COVID-19 can significantly regress. A clinical case from the practice of treating a patient with a severe course of COVID-19, significant cardiovascular comorbidity, grade 2 obesity, which was regarded as significant risk factors for an unfavorable outcome, is presented. The patient with signs of progressive respiratory failure was admitted to the intensive care unit. Pulse therapy with glucocorticosteroids and anticoagulants was started. Deterioration of the condition is regarded as the beginning of acute respiratory distress syndrome, which complicated the cytokine storm induced by the coronavirus. The patient was taken for high-flow oxygenation. An anti-cytokine therapy was prescribed. Reduction of inflammatory markers was obtained, but severe respiratory failure persisted. The bovgialuronidase azoximer was included in the treatment. The patient's condition began to stabilize then she was discharged in stable condition without oxygen support. The available data on the negative role of hyaluronic acid in the pathogenesis of acute respiratory distress syndrome in patients with COVID-19, as well as the need to reduce the likelihood of developing residual fibrous changes in the lungs in patients who have undergone acute respiratory distress syndrome, suggest the need for further studies of domestic azoximer bovgialuronidase properties in the treatment of severe forms of COVID-19.

Biosynthesis of hyaluronan in engineered Escherichia coli via the secretion of thermophilic exo-mannanase using palm kernel cake as the carbon source

The potential of palm kernel cake (PKC) wastes as feedstocks for the production of hyaluronic acid (HA) using engineered Escherichia coli has been investigated. Herein, we report a novel thermophilic exo-β-D-mannanase TFMS, which exhibits superior enzymatic properties and more efficiently hydrolyzes PKC to yield oligosaccharides composed of 1-m to 10-m monosaccharide residues. By optimizing signal peptides, we achieved high-level secretory production of TFMS in engineered chassis strain, which could effectively release oligosaccharides from PKC for cell growth. Our results confirm that the chassis strain maintains relatively high cell-growth rates and biomass in PKC media with approximately 70.5% levels of that in LB media. Furthermore, hasA, a HA synthase gene from Streptococcus zooepidemicus, was expressed in chassis strain to produce a relatively high HA accumulation of 192 mg/L from PKC. This study provides a new approach for developing feedstock utilizations of PKC wastes by constructing efficient microbial cell factories to biosynthesize value-added products.

Biomimetic stiffening of cell-laden hydrogels via sequential thiol-ene and hydrazone click reactions

Hydrogels with dynamically tunable crosslinking are invaluable for directing stem cell fate and mimicking a stiffening matrix during fibrosis or tumor development. The increases in matrix stiffness during tissue development are often accompanied by the accumulation of extracellular matrices (e.g., collagen, hyaluronic acid (HA)), a phenomenon that has received little attention in the development of dynamic hydrogels. In this contribution, we present a gelatin-based cell-laden hydrogel system capable of being dynamically stiffened while accumulating HA, a key glycosaminoglycans (GAG) increasingly deposited by stromal cells during tumor progression. Central to this strategy is the synthesis of a dually-modified gelatin macromer – gelatin-norbornene-carbohydrazide (GelNB-CH), which is susceptible to both thiol-norbornene photopolymerization and hydrazone click chemistry. We demonstrate that the crosslinking density of cell-laden thiol-norbornene hydrogels can be dynamically tuned via simple incubation with aldehyde-bearing macromers (e.g., oxidized dextran (oDex) or oHA). The GelNB-CH hydrogel system is highly cytocompatible, as demonstrated by in situ encapsulation of pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF). This unique dynamic stiffening scheme provides a platform to study tandem accumulation of HA and elevation in matrix stiffness in the pancreatic tumor microenvironment. Statement of significanceHydrogels permitting on-demand and secondary crosslinking are ideal for mimicking a stiffening tumor microenvironment (TME). However, none of the current dynamic hydrogels account for both stiffening and accumulation of hyaluronic acid (HA), a major extracellular matrix component increasingly deposited in tumor stromal tissues, including pancreatic ductal adenocarcinoma (PDAC). The current work addresses this gap by developing a dynamic hydrogel system capable of simultaneously increasing stiffness and HA accumulation. This is achieved by a new gelatin macromer permitting sequential thiol-norbornene (for primary network crosslinking) and hydrazone click chemistry (for bioinert or biomimetic stiffening with oxidized dextran (oDex) or oHA, respectively). The results of this study provide new insights into how dynamically changing physicochemical matrix properties guide cancer cell fate processes.

Interfering with hyaluronic acid metabolism suppresses glioma cell proliferation by regulating autophagy

The tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. However, the role of abnormal HA accumulation in glioma remains unclear. The present study indicated that HA, hyaluronic acid synthase 3 (HAS3), and a receptor of HA named CD44 were expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 expression or blocking CD44 inhibited glioma cell proliferation in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-methylumbelliferone (4-MU), a small competitive inhibitor of Uridine diphosphate (UDP) with the ability to penetrate the blood-brain barrier (BBB), also inhibited glioma cell proliferation in vitro and in vivo. Thus, approaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

The Fibroblast Growth Factor 9 (Fgf9) Participates in Palatogenesis by Promoting Palatal Growth and Elevation.

Cleft palate, a common global congenital malformation, occurs due to disturbances in palatal growth, elevation, contact, and fusion during palatogenesis. The Fibroblast growth factor 9 (FGF9) mutation has been discovered in humans with cleft lip and palate. Fgf9 is expressed in both the epithelium and mesenchyme, with temporospatial diversity during palatogenesis. However, the specific role of Fgf9 in palatogenesis has not been extensively discussed. Herein, we used Ddx4-Cre mice to generate an Fgf9–/– mouse model (with an Fgf9 exon 2 deletion) that exhibited a craniofacial syndrome involving a cleft palate and deficient mandibular size with 100% penetrance. A smaller palatal shelf size, delayed palatal elevation, and contact failure were investigated to be the intrinsic causes for cleft palate. Hyaluronic acid accumulation in the extracellular matrix (ECM) sharply decreased, while the cell density correspondingly increased in Fgf9–/– mice. Additionally, significant decreases in cell proliferation were discovered in not only the palatal epithelium and mesenchyme but also among cells in Meckel’s cartilage and around the mandibular bone in Fgf9–/– mice. Serial sections of embryonic heads dissected at embryonic day 14.5 (E14.5) were subjected to craniofacial morphometric measurement. This highlighted the reduced oral volume owing to abnormal tongue size and descent, and insufficient mandibular size, which disturbed palatal elevation in Fgf9–/– mice. These results indicate that Fgf9 facilitates palatal growth and timely elevation by regulating cell proliferation and hyaluronic acid accumulation. Moreover, Fgf9 ensures that the palatal elevation process has adequate space by influencing tongue descent, tongue morphology, and mandibular growth.

Helix-Loop-Helix Factor Id3 (Inhibitor of Differentiation 3): A Novel Regulator of Hyaluronan-Mediated Adipose Tissue Inflammation.

The aim of this study was to unravel mechanisms whereby deficiency of the transcription factor Id3 (inhibitor of differentiation 3) leads to metabolic dysfunction in visceral obesity. We investigated the impact of loss of Id3 on hyaluronic acid (HA) production by the 3 HAS isoenzymes (HA synthases; -1, -2, and -3) and on obesity-induced adipose tissue (AT) accumulation of proinflammatory B cells. Approach and Results: Male Id3-/- mice and respective wild-type littermate controls were fed a 60% high-fat diet for 4 weeks. An increase in inflammatory B2 cells was detected in Id3-/- epididymal AT. HA accumulated in epididymal AT of high-fat diet-fed Id3-/- mice and circulating levels of HA were elevated. Has2 mRNA expression was increased in epididymal AT of Id3-/- mice. Luciferase promoter assays showed that Id3 suppressed Has2 promoter activity, while loss of Id3 stimulated Has2 promoter activity. Functionally, HA strongly promoted B2 cell adhesion in the AT and on cultured vascular smooth muscle cells of Id3-/- mice, an effect sensitive to hyaluronidase. Our data demonstrate that loss of Id3 increases Has2 expression in the epididymal AT, thereby promoting HA accumulation. In turn, elevated HA content promotes HA-dependent binding of B2 cells and an increase in the B2 cells in the AT, which contributes to AT inflammation.

Over-expression of Chitotriosidase Modulates ECM Distribution in Atherosclerotic Plaques of Mice

Abstract Macrophage is the predominant cell type in all phases of atherosclerosis and it plays a major role in disease progression. Although mammals have no endogenous chitin, macrophages produce chitotriosidase-1 (CHIT1) as part of the innate immune response in various inflammatory conditions including atherosclerosis. We aimed to investigate mechanisms by which CHIT1 may modulate the progression of atherosclerosis using CHIT1-overexpressing, Ldlr−/− mice, and also to determine whether overexpression of CHIT1 by macrophages affects the morphology of atherosclerotic plaques. We developed an atherosclerosis-prone, conditional CHIT1 over-expressing (CHIT1-OE) mouse model which allowed us to study the effects of macrophage CHIT1 over-expression on morphological characteristics as well as extracellular matrix (ECM) formation in vivo. Further, bone marrow-derived macrophages (BMDM) from CHIT1-OE mice and littermate controls were used to determine the cellular mechanisms of non-enzymatic CHIT1 signaling in vitro. Over-expression of CHIT1 in Ldlr−/− mice after 12 weeks of high-fat diet modulated the ECM by altering the accumulation and localization of hyaluronic acid (HA) and collagen in atherosclerotic plaques observed within the aortic sinus. We also found, in vitro, that macrophage invasiveness was significantly enhanced in BMDM from CHIT1-OE mice upon addition of IL-13 as an immunoregulator. Our findings indicate that CHIT1 over-expression modulates ECM properties in the atherosclerotic plaques of Ldlr−/− mice and macrophage behavior in vitro, which may be atheroprotective and promote plaque stability.

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

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