Glycosaminoglycans Research Articles

Adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the AAV vectors expressing GALNS enzyme and a C-type natriuretic peptide (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

Polysaccharide sulfotransferases: the identification of putative sequences and respective functional characterisation.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

Effect of Chitosan on Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts.

Previously, chitosan reduces the senescence-related phenotypes in human foreskin fibroblasts through the transforming growth factor beta (TGF-β) pathway, and enhances the proliferation and migration capabilities of these cells are demonstrated. In this study, we examined whether the senescence-delaying effect of chitosan could be applied to primary knee-related fibroblasts, such as human synovial membrane derived cells (SCs) and anterior cruciate ligament fibroblasts (ACLs). These two types of cells were obtained from donors who needed ACL reconstruction or knee replacement. We found that chitosan treatment effectively reduced aging-associated β-galactosidase (SA-β-gal)-positive cells, downregulated the expression of senescence-related proteins pRB and p53, and enhanced the 5-bromo-2'-deoxyuridine (BrdU) incorporation ability of SCs and ACLs. Moreover, chitosan could make SCs secret more glycosaminoglycans (GAGs) and produce type I collagen. The ability of ACLs to close the wound was also enhanced, and the TGF-β and alpha smooth muscle actin (αSMA) protein expression decreased after chitosan treatment. In summary, chitosan not only delayed the senescence but also enhanced the functions of SCs and ACLs, which is beneficial to the application of chitosan in cell expansion in vitro and cell therapy.

Compositional, Structural, and Biomechanical Properties of Three Different Soft Tissue-Hard Tissue Insertions: A Comparative Review.

Connective tissue attaches to bone across an insertion with spatial gradients in components, microstructure, and biomechanics. Due to regional stress concentrations between two mechanically dissimilar materials, the insertion is vulnerable to mechanical damage during joint movements and difficult to repair completely, which remains a significant clinical challenge. Despite interface stress concentrations, the native insertion physiologically functions as the effective load-transfer device between soft tissue and bone. This review summarizes tendon, ligament, and meniscus insertions cross-sectionally, which is novel in this field. Herein, the similarities and differences between the three kinds of insertions in terms of components, microstructure, and biomechanics are compared in great detail. This review begins with describing the basic components existing in the four zones (original soft tissue, uncalcified fibrocartilage, calcified fibrocartilage, and bone) of each kind of insertion, respectively. It then discusses the microstructure constructed from collagen, glycosaminoglycans (GAGs), minerals and others, which provides key support for the biomechanical properties and affects its physiological functions. Finally, the review continues by describing variations in mechanical properties at the millimeter, micrometer, and nanometer scale, which minimize stress concentrations and control stretch at the insertion. In summary, investigating the contrasts between the three has enlightening significance for future directions of repair strategies of insertion diseases and for bioinspired approaches to effective soft-hard interfaces and other tough and robust materials in medicine and engineering.

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GENETIC VARIANCE IN HEPARAN SULFATION IS ASSOCIATED WITH SALT-SENSITIVITY: A GENE-ENVIRONMENT ANALYSIS

Objective: The high heritability of salt sensitivity suggests an essential role for genetic variants in the relationship between sodium intake and blood pressure (BP). Candidate gene studies have mainly focused on renal and vascular pathways, but the role of glycosaminoglycan (GAG) genes, which are crucial for salinity tolerance, remains to be elucidated. Design and method: Interactions between 54,126 variants in 130 GAG genes and daily sodium excretion on BP were explored in 20,420 EPIC-Norfolk subjects. UK Biobank (n=414,132) and the multi-ethnic HELIUS study (n=2,239) were used for validation. In HELIUS sodium intake was estimated with Food Frequency Questionnaires (FFQ). Urinary GAG composition in 57 Dutch origin HELIUS participants stratified by genotype were analyzed to assess the association between SNP and GAG difference. Also, urinary GAG composition in 12 healthy volunteers after a sodium intervention (7-day low (50 mmol Na+/day) and 7-day high (200 mmol Na+/day) diet) were studied to assess if differences in genotype are associated with different GAG expression. Results: The intron variant rs2892799, mapped to NDST3, showed the strongest interaction with sodium on mean arterial pressure (MAP) (FDR 0.03), and the intron variant rs9654628 mapped to HS3ST5 with sodium on systolic BP (FDR 0.03). These interactions were multi-ethnically validated (Fig 1A, B). When stratified for rs2892799 genotype, T-allele carriers had a significantly higher urinary expression of N-sulfated heparan sulfate epitope D0S0 (Fig1C). Conversely, upon dietary salt loading, urinary D0S0 expression differed substantially between salt-sensitive and salt-resistant individuals (Fig1D). Sodium-induced effects on this epitope were opposite in salt-sensitive in salt-resistant individuals. Conclusions: Genetic variants associated with NDST3 and HS3ST5 interact with relation between sodium consumption and BP. rs2892799 (NDST3) genotype results in a different expression of the N-sulfated D0S0 epitope. In turn, D0S0 expression is associated with salt-mediated BP increase, indicating that genetic GAG variation and metabolic GAG adaptation to a high salt environment might be important for human BP regulation.

Elevated Circulating Cell-Free Hemoglobin Drives Endothelial Glycocalyx Destruction and Inflammation in Sepsis

Rationale: Endothelial injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS). Endothelial barrier integrity is regulated by the endothelial glycocalyx, a matrix of proteoglycans and glycosaminoglycans (GAGs) that lines the vascular lumen. In sepsis, this glycocalyx is degraded by heparanase, which cleaves heparan sulfate, the primary glycocalyx GAG. Glycocalyx breakdown is linked to worse sepsis outcomes, but the mechanisms inducing heparanase activity in sepsis are unknown. We previously found that plasma levels of hemoglobin released from red blood cells (cell-free hemoglobin, CFH) are elevated in clinical sepsis and correlate with higher rates of ARDS and death. CFH also worsens endothelial injury and microvascular permeability in murine sepsis. We therefore hypothesized that CFH upregulates heparanase production to drive glycocalyx shedding and inflammation in sepsis. Methods: To test the association between CFH levels, heparanase expression, and glycocalyx degradation, circulating levels of heparanase, heparan sulfate, and total GAGs were measured in 77 sepsis patients and compared across CFH quartiles. In mice with intraabdominal polymicrobial sepsis with elevated CFH (CS [cecal slurry] + intravenous [IV] CFH), plasma heparanase, total GAGs, and syndecan-1 were quantified. Whole lung and plasma pro-inflammatory cytokines were also determined to investigate the impact of CFH on inflammation in sepsis. Results: In sepsis patients, higher circulating CFH levels were associated with increased plasma heparanase (p=0.0304), total GAGs (p=0.0178), and heparan sulfate (p=0.0112). CFH also exacerbated glycocalyx degradation in murine sepsis; circulating syndecan-1 (p=0.0152) and total GAGs (p=0.0094) were elevated in animals that received CS+IV CFH compared to CS alone. This effect may be mediated by upregulation of active heparanase, which was higher in the plasma of CS+IV CFH treated animals (p=0.1333). CFH also worsened illness severity, as CS+IV CFH animals had worse sepsis severity scores (p=0.0074). Whole lung tumor necrosis factor-alpha (TNF-a; p=0.0129) and interferon-gamma (IFN-g; p=0.0264) transcript levels were increased in the CS+IV CFH group, indicating greater lung inflammation. Systemic inflammation was also enhanced in the CS+IV CFH animals, demonstrated by higher plasma TNF-a (p<0.001) and IFN-g (p=0.0221) concentrations. Conclusions: In sepsis, CFH is associated with upregulation of heparanase and endothelial glycocalyx degradation, which may disrupt the endothelial barrier, causing downstream organ injury. Future studies to define the molecular pathways that underlie these findings and their effect on endothelial permeability will be an important next step towards identifying novel treatment targets to improve outcomes for patients with sepsis-associated ARDS. Funding F30HL170483, R01HL164937, R01HL158906, R01HL150783, R01HL125371. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Podocyte glycocalyx plays an essential role in the function of GFB and the development of albumin leakage

The role of the cell surface layer, glycocalyx is emerging in various areas of cell biology including signaling, metabolism, and immune response. Although a decrease of podocyte glycocalyx depth in glomerular pathologies has been reported before, the specific role of specialized glycocalyx between the podocyte foot processes and the glomerular basement membrane (GBM) in the function and dysfunction of the glomerular filtration barrier (GFB) is elusive. Here we aimed to develop podocyte or endothelial glycocalyx targeting strategies to gain mechanistic understanding of the specific roles of the glycocalyx in the function of GFB and in the development of albumin leakage. Our overall hypothesis is that the specialized podocyte glycocalyx forms an important additional layer of the GFB and changes in the structure and composition of podocyte glycocalyx leads to GFB dysfunction and the development of albumin leakage. New genetic mouse models were generated by the knock-in of pCAG-Lox-STOP-Lox-EGFP-Sdc1 to generate animals with Cre-dependent expression of a syndecan-1 (Sdc1) fusion protein with N-terminal, extracellular GFP. Sdc1-GFPflox animals were crossed with either Podocin-Cre or Cdh5-Cre mice for the specific genetic labeling of the glycocalyx in podocytes or endothelial cells, respectively. Intravital multiphoton microscopy (MPM) was used to measure changes in the depth of glycocalyx and corresponding changes in GFB function in vivo over time. Endoglycosidase enzymes, such as hyaluronidase (50U/animal), heparinase (0.7 U/mouse), and chondroitinase (1 U/mouse) were used to acutely modify the composition of glycocalyx. Alexa-680 conjugated wheat germ agglutinin (WGA) lectin was used to label the glycosaminoglycan (GAG) component of the glycocalyx; Alexa-594 conjugated albumin was used to visualize GFB permeability. Classic phenotyping of Pod and Cdh5 Sdc1-GFP animals showed no significant difference in terms of systolic blood pressure, glomerular filtration rate, body and kidney weight between the Sdc1-GFP and wild type littermates. The co-localization of the genetic GFP and anti-podocin immunofluorescence co-staining confirmed the podocyte-specific expression of Sdc1-GFP. In vivo MPM imaging of Pod-Sdc1-GFP mouse kidneys revealed intense linear labeling of the podocyte cell surface including the apical and basolateral surfaces of the cell body as well as the surfaces of primary, secondary, and foot processes. Interestingly, the thickness and fluorescence intensity of Sdc1-GFP labeled glycocalyx was greater on the basal surface of the podocyte at the GBM as compared to the apical surface implying the role of glycocalyx in the function of GFB. In addition, labeling of the basolateral surface of the podocyte cell body and podocyte foot processes enabled the visualization of sub-podocyte space. Acute injection of endoglycosidase enzymes significantly (3-fold) reduced Sdc1-GFP fluorescence intensity and the thickness of the linear labeling suggesting the acute shedding of podocyte glycocalyx. Importantly, increased albumin permeability of the GFB was observed simultaneously with the shedding of podocyte glycocalyx, indicating the disruption of GFB charge and size-selective functions. In conclusion, the specialized podocyte glycocalyx at the GBM may function as an important new layer of the GFB and may play an important role in the disruption of GFB functions in disease. ASN Carl W. Gottschalk Research Scholar Grant University of Southern California Dean's Pilot Project Award DK123564. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Injectable biomimetic hybrid nanofibers for targeting cartilage in early osteoarthritis treatment

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and limited effective treatment options for regeneration. To address this clinical challenge, an innovative injectable biomimetic hybrid nanofiber, namely hyaluronic acid-graft-poly(2-methacryloyloxyethyl phosphorylcholine)-chondrocyte affinity peptides (HA-PMPC-CAP), was designed and synthesized through a ultraviolet light (UV)-initiated reaction process for intervention of early osteoarthritis (OA). Comprehensive in vitro assessments and molecular docking experiments have confirmed its non-cytotoxicity, exceptional lubrication properties, favorable biocompatibility and high binding affinity to cartilage proteins. Utilizing an OA rat model, the therapeutic potential of HA-PMPC-CAP was explored through intra-articular injection, with a focus on its impact on cartilage regeneration. During an 8-week period, macroscopic, histological, immunohistochemical, and microscopic CT evaluations were conducted on the treated joints. The findings were remarkable: HA-PMPC-CAP established a durable lubricating layer on the surface of cartilage, significantly reduced cartilage deterioration, replenished glycosaminoglycan (GAG) levels, lowered the scores of Osteoarthritis Research Society International (OARSI), and curtailed the development of osteophytes, which was superior to the untreated OA control groups. These results position HA-PMPC-CAP as an effective biomimetic therapy, offering a novel strategy for cartilage regeneration and lubrication in the early stages of OA, which has promising implications for future clinical applications.

Mature neurons from iPSCs unveil neurodegeneration-related pathways in mucopolysaccharidosis type II: GSK-3β inhibition for therapeutic potential

Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/β-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3β inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.

Structural Elucidation of Glycosaminoglycans in the Tissue of Flounder and Isolation of Chondroitin Sulfate C.

Glycosaminoglycans (GAGs) are valuable bioactive polysaccharides with promising biomedical and pharmaceutical applications. In this study, we analyzed GAGs using HPLC-MS/MS from the bone (B), muscle (M), skin (S), and viscera (V) of Scophthalmus maximus (SM), Paralichthysi (P), Limanda ferruginea (LF), Cleisthenes herzensteini (G), Platichthys bicoloratus (PB), Pleuronichthys cornutus (PC), and Cleisthenes herzensteini (CH). Unsaturated disaccharide products were obtained by enzymatic hydrolysis of the GAGs and subjected to compositional analysis of chondroitin sulfate (CS), heparin sulfate (HS), and hyaluronic acid (HA), including the sulfation degree of CS and HS, as well as the content of each GAG. The contents of GAGs in the tissues and the sulfation degree differed significantly among the fish. The bone of S. maximus contained more than 12 μg of CS per mg of dry tissue. Although the fish typically contained high levels of CSA (CS-4S), some fish bone tissue exhibited elevated levels of CSC (CS-6S). The HS content was found to range from 10-150 ug/g, primarily distributed in viscera, with a predominant non-sulfated structure (HS-0S). The structure of HA is well-defined without sulfation modification. These analytical results are independent of biological classification. We provide a high-throughput rapid detection method for tissue samples using HPLC-MS/MS to rapidly screen ideal sources of GAG. On this basis, four kinds of CS were prepared and purified from flounder bone, and their molecular weight was determined to be 23-28 kDa by HPGPC-MALLS, and the disaccharide component unit was dominated by CS-6S, which is a potential substitute for CSC derived from shark cartilage.

On the Mechanism of the Lysosomal Enzyme Iduronate‐2‐sulfatase. A Multiscale Approach

AbstractIduronate‐2‐sulfatase (IDS) is a Ca2+‐dependent enzyme belonging to the family of sulfatases that catalyzes the hydrolysis of sulphurylated glycosaminoglycans (GAGs), like dermatan and heparan sulphate. Its deficiency or modification leads to the accumulation of GAGs in the human body and to the occurrence of severe conditions, such as Hunter disease, or Mucopolysaccharidosis type II. Due to its involvement in this syndrome, it is of interest to understand the action mechanism of the enzyme to design new drugs for more efficient medical strategies. In the present work, we carried out a detailed multiscale modelling‐based investigation, adopting molecular dynamics simulation (MDs) and QM/MM calculations to study the enzyme‐dermatan sulfate interactions and to investigate the reaction mechanism of IDS. The analysis of molecular dynamics trajectories helped to shed light on the contribution of the individual residues of the active site in the recognition of dermatan sulfate. The role of FGly84 and His229, investigated in both neutral and protonated states in the case of the latter, is highlighted for the binding of the substrate. QM/MM calculations demonstrated that the reaction mechanism is a two‐steps process occurring via a sulphurylation‐desulphurylation mechanism where the FGly84 first attacks the sulphate group of the dermatan sulphate (DS), and later is desulphurylated.

Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10μM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Glycosaminoglycan-Mediated Interactions in Articular, Auricular, Meniscal, and Nasal Cartilage.

Glycosaminoglycans (GAGs) are ubiquitous components in the cartilage extracellular matrix (ECM). Ultrastructural arrangement of ECM and GAG-mediated interactions with collagen are known to govern the mechanics in articular cartilage, but these interactions are less clear in other cartilage types. Therefore, this article reviews the current literature on ultrastructure of articular, auricular, meniscal, and nasal septal cartilage, seeking insight into GAG-mediated interactions influencing mechanics. Ultrastructural features of these cartilages are discussed to highlight differences between them. GAG-mediated interactions are reviewed under two categories: interactions with chondrocytes and interactions with other fibrillar macromolecules of the ECM. Moreover, efforts to replicate GAG-mediated interactions to improve mechanical integrity of tissue-engineered cartilage constructs are discussed. In conclusion, studies exploring cartilage specific GAGs are poorly represented in the literature, and the ultrastructure of nasal septal and auricular cartilage is less studied compared with articular and meniscal cartilages. Understanding the contribution of GAGs in cartilage mechanics at the ultrastructural level and translating that knowledge to engineered cartilage will facilitate improvement of cartilage tissue engineering approaches.

Chondroitin Sulfate/Hyaluronic Acid-Blended Hydrogels Suppress Chondrocyte Inflammation under Pro-Inflammatory Conditions.

Osteoarthritis is characterized by enzymatic breakdown of the articular cartilage via the disruption of chondrocyte homeostasis, ultimately resulting in the destruction of the articular surface. Decades of research have highlighted the importance of inflammation in osteoarthritis progression, with inflammatory cytokines shifting resident chondrocytes into a pro-catabolic state. Inflammation can result in poor outcomes for cells implanted for cartilage regeneration. Therefore, a method to promote the growth of new cartilage and protect the implanted cells from the pro-inflammatory cytokines found in the joint space is required. In this study, we fabricate two gel types: polymer network hydrogels composed of chondroitin sulfate and hyaluronic acid, glycosaminoglycans (GAGs) known for their anti-inflammatory and prochondrogenic activity, and interpenetrating networks of GAGs and collagen I. Compared to a collagen-only hydrogel, which does not provide an anti-inflammatory stimulus, chondrocytes in GAG hydrogels result in reduced production of pro-inflammatory cytokines and enzymes as well as preservation of collagen II and aggrecan expression. Overall, GAG-based hydrogels have the potential to promote cartilage regeneration under pro-inflammatory conditions. Further, the data have implications for the use of GAGs to generally support tissue engineering in pro-inflammatory environments.

Role of proteoglycan synthesis genes in osteosarcoma stem cells.

Osteosarcoma stem cells (OSCs) contribute to the pathogenesis of osteosarcoma (OS), which is the most common malignant primary bone tumor. The significance and underlying mechanisms of action of proteoglycans (PGs) and glycosaminoglycans (GAGs) in OSC phenotypes and OS malignancy are largely unknown. This study aimed to investigate the role of PG/GAG biosynthesis and the corresponding candidate genes in OSCs and poor clinical outcomes in OS using scRNA-seq and bulk RNA-seq datasets of clinical OS specimens, accompanied by biological validation by in vitro genetic and pharmacological analyses. The expression of β-1,3-glucuronyltransferase 3 (B3GAT3), one of the genes responsible for the biosynthesis of the common core tetrasaccharide linker region of PGs, was significantly upregulated in both OSC populations and OS tissues and was associated with poor survival in patients with OS with high stem cell properties. Moreover, the genetic inactivation of B3GAT3 by RNA interference and pharmacological inhibition of PG biosynthesis abrogated the self-renewal potential of OSCs. Collectively, these findings suggest a pivotal role for B3GAT3 and PG/GAG biosynthesis in the regulation of OSC phenotypes and OS malignancy, thereby providing a potential target for OSC-directed therapy.

The glycosaminoglycan-binding chemokine fragment CXCL9(74-103) reduces inflammation and tissue damage in mouse models of coronavirus infection.

Pulmonary diseases represent a significant burden to patients and the healthcare system and are one of the leading causes of mortality worldwide. Particularly, the COVID-19 pandemic has had a profound global impact, affecting public health, economies, and daily life. While the peak of the crisis has subsided, the global number of reported COVID-19 cases remains significantly high, according to medical agencies around the world. Furthermore, despite the success of vaccines in reducing the number of deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a gap in the treatment of the disease, especially in addressing uncontrolled inflammation. The massive recruitment of leukocytes to lung tissue and alveoli is a hallmark factor in COVID-19, being essential for effectively responding to the pulmonary insult but also linked to inflammation and lung damage. In this context, mice models are a crucial tool, offering valuable insights into both the pathogenesis of the disease and potential therapeutic approaches. Here, we investigated the anti-inflammatory effect of the glycosaminoglycan (GAG)-binding chemokine fragment CXCL9(74-103), a molecule that potentially decreases neutrophil transmigration by competing with chemokines for GAG-binding sites, in two models of pneumonia caused by coronavirus infection. In a murine model of betacoronavirus MHV-3 infection, the treatment with CXCL9(74-103) decreased the accumulation of total leukocytes, mainly neutrophils, to the alveolar space and improved several parameters of lung dysfunction 3 days after infection. Additionally, this treatment also reduced the lung damage. In the SARS-CoV-2 model in K18-hACE2-mice, CXCL9(74-103) significantly improved the clinical manifestations of the disease, reducing pulmonary damage and decreasing viral titers in the lungs. These findings indicate that CXCL9(74-103) resulted in highly favorable outcomes in controlling pneumonia caused by coronavirus, as it effectively diminishes the clinical consequences of the infections and reduces both local and systemic inflammation.

Sulfated glyco-based hydrogels as self-healing, adhesive, and anti-inflammatory dressings for wound healing

Hydrogels have emerged as a new type of wound dressing materials that involved in different stages of the healing processes. However, most of the existing wound dressings mainly offer a protective and moisturizing layer to prevent cross-infection, while the anti-inflammatory and anti-oxidative properties are frequently induced by extra addition of other bioactive molecules. Here, a novel type of sulfated glyco-functionalized hydrogels for wound dressing was prepared through the hybrid supramolecular co-assembly of carbohydrate segments (FG, FGS and FG3S), fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF), and diphenylalanine-dopamine (FFD). Implanting sulfated carbohydrates can mimic the structure of glycosaminoglycans (GAGs), promoting cell proliferation and migration, along with anti-inflammatory effects. In situ polymerization of FFD introduced a secondary covalent network to the hydrogel, meanwhile, providing anti-oxidation and adhesion properties to wound surfaces. Furthermore, the dynamic supramolecular interactions within the hydrogels also confer self-healing capabilities to the wound dressing materials. In vivo experiments further demonstrated significantly accelerated healing rates with the multifunctional hydrogel FG3S-FFD, indicating high application potential.

Chemical Modifications in Hyaluronic Acid-Based Electrospun Scaffolds.

Hyaluronic acid (HA) is a natural, non-sulfated glycosaminoglycan (GAG) present in ECM. It is involved in different biological functions with appealing properties in cosmetics and pharmaceutical preparations as well as in tissue engineering. Generally, HA has been electrospun in blends with natural or synthetic polymers to produce fibers having diameters in the order of nano and micro-scale whose pores can host cells able to regenerate damaged tissues. In the last decade, a rich literature on electrospun HA-based materials arose. Chemical modifications were generally introduced in HA scaffolds to favour crosslinking or conjugation with bioactive molecules. Considering the high solubility of HA in water, HA-based electrospun scaffolds are cross-linked to increase the stability in biological fluids. Crosslinking is necessary also to avoid the release of HA from the hybrid scaffold when implanted in-vivo. Furthermore, to endow the HA based scaffolds with new chemical or biological properties, conjugation of bioactive molecules to HA was widely reported. Herein, we review the existing research classifying chemical modifications on HA and HA-based electrospun fibers into three categories: i) in-situ crosslinking of electrospun HA-based scaffolds ii) off-site crosslinking of electrospun HA-based scaffolds; iii) conjugation of biofunctional molecules to HA with focus on peptides.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary Oligosaccharides and Glycoamino Acids for the Diagnosis of Mucopolysaccharidosis and Glycoproteinosis.

Mucopolysaccharidosis (MPS) and glycoproteinosis are 2 groups of heterogenous lysosomal storage disorders (LSDs) caused by defective degradation of glycosaminoglycans (GAGs) and glycoproteins, respectively. Oligosaccharides and glycoamino acids have been recognized as biomarkers for MPS and glycoproteinosis. Given that both groups of LSDs have overlapping clinical features, a multiplexed assay capable of unambiguous subtyping is desired for accurate diagnosis, and potentially for severity stratification and treatment monitoring. Urinary oligosaccharides were derivatized with 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP) and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) together with the underivatized glycoamino acids. Novel biomarkers were identified with a semi-targeted approach with precursor mass scanning, the fragmentation pattern (if applicable), and the biochemical basis of the condition. A UPLC-MS/MS analysis with improved chromatographic separation was developed. Novel biomarkers for MPS-IIIA, IIIB, IIIC, and VII were identified and validated. A total of 28 oligosaccharides, 2 glycoamino acids, and 2 ratios were selected as key diagnostic biomarkers. Validation studies including linearity, lower limit of quantitation (LLOQ), and precision were carried out with the assay performance meeting the required criteria. Age-specific reference ranges were collected. In the 76 untreated patients, unambiguous diagnosis was achieved with 100% sensitivity and specificity. Additionally, the levels of disease-specific biomarkers were substantially reduced in the treated patients. A multiplexed UPLC-MS/MS assay for urinary oligosaccharides and glycoamino acids measurement was developed and validated. The assay is suitable for the accurate diagnosis and subtyping of MPS and glycoproteinosis, and potentially for severity stratification and monitoring response to treatment.