Glycosaminoglycans Research Articles

Mucopolysaccharidosis-Plus Syndrome: Is This a Type of Mucopolysaccharidosis or a Separate Kind of Metabolic Disease?

Several years ago, dozens of cases were described in patients with symptoms very similar to mucopolysaccharidosis (MPS). This new disease entity was described as mucopolysaccharidosis-plus syndrome (MPSPS). The name of the disease indicates that in addition to the typical symptoms of conventional MPS, patients develop other features such as congenital heart defects and kidney and hematopoietic system disorders. The symptoms are highly advanced, and patients usually do not survive past the second year of life. MPSPS is inherited in an autosomal recessive manner and is caused by a homozygous-specific mutation in the gene encoding the VPS33A protein. To date, it has been described in 41 patients. Patients with MPSPS exhibited excessive excretion of glycosaminoglycans (GAGs) in the urine and exceptionally high levels of heparan sulfate in the plasma, but the accumulation of substrates is not caused by a decrease in the activity of any lysosomal enzymes. Here, we discuss the pathomechanisms and symptoms of MPSPS, comparing them to those of MPS. Moreover, we asked the question whether MPSPS should be classified as a type of MPS or a separate disease, as contrary to 'classical' MPS types, despite GAG accumulation, no defects in lysosomal enzymes responsible for degradation of these compounds could be detected in MPSPS. The molecular mechanism of the appearance of GAG accumulation in MPSPS is suggested on the basis of results available in the literature.

Корекція пробіотиком пародонтального синдрому у тварин за умов ожиріння і хронічного стресу

The aim of this study was to investigate the effect of obesity and chronic stress on periodontal tissues, both separately and in the combined comorbidity of these conditions in rats, and the role of the probiotic Lactobacillus casei in the correction of periodontal syndrome under these conditions. Methods. The development of periodontal syndrome and the effectiveness of the probiotic Lactobacillus casei IMV B-7280, administered to rats from one month of age intragastrically in a volume of 1 mL (5 · 108 CFU) in two-week courses with breaks of 2 weeks, were studied in a model of glutamate-induced obesity and chronic stress - in combination and separately. In periodontal tissues, the total proteolytic and antitryptic activity, free fucose and glycosaminoglycans (GAG) content, catalase activity, thiobarbiturate active products (TBA reagents), and the content of oxidatively modified proteins (OMP) and molecules of average mass (AMM) were determined; in bone tissue, the molar root exposure coefficient was calculated. The anthropometric parameters of the animals and the Selye triad were studied. Results. We have substantiated the development of periodontal syndrome in animals with glutamate-induced obesity against the background of chronic stress, as evidenced by the activation of free radical oxidation of lipids and proteins, depolymerization of fucoproteins and proteoglycans of periodontal connective tissue, which causes exposure of molar roots by almost 65%. The administration of the probiotic Lactobacillus casei in two-week courses with intervals of 2 weeks to obese rats with chronic stress prevented the development of obesity, as evidenced by a significant decrease in visceral fat mass, Li index, and BMI, as well as the severity of stress syndrome, as evidenced by a decrease in ulcer formation, thymus involution, and adrenal hypertrophy. The periodontoprotective effect of Lactobacillus casei under the combined influence of obesity and chronic stress was proved by significant changes in oxidative stress, free fucose, and GAG content and a 1.5-times decrease in the molar root exposure ratio. Conclusions. Periodic administration of Lactobacillus casei to rats treated with sodium glutamate in the neonatal period against the background of chronic stress prevented the development of obesity, the severity of stress syndromeб and damage to periodontal tissues, as evidenced by the prevention of oxidative stress, increased catabolism of extracellular connective tissue proteins and antiresorptive effect.

Abstract 72: Genetic variance in heparan sulfation is associated with salt-sensitivity: a gene-environment analysis in EPIC-Norfolk, UK biobank, and HELIUS

Background: High heritability of salt sensitivity suggests an essential role for genetics in the relationship between sodium intake and blood pressure (BP). The role of glycosaminoglycan (GAG) genes, which are crucial for salinity tolerance througout evolution, remains to be elucidated. Methods: 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, comprising a balanced representation of people from European, Subsaharan, South Asian, Turkish, and Moroccan descent) were used for validation. Afterwards, urinary GAG composition was studied in HELIUS participants (n=57) stratified by genotype, and upon dietary sodium loading in a time-controlled crossover intervention study (n=12). Results: rs2892799 (NDST3) showed the strongest interaction with sodium on mean arterial pressure (MAP) (FDR 0.03), with higher MAP for the C-allele in high sodium conditions. Also, rs9654628 (HS3ST5) showed an interaction with sodium on systolic BP (FDR 0.03). These interactions were multi-ethnically validated (figures A and B, respectively). Stratifying for rs2892799 genotype, showed higher urinary expression of N-sulfated heparan sulfate epitope D0S0 for the T-allele. Conversely, upon dietary sodium loading, urinary D0S0 expression was higher in participants with stable BP after sodium loading, and sodium-induced effects on this epitope were opposite in individuals with and without BP response to sodium. Conclusion: The C-allele of rs2892799 (NDST3) exhibits higher BP in high sodium conditions when compared to low sodium conditions, whereas no differences were detected for the T-allele. Concomitantly, both alleles demonstrate distinct expressions of D0S0, which, in turn, correlates with sodium-mediated BP elevation. These findings underscore the potential significance of genetic GAG variation in human BP regulation.

Synergistic effects of biological stimuli and flexion induce microcavities promote hypertrophy and inhibit chondrogenesis during in vitro culture of human mesenchymal stem cell aggregates.

Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.

Novel Cosmetic Ingredient CS-AA Polyion Complex and Skin Moisturizing Effect.

The study explored the enhanced skin moisturizing capabilities and moisture retention effects achieved by forming a polyion complex using sulfated glycosaminoglycan (GAG), specifically chondroitin sulfate (CS), and amino acids (AA) such as glutamine (Q) and arginine (R). The overall hydration effect of this CS-AA complex was examined. After analyzing the CS-AA polyion complex structure using spectroscopic methods, the ex vivo moisture retention ability was assessed under dry conditions using porcine skin samples. Additionally, the efficacy of the CS-AA polyion complex in reducing transepidermal water loss (TEWL) and improving skin hydration was evaluated on human subjects using a digital evaporimeter and a corneometer, respectively. Validating a systematic reduction in particle size, the following order was observed: CS>CS/AA simple mixture>CS-AA complex based on dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis. Furthermore, observations revealed that the CS-AA complex exhibits negligible surface charge. Additionally, Fourier-transform infrared spectroscopy (FT-IR) analysis demonstrated a distinct peak shift in the complex, confirming the successful formation of the CS-AA complex. Subsequently, the water-holding effect through porcine skin was assessed, revealing a notable improvement in moisture retention (weight loss) for the CS-Q complex: 40.6% (1h), 20.5% (2h), and 18.7% (4h) compared to glycerin. Similarly, the CS-R complex demonstrated enhancements of 50.2% (1h), 37.5% (2h), and 33% (4h) compared to glycerin. Furthermore, TEWL improvement efficacy on human skin demonstrated approximately 25% improvement for both the CS-Q complex and CS-R complex, surpassing the modest 12.5% and 18% improvements witnessed with water and glycerin applications, respectively. Finally, employing a corneometer, hydration changes in the skin were monitored over 4 weeks. Although CS alone exhibited nominal alterations, the CS-Q complex and CS-R complex showed a significant increase in moisture levels after 4weeks of application. In this study, polyion complexes were successfully formed between CS, a sulfated GAG, and AA. Comparisons with glycerin, a well-known moisturizing agent, confirmed that the CS-AA complex exhibits superior moisturizing effects in various aspects. These findings suggest that the CS-AA complex is a more effective ingredient than CS or AA alone in terms of efficacy.

BMP2 Binds Non-Specifically to PEG-Passivated Biomaterials and Induces pSMAD 1/5/9 Signalling.

Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore how extracellular matrix components influence cellular response. In this study, the previously developed streptavidin platforms is aimed to use to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, it is observed that the interpretation of findings is skewed due to the GAG-unrelated, non-specific binding of BMP2 on components of biomaterials. Non-specific adsorption of proteins is a recurrent and challenging issue for biomaterial studies. Despite the initial incorporation of anti-fouling polyethylene glycol (PEG) chains within biomaterials, the residual non-specific BMP2 adsorption still triggered BMP2 signaling within the same range as conditions of interest. The various options are explored to prevent BMP2 non-specific adsorption and a successful blocking condition involving a combination of bovine serum albumin and trehalose are identified. Furthermore, the effect of this blocking step improved when using gold platforms instead of glass, particularly with Chinese hamster ovary (CHO) cells. With this specific example, it is suggested that non-specific adsorption of BMPs on biomaterials may be a general concern - often undetected by classical surface-sensitive techniques - that needs to be addressed to better interpret cellular responses.

Novel simultaneous analysis of 18 types of glycosaminoglycan-derived disaccharides using 4-aminobenzoic acid ethyl ester derivatization by HPLC with fluorescence detection.

Glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), heparan sulfate (HS)/heparin (HP), and keratan sulfate (KS), play pivotal roles in living organisms. Generally, GAGs are analyzed after enzymatic digestion into unsaturated or saturated disaccharides. Due to high structural similarity between disaccharides, however, separation during analysis is challenging. Additionally, little is known about the structures of GAGs and their functional relationships. Elucidating the function of GAGs requires highly sensitive quantitative analytical methods. We developed a method for the simultaneous analysis of 18 types of disaccharides derived from HA (1 type), CS/DS (7 types), HS/HP (8 types), and KS (2 types) potentially detectable in analyses of human urine. The simple method involves HPLC separation with fluorescence detection following derivatization of GAG-derived disaccharides using 4-aminobenzoic acid ethyl ester (ABEE) as a pre-labeling agent and 2-picoline borane as a reductant. The ABEE derivatization reaction can be performed under aqueous conditions, and excess derivatization reagents can be easily, rapidly, and safely removed. This method enables highly sensitive simultaneous analysis of the 18 abovementioned types of GAG-derived disaccharides using HPLC with fluorescence detection in small amounts of urine (1mL) in a single run. The versatile method described here could be applied to the analysis of GAGs in other biological samples.

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT)

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT).

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

Hunter’s syndrome: a rare case report

Hunter’s syndrome also known as mucopolysaccridosis-II (MPS-II) is a very rare X-linked recessive disease. It is a chronic progressive metabolic disease. The incidence of this condition is 1 in 1, 62,000 live births, mostly males. There is excessive accumulation of glycosaminoglycan (GAGs) in the lysosome in various parts of body due to the deficiency of enzyme iduronate sulphatase (IDS) and manifests with variety of clinical signs and symptoms. Four novel mutations in IDS gene are reported among families of Hunter syndrome tested by Sanger sequencing of IDS gene.

Cant1 Affects Cartilage Proteoglycan Properties: Aggrecan and Decorin Characterization in a Mouse Model of Desbuquois Dysplasia Type 1.

Desbuquois dysplasia type 1 (DBQD1) is a recessive chondrodysplasia caused by mutations in the CANT1 gene, encoding for the Golgi Calcium-Activated Nucleotidase 1 (CANT1). The enzyme hydrolyzes UDP, the by-product of glycosyltransferase reactions, but it might play other roles in different cell types. Using a Cant1 knock-out mouse, we demonstrated that CANT1 is crucial for glycosaminoglycan (GAG) synthesis; however, its impact on the biochemical properties of cartilage proteoglycans remains unknown. Thus, in this work, we characterized decorin and aggrecan from primary chondrocyte cultures and cartilage biopsies of mutant mice at post-natal day 4 by Western blots and further investigated their distribution in the cartilage extracellular matrix (ECM) by immunohistochemistry. We demonstrated that the GAG synthesis defect caused by CANT1 impairment led to the synthesis and secretion of proteoglycans with shorter GAG chains compared with wild-type animals. However, this alteration did not result in the synthesis and secretion of decorin and aggrecan in the unglycanated form. Interestingly, the defect was not cartilage-specific since also skin decorin showed a reduced hydrodynamic size. Finally, immunohistochemical studies in epiphyseal sections of mutant mice demonstrated that the proteoglycan structural defect moderately affected decorin distribution in the ECM.

Heparan Sulfate-Collagen Surface Multilayers Support Serum-Free Microcarrier Culture of Mesenchymal Stem Cells.

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (μCs) cultures. Additionally, rHS surfaces reduced β-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.

Structural Insights into Endostatin-Heparan Sulfate Interactions Using Modeling Approaches.

Glycosaminoglycans (GAGs) play a key role in a variety of biological processes in the extracellular matrix (ECM) via interactions with their protein targets. Due to their high flexibility, periodicity and electrostatics-driven interactions, GAG-containing complexes are very challenging to characterize both experimentally and in silico. In this study, we, for the first time, systematically analyzed the interactions of endostatin, a proteolytic fragment of collagen XVIII known to be anti-angiogenic and anti-tumoral, with heparin (HP) and representative heparan sulfate (HS) oligosaccharides of various lengths, sequences and sulfation patterns. We first used conventional molecular docking and a docking approach based on a repulsive scaling-replica exchange molecular dynamics technique, as well as unbiased molecular dynamic simulations, to obtain dynamically stable GAG binding poses. Then, the corresponding free energies of binding were calculated and the amino acid residues that contribute the most to GAG binding were identified. We also investigated the potential influence of Zn2+ on endostatin-HP complexes using computational approaches. These data provide new atomistic details of the molecular mechanism of HP's binding to endostatin, which will contribute to a better understanding of its interplay with proteoglycans at the cell surface and in the extracellular matrix.

TNFα-Induced Inflammation Model-Evaluation of Concentration and Passage-Dependent Effects on Bovine Chondrocytes.

Inflammation models are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis. TNFα (tumor necrosis factor alpha) plays an important role in the inflammatory process. Current inflammation models lack uniformity and make comparisons difficult. Therefore, this study aimed to systematically investigate whether the effects of TNFα are concentration-dependent and whether chondrocyte expansion has an effect on the inflammatory model. Bovine chondrocytes were enzymatically isolated, expanded to passages 1-3, and transferred into a 3D pellet culture. Chondrocyte pellets were stimulated with recombinant bovine TNFα at different concentrations for 48 h to induce inflammation. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP)), catabolic (matrix metalloproteinases (MMP3, MMP13)), dedifferentiation (collagen 1) markers, inflammation markers (interleukin-6 (IL-6), nuclear factor kappa B (NFkB), cyclooxygenase-2 (COX), prostaglandin-E-synthase-2 (PTGES2)), and the apoptosis marker caspase 3 was determined. At the protein level, concentrations of IL-6, nitric oxide (NO), and sulfated glycosaminoglycans (GAG) were evaluated. Statistical analysis was performed using the independent t-test, and significance was defined as p < 0.05. In general, TNFα caused a decrease in anabolic markers and an increase in the expression of catabolic and inflammatory markers. There was a concentration-dependent threshold of 10 ng/mL to induce significant inflammatory effects. Most of the markers analyzed showed TNFα concentration-dependent effects (COMP, PRG4, AGN, Col1, MMP3, and NFkB). There was a statistical influence of selected gene expression markers from different passages on the TNFα chondrocyte inflammation model, including Col2, MMP13, IL-6, NFkB, COX2, and PTGES2. Considering the expression of collagen 2 and MMP3, passage 3 chondrocytes showed a higher sensitivity to TNFα stimulation compared to passages 1 and 2. On the other hand, MMP13, IL-6, NFkB, and caspase 3 gene expression were lower in P3 chondrocytes compared to the other passages. On the protein level, inflammatory effects showed a similar pattern, with cytokine effects starting at 10 ng/mL and differences between the passages. TNFα had a detrimental effect on cartilage, with a clear threshold observed at 10 ng/mL. Although TNFα effects showed concentration-dependent patterns, this was not consistent for all markers. The selected passage showed a clear influence, especially on inflammation markers. Further experiments were warranted to explore the effects of TNFα concentration and passage in long-term stimulation.

Urinary Free Glycosaminoglycans Identify Adults at High Risk of Developing Early-stage High-grade Bladder Cancer

Background and objectiveScreening for bladder cancer (BCa) could reduce mortality via early detection of early-stage high-grade (Ta/T1 N0 M0 grade 2–3) disease. Noninvasive biomarkers could aid in screening, but current markers lack the specificity required. The urinary free glycosaminoglycan profile (GAGome) is a promising biomarker for early detection of BCa metabolism. MethodsIn a prospective case-control development study, we included patients with BCa or no evidence of disease (NED) and measured the urinary GAGome. We then developed a score to predict the probability of BCa using GAGome features that correlated with BCa versus NED according to Bayesian regression. Next, in a retrospective, population-based, case-control study, we included adults from the Lifelines Cohort Study who were presumed healthy at baseline. All cases with BCa confirmed in the cancer registry by the 2-yr or 6-yr study visit were matched to randomly selected control subjects. We developed a reference logistic regression model using age and sex to predict BCa at 7 yr after baseline. We then added the GAGome score to the model and assessed model improvement using the likelihood ratio test. We dichotomized outputs for the reference model and saturated model (reference + GAGome score) into high-risk versus low-risk categories using a 99% specificity cutoff and estimated the sensitivity for association with BCa at 7 yr. Key findings and limitationsWe prospectively included 51 individuals with BCa and 38 with NED and observed alterations in three GAGome features compatible with BCa. We developed a score that discriminated BCa with an area under the receiver operating characteristic curve of 0.77 (95% confidence interval [CI] 0.67–0.87). We retrospectively selected a cohort of 1088 presumed healthy adults (median age 48 yr, 56% females), of whom 48 had developed BCa by 7 yr after baseline (median time to diagnosis 1.4 yr). The GAGome score was an independent predictor of BCa at 7 yr when added to the reference model (p < 0.001). The sensitivity for BCa at 7 yr for high-risk subjects was 31% (95% CI 20–43%) using the saturated model and 17% (95% CI 4.7–29%) using the reference model at 99% specificity (95% CI 98–99%). Conclusions and clinical implicationsThe urinary free GAGome is specifically altered in BCa and can be used for noninvasive identification of adults at high risk of developing BCa, independent of age and sex. This information could be useful for the design of risk-stratified targeted screening programs for BCa. Patient summaryWe tested whether measurement of a class of sugars called glycosaminoglycans (GAGs) in urine could be used for early detection of bladder cancer. Our results show that GAG levels in urine can distinguish people at high risk of developing bladder cancer within 7 years, even if they are healthy at the time of the urine sampling.

Cell-free decellularized skin matrix scaffolds: A promising approach for meniscus regeneration in a rabbit meniscectomy model

Tissue engineering presents a promising approach for the treatment of meniscal injuries, yet the development of meniscal scaffolds that exhibit both superior biomechanical properties and biocompatibility remains a considerable challenge. In this study, decellularized skin matrix (DSM) scaffolds were first prepared using porcine skin through decellularization and freeze-drying techniques. The DSM scaffold has favorable porosity, hydrophilicity, and biocompatibility. Importantly, the collagen content and tensile modulus of the scaffold are comparable to those of native meniscus (44.13 ± 2.396 mg/g vs. 42.41 ± 2.40 mg/g and 103.30 ± 2.98 MPa vs. 128.80 ± 9.115 MPa). Subsequently, the peptide PFSSTKT (PFS) with mesenchymal stem cells (MSCs) recruitment capability was used to modify DSM to construct DSM-PFS scaffolds. Compared to the DSM scaffold, the optimized DSM-PFS scaffold enhanced in vitro collagen and glycosaminoglycan (GAG) production and upregulated the expression of cartilage-specific genes. Furthermore, the DSM-PFS scaffold was more effective in recruiting MSCs in vitro. In vivo studies in rabbit models showed that the DSM-PFS scaffold successfully promoted meniscus tissue regeneration. Three months post-implantation, meniscus tissue formation can be observable, and after six months, the neo-meniscus exhibited tissue structure and tensile properties similar to the native meniscus. Notably, the DSM-PFS scaffold exhibited significant chondroprotective effects, slowing osteoarthritis (OA) progression. In conclusion, the DSM-PFS scaffold may represent a promising candidate for future applications in meniscus tissue engineering. Statement of significanceWe developed a decellularized skin matrix (DSM) meniscus scaffold using whole-layer porcine skin, demonstrating superior biomechanical strength and biocompatibility. Following modification with the stem cell-recruiting peptide PFS, the optimized DSM-PFS scaffold outperformed the DSM scaffold in cell attraction, collagen and glycosaminoglycan production, and cartilage-specific gene expression. Implanted into rabbit knee joints, the cell-free DSM-PFS scaffold induced meniscal tissue formation within three months, achieving the histological structure and tensile strength of the native meniscus by six months. Moreover, it significantly protected the cartilage. Our findings provide new insights into the fabrication of scaffolds for meniscal tissue engineering, with the DSM-PFS scaffold emerging as an ideal candidate for future applications.

Improving the Purity of Extracellular Vesicles by Removal of Lipoproteins from Size Exclusion Chromatography- and Ultracentrifugation-Processed Samples Using Glycosaminoglycan-Functionalized Magnetic Beads.

Extracellular vesicles (EVs) are present in blood at much lower concentrations (5-6 orders of magnitude) compared to lipoprotein particles (LP). Because LP and EV overlap in size and density, isolating high-purity EVs is a significant challenge. While the current two-step sequential EV isolation process using size-expression chromatography (SEC) followed by a density gradient (DG) achieves high purity, the time-consuming ultracentrifugation (UC) step in DG hinders workflow efficiency. This paper introduces an optimized magnetic bead reagent, LipoMin, functionalized with glycosaminoglycans (GAGs), as a rapid alternative for LP removal during the second-step process in about 10 minutes. We evaluated LipoMin's efficacy on two sample types: (a) EV fractions isolated by size exclusion chromatography (SEC + LipoMin) and (b) the pellet obtained from ultracentrifugation (UC + LipoMin). The workflow is remarkably simple, involving a 10 min incubation with LipoMin followed by magnetic separation of the LP-depleted EV-containing supernatant. Results from enzyme-linked immunosorbent assay (ELISA) revealed that LipoMin removes 98.2% ApoB from SEC EV fractions, comparable to the LP removal ability of DG in the SEC + DG two-step process. Importantly, the EV yield (CD81 ELISA) remained at 93.0% and Western blot analysis confirmed that key EV markers, flotillin and CD81, were not compromised. Recombinant EV (rEV), an EV reference standard, was spiked into SEC EV fractions and recovered 89% of CD81 protein. For UC + LipoMin, ApoA1 decreased by 76.5% while retaining 90.7% of CD81. Notably, both colorectal cancer (CRC) and Alzheimer's disease (AD) samples processed by SEC + LipoMin and UC + LipoMin displayed clear expression of relevant EV and clinical markers. With a 10 min workflow (resulting in a 96% time saving compared to the traditional method), the LipoMin reagent offers a rapid and efficient alternative to DG for LP depletion, paving the way for a streamlined SEC + LipoMin two-step EV isolation process.

Kinetics and Retention of Polystyrenesulfonate for Proteoglycan Replacement in Cartilage.

Tissue hydration provides articular cartilage with dynamic viscoelastic properties. Early stage osteoarthritis (OA) is marked by loss of proteoglycans and glycosaminoglycans (GAG), lowering fixed charge density, and impairing tissue osmotic function. The most common GAG replacement, chondroitin sulfate (CS), has failed to show effectiveness. Here, we investigated a synthetic polyelectrolyte, poly(styrenesulfonate) (PSS), both as a model compound to investigate polyelectrolyte transport in cartilage, and as a potential candidate to restore bulk fixed charge density in cartilage with GAG loss. Through bovine explants and histology, we determined zonal-based effective diffusion coefficients for three different molecular weights of PSS. Compared to CS, PSS was retained longer in GAG-depleted cartilage in static and compression-based desorption experiments. We explained enhanced solute performance of PSS by its more compact morphology and higher charge density by small-angle X-ray scattering. This study may improve design of GAG mimetic molecules for repairing osmotic function in OA cartilage.

Synovium friction properties are influenced by proteoglycan content

The synovium plays a crucial role in diarthrodial joint health, and its study has garnered appreciation as synovitis has been linked to osteoarthritis symptoms and progression. Quantitative synovium structure–function data, however, remain sparse. In the present study, we hypothesized that tissue glycosaminoglycan (GAG) content contributes to the low friction properties of the synovium. Bovine and human synovium tribological properties were evaluated using a custom friction testing device in two different cases: (1) proteoglycan depletion to isolate the influence of tissue GAGs in the synovium friction response and (2) interleukin-1 (IL) treatment to observe inflammation-induced structural and functional changes. Following proteoglycan depletion, synovium friction coefficients increased while GAG content decreased. Conversely, synovium explants treated with the proinflammatory cytokine IL exhibited elevated GAG concentrations and decreased friction coefficients. For the first time, a relationship between synovium friction coefficient and GAG concentration is demonstrated. The study of synovium tribology is necessary to fully understand the mechanical environment of the healthy and diseased joint.

Mucopolysaccharidosis Type IIIE: A Real Human Disease or a Diagnostic Pitfall?

Mucopolysaccharidoses (MPS) comprise a group of 12 metabolic disorders where defects in specific enzyme activities lead to the accumulation of glycosaminoglycans (GAGs) within lysosomes. This classification expands to 13 when considering MPS IIIE. This type of MPS, associated with pathogenic variants in the ARSG gene, has thus far been described only in the context of animal models. However, pathogenic variants in this gene also occur in humans, but are linked to a different disorder, Usher syndrome (USH) type IV, which is sparking increasing debate. This paper gathers, discusses, and summarizes arguments both for and against classifying dysfunctions of arylsulfatase G (due to pathogenic variants in the ARSG gene) in humans as another subtype of MPS, called MPS IIIE. Specific difficulties in diagnostics and the classification of some inherited metabolic diseases are also highlighted and discussed.