Glycosaminoglycans Research Articles

Studying specificity in protein-glycosaminoglycan recognition with umbrella sampling.

In the past few decades, glycosaminoglycan (GAG) research has been crucial for gaining insights into various physiological, pathological, and therapeutic aspects mediated by the direct interactions between the GAG molecules and diverse proteins. The structural and functional heterogeneities of GAGs as well as their ability to bind specific proteins are determined by the sugar composition of the GAG, the size of the GAG chains, and the degree and pattern of sulfation. A deep understanding of the interactions in protein-GAG complexes is essential to explain their biological functions. In this study, the umbrella sampling (US) approach is used to pull away a GAG ligand from the binding site and then pull it back in. We analyze the binding interactions between GAGs of three types (heparin, desulfated heparan sulfate, and chondroitin sulfate) with three different proteins (basic fibroblast growth factor, acidic fibroblast growth factor, and cathepsin K). The main focus of our study was to evaluate whether the US approach is able to reproduce experimentally obtained structures, and how useful it can be for getting a deeper understanding of GAG properties, especially protein recognition specificity and multipose binding. We found that the binding free energy landscape in the proximity of the GAG native binding pose is complex and implies the co-existence of several binding poses. The sliding of a GAG chain along a protein surface could be a potential mechanism of GAG particular sequence recognition by proteins.

In vitro differentiation of human induced pluripotent stem cells into temporomandibular joint disc like cells

Temporomandibular joint discs (TMJ discs) are unable to repair themselves in disease states, while induced stem cell differentiation is a common method to repair tissue defects. Nowadays, kinds of stem cells are attempted for tissue regeneration of TMJ disc, but these methods have several downsides, which limit their wide application. The proliferation and differentiation ability of human induced pluripotent stem cells (hiPSC) provides a new research direction for TMJ disc tissue regeneration. In this study, we investigated the feasibility of induced differentiation of hiPSC into TMJ disc cells in vitro and the differentiation efficiency of different methods to clarify the possibility and conditions of hiPSC application in TMJ disc tissue engineering. We collected sheep TMJ disc cells cultures for adding in hiPSC culture environment and treated hiPSC by both direct induction and Transwell co-culture for 7 days, 14 days and 21 days. The secretion of extracellular matrix in TMJ disc cells was detected by Sirius Red and Safranin O staining. Collagen Ⅰ and Collagen Ⅱ were qualitatively detected by immunohistochemical staining. The expression of extracellular matrix genes (type I collagen (COL1A1), type II collagen(COL2), glycosaminoglycan (GAG)), chondrogenic differentiation gene SOX9 and pluripotency gene OCT4 were detected by RT-qPCR. Our results showed that hiPSC had the ability to differentiate to TMJ disc cells by direct induction in TMJ disc cell culture medium and by Transwell co-culture method. The highest degree of differentiation was observed after 14 days of direct induction, while Transwell co-culture showed significant differentiation at different times and with different major directions. Meanwhile, Transwell co-culture not only differentiates hiPSC but also promotes the growth and proliferation of TMJ disc cells. Our study is valuable to investigate the possibility of differentiation of hiPSC toward TMJ disc cells and to determine the time of differentiation. It provides new ideas for the selection of seed cells for TMJ disc tissue engineering.

Chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially modify the biophysical properties of collagen-based hydrogels

Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. This study focuses on the GAG molecules chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA). CS and DS are stereoisomers while HA is the only non-sulfated GAG. We characterized and decoupled the effects of these GAG molecules on the stiffness, transport, and matrix microarchitecture properties of type I collagen hydrogels using mechanical indentation testing, microfluidics, and confocal reflectance imaging, respectively. We complement these biophysical measurements with turbidity assays to profile collagen aggregate formation. Surprisingly, only HA enhanced the ECM indentation modulus, while all three GAGs had no effect on hydraulic permeability. Strikingly, we show that CS, DS, and HA differentially regulate the matrix microarchitecture of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs define key physical properties of the ECM, this work shows new ways in which stiffness measurements, microfluidics, microscopy, and turbidity kinetics can be used complementarily to reveal details of collagen self-assembly and structure. Statement of SignificanceCollagen and glycosaminoglycans (GAGs) are integral to the structure, function, and bioactivity of the extracellular matrix (ECM). Despite widespread interest in collagen-GAG composite hydrogels, there is a lack of quantitative understanding of how different GAGs alter the biophysical properties of the ECM across tissue, cellular, and subcellular length scales. Here we show using mechanical, microfluidic, microscopy, and analytical methods and measurements that the GAG molecules chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially regulate the mechanical, transport, and microstructural properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. As such, these results will inform improved design and utilization of collagen-based scaffolds of tailored composition, mechanical properties, molecular availability due to mass transport, and microarchitecture.

Sweet but Challenging: Tackling the Complexity of GAGs with Engineered Tailor-Made Biomaterials.

Glycosaminoglycans (GAGs) play a crucial role in tissue homeostasis by regulating the activity and diffusion of bioactive molecules. Incorporating GAGs into biomaterials has emerged as a widely adopted strategy in medical applications, owing to their biocompatibility and ability to control the release of bioactive molecules. Nevertheless, immobilized GAGs on biomaterials can elicit distinct cellular responses compared to their soluble forms, underscoring the need to understand the interactions between GAG and bioactive molecules within engineered functional biomaterials. By controlling critical parameters such as GAG type, density, and sulfation, it becomes possible to precisely delineate GAG functions within a biomaterial context and to better mimic specific tissue properties, enabling tailored design of GAG-based biomaterials for specific medical applications. However, this requires access to pure and well-characterized GAG compounds, which remains challenging. This review focuses on different strategies for producing well-defined GAGs and explores high-throughput approaches employed to investigate GAG-growth factor interactions and to quantify cellular responses on GAG-based biomaterials. These automated methods hold considerable promise for improving the understanding of the diverse functions of GAGs. In perspective, the scientific community is encouraged to adopt a rational approach in designing GAG-based biomaterials, taking into account the in vivo properties of the targeted tissue for medical applications.

Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry

The human sulfatase HSulf-2 is one of only two known endosulfatases that play a decisive role in modulating the binding properties of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Recently, HSulf-2 was shown to exhibit an unusual post-translational modification consisting of a sulfated glycosaminoglycan chain. This study describes the structural characterization of this glycosaminoglycan (GAG) and provides new data on its impact on the catalytic properties of HSulf-2. The unrevealed nature of this GAG chain is identified as a chondroitin/dermatan sulfate (CS/DS) mixed chain, as shown by mass spectrometry combined with NMR analysis. It consists primarily of 6-O and 4-O monosulfated disaccharide units, with a slight predominance of the 4-O-sulfation. Using atomic force microscopy, we show that this unique post-translational modification dramatically impacts the enzyme hydrodynamic volume. We identified human hyaluronidase-4 as a secreted hydrolase that can digest HSulf-2 GAG chain. We also showed that HSulf-2 is able to efficiently 6-O-desulfate antithrombin III binding pentasaccharide motif, and that this activity was enhanced upon removal of the GAG chain. Finally, we identified five N-glycosylation sites on the protein and showed that, although required, reduced N-glycosylation profiles were sufficient to sustain HSulf-2 integrity.

Anti-Apoptosis Therapy for Meniscal Avascular Zone Repair: A Proof-of-Concept Study in a Lapine Model.

In the present study, 24 rabbits were firstly used to evaluate the apoptosis index and matrix degeneration after untreated adult meniscal tears. Vertical tears (0.25 cm in length) were prepared in the avascular zone of the anterior horn. Specimens were harvested at 1, 3, 6, 12 weeks postoperatively. The apoptosis index around tear sites stayed at a high level throughout the whole follow-up period. The depletion of glycosaminoglycans (GAG) and aggrecan at the tear site was observed, while the deposition of COL I and COL II was not affected, even at the last follow-up of 12 weeks after operation. The expression of SOX9 decreased significantly; no cellularity was observed at the wound interface at all timepoints. Secondly, another 20 rabbits were included to evaluate the effects of anti-apoptosis therapy on rescuing meniscal cells and enhancing meniscus repair. Longitudinal vertical tears (0.5 cm in length) were made in the meniscal avascular body. Tears were repaired by the inside-out suture technique, or repaired with sutures in addition to fibrin gel and blank silica nanoparticles, or silica nanoparticles encapsulating apoptosis inhibitors (z-vad-fmk). Samples were harvested at 12 months postoperatively. We found the locally administered z-vad-fmk agent at the wound interface significantly alleviated meniscal cell apoptosis and matrix degradation, and enhanced meniscal repair in the avascular zone at 12 months after operation. Thus, local administration of caspase inhibitors (z-vad-fmk) is a promising therapeutic strategy for alleviating meniscal cell loss and enhancing meniscal repair after adult meniscal tears in the avascular zone.

Resveratrol-loaded decellularized ovine pericardium: ECM introduced for tissue engineering.

An ideal scaffold for skin tissue engineering should have a suitable potential for antibacterial activity, no hemolysis, sufficient porosity for air exchange, water retention capacity, and a suitable swelling rate to maintain tissue moisture. Considering this issue, our study used decellularized ovine pericardial tissue's extracellular matrix (ECM). These scaffolds were decellularized with sodium dodecyl sulfate (SDS) and sodium deoxycholate (SD) detergents along with vacuum methods. Following imaging with scanning electron microscopy (SEM), analysis of the mechanical properties, and the measurement of the amount of DNA, collagen, and glycosaminoglycan (GAG), our study observed that the three-dimensional (3D) structure of ECM was largely preserved. Resveratrol (RES) 400µg/µL was loaded into the above scaffold, and analysis revealed that scaffolds containing RES and with vacuum reported higher antibacterial properties, a higher swelling rate, and increased water retention capacity. The biocompatibility and hemocompatibility properties of the above scaffolds also reported a significant difference between methods of decellularization.

Glycosaminoglycans' Ability to Promote Wound Healing: From Native Living Macromolecules to Artificial Biomaterials.

Glycosaminoglycans (GAGs) are important for the occurrence of signaling molecules and maintenance of microenvironment within the extracellular matrix (ECM) in living tissues. GAGs and GAG-based biomaterial approaches have been widely explored to promote in situ tissue regeneration and repair by regulating the wound microenvironment, accelerating re-epithelialization, and controlling ECM remodeling. However, most approaches remain unacceptable for clinical applications. To improve insights into material design and clinical translational applications, this review highlights the innate roles and bioactive mechanisms of native GAGs during in situ wound healing and presents common GAG-based biomaterials and the adaptability of application scenarios in facilitating wound healing. Furthermore, challenges before the widespread commercialization of GAG-based biomaterials are shared, to ensure that future designed and constructed GAG-based artificial biomaterials are more likely to recapitulate the unique and tissue-specific profile of native GAG expression in human tissues. This review provides a more explicit and clear selection guide for researchers designing biomimetic materials, which will resemble or exceed their natural counterparts in certain functions, thereby suiting for specific environments or therapeutic goals.

Differential heparan sulfate dependency of the Drosophila glypicans

Heparan sulfate proteoglycans (HSPGs) are composed of a core-protein and glycosaminoglycan (GAG) chains and serve as co-receptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the PG function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment Serine residues are substituted to Alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core-proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require HS chains and that need both core-protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS-dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

B3GALT6 promotes dormant breast cancer cell survival and recurrence by enabling heparan sulfate-mediated FGF signaling

Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. Invivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.

Mapping microarchitectural degeneration in the dilated ascending aorta with ex vivo diffusion tensor imaging.

Thoracic aortic aneurysms (TAAs) carry a risk of catastrophic dissection. Current strategies to evaluate this risk entail measuring aortic diameter but do not image medial degeneration, the cause of TAAs. We sought to determine if the advanced magnetic resonance imaging (MRI) acquisition strategy, diffusion tensor imaging (DTI), could delineate medial degeneration in the ascending thoracic aorta. Porcine ascending aortas were subjected to enzyme microinjection, which yielded local aortic medial degeneration. These lesions were detected by DTI, using a 9.4 T MRI scanner, based on tensor disorientation, disrupted diffusion tracts, and altered DTI metrics. High-resolution spatial analysis revealed that fractional anisotropy positively correlated, and mean and radial diffusivity inversely correlated, with smooth muscle cell (SMC) and elastin content (P < 0.001 for all). Ten operatively harvested human ascending aorta samples (mean subject age 61.6 ± 13.3 years, diameter range 29-64 mm) showed medial pathology that was more diffuse and more complex. Nonetheless, DTI metrics within an aorta spatially correlated with SMC, elastin, and, especially, glycosaminoglycan (GAG) content. Moreover, there were inter-individual differences in slice-averaged DTI metrics. Glycosaminoglycan accumulation and elastin degradation were captured by reduced fractional anisotropy (R2 = 0.47, P = 0.043; R2 = 0.76, P = 0.002), with GAG accumulation also captured by increased mean diffusivity (R2 = 0.46, P = 0.045) and increased radial diffusivity (R2 = 0.60, P = 0.015). Ex vivo high-field DTI can detect ascending aorta medial degeneration and can differentiate TAAs in accordance with their histopathology, especially elastin and GAG changes. This non-destructive window into aortic medial microstructure raises prospects for probing the risks of TAAs beyond lumen dimensions.

Investigation of Glycosaminoglycans in Urine and Their Alteration in Patients with Juvenile Idiopathic Arthritis.

(1) Background: In this study, we evaluated the modulation of urine glycosaminoglycans (GAGs), which resulted from etanercept (ETA) therapy in patients with juvenile idiopathic arthritis (JIA) in whom methotrexate therapy failed to improve their clinical condition. (2) Methods: The sulfated GAGs (sGAGs, by complexation with blue 1,9-dimethylmethylene), including chondroitin-dermatan sulfate (CS/DS) and heparan sulfate (HS), as well as non-sulfated hyaluronic acid (HA, using the immunoenzymatic method), were determined in the blood of 89 children, i.e., 30 healthy children and 59 patients with JIA both before and during two years of ETA treatment. (3) Results: We confirmed the remodeling of the urinary glycan profile of JIA patients. The decrease in the excretion of sGAGs (p < 0.05), resulting from a decrease in the concentration of the dominant fraction in the urine, i.e., CS/DS (p < 0.05), not compensated by an increase in the concentration of HS (p < 0.000005) and HA (p < 0.0005) in the urine of patients with the active disease, was found. The applied biological therapy, leading to clinical improvement in patients, at the same time, did not contribute to normalization of the concentration of sGAGs (p < 0.01) in the urine of patients, as well as CS/DS (p < 0.05) in the urine of sick girls, while it promoted equalization of HS and HA concentrations. These results indicate an inhibition of the destruction of connective tissue structures but do not indicate their complete regeneration. (4) Conclusions: The metabolisms of glycans during JIA, reflected in their urine profile, depend on the patient's sex and the severity of the inflammatory process. The remodeling pattern of urinary glycans observed in patients with JIA indicates the different roles of individual types of GAGs in the pathogenesis of osteoarticular disorders in sick children. Furthermore, the lack of normalization of urinary GAG levels in treated patients suggests the need for continued therapy and continuous monitoring of its effectiveness, which will contribute to the complete regeneration of the ECM components of the connective tissue and thus protect the patient against possible disability.

Pleiotropic effects of hyaluronic acid and silver sulfadiazine dressings in the treatment of acute and chronic skin lesions.

Hyaluronic acid (HA) is a natural unbranched polymer that belongs to a group of heteropolysaccharide glycosaminoglycans (GAGs) that are major components of the extracellular matrix (ECM), while silver sulfadiazine exerts antibacterial activity. In this study, we evaluated the efficacy and safety of dressings with hyaluronic acid and silver sulfadiazine in acute and chronic lesions, according to the wound bed preparation and TIME principles. Thirty-two patients with acute and chronic injuries participated in the study. After collecting their personal histories and making a differential diagnosis by evaluating their ankle/arm index, patients with a Winsor Index below 0.8 underwent lower extremity color Doppler echocardiography. The dressing protocol followed the principles of wound bed preparation, identifying the prevailing clinical sign by evaluating the lesion background, margins, and perilesional skin. A product containing low molecular weight hyaluronic acid (200 kDa) and silver sulfadiazine 1% was used for the dressing. In the acute lesion group, the protocol we applied resulted in all injuries being healed. Of the 20 treated wounds in the chronic lesion group, 10 healed at the end of 8 weeks of treatment and 8 saw an improvement with a reduction in the lesion area. Consisting of a combination of hyaluronic acid and silver sulfadiazine, the dressing is widely used in the management of acute and chronic skin wounds. In the present study, the healing rate of acute wounds was 100%; in chronic wounds, healing was reported in 50% of cases while in 40% of the remainder, we found a 40% reduction in the lesion area.

Discovery of Novel Chromenopyridine Derivatives as Readthrough-Inducing Drugs.

Hurler syndrome, a type of Mucopolysaccharidosis type I, is an inherited disorder caused by the accumulation of glycosaminoglycans (GAG) due to a deficiency in lysosomal α-L-iduronidase (IDUA), resulting in multiorgan dysfunction. In many patients with Hurler syndrome, IDUA proteins are not produced due to nonsense mutations in their genes; therefore, readthrough-inducing compounds, such as gentamycin, are expected to restore IDUA proteins by skipping the premature termination codon. In the present study, we synthesized a series of chromenopyridine derivatives to identify novel readthrough-inducing compounds. The readthrough-inducing activities of synthesized compounds were examined by measuring cellular IDUA activities and GAG concentrations in Hurler syndrome patient-derived cells. Compounds with a difluorophenyl group at the 2-position of chromenopyridine, a cyclobutyl group at the 3-position, and a basic side chain or basic fused ring exhibited excellent readthrough-inducing activities. KY-640, a chromenopyridine derivative with a tetrahydroisoquinoline sub-structure, increased the cellular IDUA activities of patient-derived cells by 3.2-fold at 0.3 µM and significantly reduced GAG concentrations, and also significantly increased enzyme activity in mouse models, suggesting its therapeutic potential in patients with Hurler syndrome.

Characterization of the Age-Related Differences in Porcine Acetabulum and Femoral Head Articular Cartilage.

The use of porcine animal models for cartilage injury has increased recently due to their similarity with humans with regard to cartilage thickness, limited intrinsic healing of chondral defects, and joint loading biomechanics. However, variations in the mechanical and biochemical properties of porcine hip articular cartilage among various tissue ages and weightbearing (WB) regions are still unknown. This study's aim was to characterize the mechanical and biochemical properties of porcine hip articular cartilage across various ages and WB regions. Articular cartilage explants were harvested from WB and non-weightbearing (NWB) surfaces of the femoral head and acetabulum of domesticated pigs (Sus scrofa domesticus) at fetal (gestational age: 80 days), juvenile (6 months), and adult (2 years) ages. Explants underwent compressive stress-relaxation mechanical testing, biochemical analysis for total collagen and glycosaminoglycan (GAG) content, and histological staining. Juvenile animals consistently had the highest mechanical properties, with 2.2- to 7.6-time increases in relaxation modulus, 1.3- to 2.3-time increases in instantaneous modulus, and 4.1- to 14.2-time increases in viscosity compared with fetal cartilage. Mechanical properties did not significantly differ between the WB and NWB regions. Collagen content was highest in the NWB regions of the juvenile acetabulum (65.3%/dry weight [DW]) and femoral head (75.4%/DW) cartilages. GAG content was highest in the WB region of the juvenile acetabulum (23.7%/DW) and the WB region of the fetal femoral head (27.5%/DW) cartilages. Histological staining for GAG and total collagen content followed the trends from the quantitative biochemical assays. This study provides a benchmark for the development and validation of preclinical porcine models for hip cartilage pathologies.

Glycosaminoglycan-mimetic infernan grafted with poly(N-isopropylacrylamide): Toward a thermosensitive polysaccharide

Glycosaminoglycans (GAGs) are essential constituents of the cell surface and extracellular matrix, where they are involved in several cellular processes through their interactions with various proteins. For successful tissue regeneration, developing an appropriate matrix supporting biological activities of cells in a similar manner than GAGs remains still challenging. In this context, this study aims to design a thermosensitive polysaccharide that could further be used as hydrogel for tissue engineering applications. For this purpose, infernan, a marine bacterial exopolysaccharide (EPS) endowed with GAG-mimetic properties was grafted with a thermosensitive polymer, poly(N-isopropylacrylamide) (pNIPAM). Eight grafted polysaccharides were obtained by varying EPS/pNIPAM molar ratio and the molecular weight of pNIPAM. Their physicochemical characteristics and their thermosensitive properties were determined using a multi-technique, experimental approach. In parallel, molecular dynamics and Monte Carlo simulations were applied at two different scales to elucidate, respectively, the molecular conformation of grafted infernan chain and their ability to form an infinite network undergoing a sol-gel transition near the percolation, a necessary condition in hydrogel formation. It comes out from this study that thermosensitive infernan was successfully developed and its potential use in tissue regeneration as a hydrogel scaffold will further be assessed.

Zinc-a2-Glycoprotein Acts as a Component of PNN to Protect Hippocampal Neurons from Apoptosis.

In the adult mouse brain, perineuronal net (PNN), a highly structured extracellular matrix, surrounds subsets of neurons. The AZGP1 gene encodes zinc-2-glycoprotein (ZAG) is a lipid-mobilizing factor. However, its expression and distribution in the adult brain have been controversial. Here, for the first time, we demonstrate that the secreted ZAG is localized to Wisteria floribunda agglutinin (WFA)-positive PNNs around parvalbumin (PV)-expressing interneurons in the hippocampus, cortex, and a number of other PNN-bearing neurons and co-localizes with aggrecan, one of the components of PNNs. Few ZAG-positive nets were seen in the area without WFA staining by chondroitinase ABC (ChABC) which degrades glycosaminoglycans (GAGs) from the chondroitin sulfate proteoglycans (CSPGs) in the PNN. Reanalysis of single-cell sequencing data revealed that ZAG mRNA was mainly expressed in oligodendrocyte lineages, specifically in olfactory sheathing cells. The ZAG receptor β3 adrenergic receptor (β3AR) is also selectively co-localized with PV interneurons and CA2 pyramidal neurons in the hippocampus. In addition, molecular docking provides valuable new insights on how GAGs interfere with ZAG and ZAG/β3AR complex. Finally, our results indicated that human recombinant ZAG could significantly inhibit serum derivation-induced cell apoptosis in HT22 cells. Our combined experimental and theoretical approach raises a unique hypothesis namely that ZAG may be a crucial functional attribute of PNNs in the brain to protect neuronal cell from apoptosis.

Zn2+ Differentially Influences the Neutralisation of Heparins by HRG, Fibrinogen, and Fibronectin.

For coagulation to be initiated, anticoagulant glycosaminoglycans (GAGs) such as heparins need to be neutralised to allow fibrin clot formation. Platelet activation triggers the release of several proteins that bind GAGs, including histidine-rich glycoprotein (HRG), fibrinogen, and fibronectin. Zn2+ ions are also released and have been shown to enhance the binding of HRG to heparins of a high molecular weight (HMWH) but not to those of low molecular weight (LMWH). The effect of Zn2+ on fibrinogen and fibronectin binding to GAGs is unknown. Here, chromogenic assays were used to measure the anti-factor Xa and anti-thrombin activities of heparins of different molecular weights and to assess the effects of HRG, fibrinogen, fibronectin, and Zn2+. Surface plasmon resonance was also used to examine the influence of Zn2+ on the binding of fibrinogen to heparins of different molecular weights. Zn2+ had no effect on the neutralisation of anti-factor Xa (FXa) or anti-thrombin activities of heparin by fibronectin, whereas it enhanced the neutralisation of unfractionated heparin (UFH) and HMWH by both fibrinogen and HRG. Zn2+ also increased neutralisation of the anti-FXa activity of LMWH by fibrinogen but not HRG. SPR showed that Zn2+ increased fibrinogen binding to both UFH and LMWH in a concentration-dependent manner. The presented results reveal that an increase in Zn2+ concentration has differential effects upon anticoagulant GAG neutralisation by HRG and fibrinogen, with implications for modulating anti-coagulant activity in plasma.

Gastroprotective Effects of Oral Glycosaminoglycans with Sodium Alginate in an Indomethacin-Induced Gastric Injury Model in Rats.

The gastrointestinal (GI) mucosal barrier is often exposed to inflammatory and erosive insults, resulting in gastric lesions. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), chondroitin sulfate (CS), and N-acetylglucosamine (NAG) have shown potential beneficial effects as GI protectants. This study aimed to evaluate the gastroprotective effects of oral GAGs in rats with indomethacin-induced GI lesions. Forty-five Sprague-Dawley rats (8-9 weeks-old, 228 ± 7 g) were included in the study, divided into five study groups, and given, administered orally, either sucralfate (positive control group; PC), NAG (G group), sodium alginate plus HA and CS (AHC group), sodium alginate plus HA, CS, and NAG (AHCG group), or no treatment (negative control group; NC). Animals were administered 12.5 mg/kg indomethacin orally 15 min after receiving the assigned treatment. After 4 h, stomach samples were obtained and used to perform a macroscopic evaluation of gastric lesions and to allow histological assessment of the gastric wall (via H/E staining) and mucous (via PAS staining). The AHCG group showed significant gastroprotective improvements compared to the NC group, and a similar efficacy to the PC group. This combination of sodium alginate with GAGs might, therefore, become a safe and effective alternative to prescription drugs for gastric lesions, such as sucralfate, and have potential usefulness in companion animals.