Sulfated Glycosaminoglycans Research Articles

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.

Evaluation of morphology, physical and mechanical properties of hyaluronic acid matrices of various molecular weights by atomic force microscopy

Using atomic force microscopy the morphology of 1 and 2 % sodium hyaluronate solutions matrix with low molecular weight (LM) and high molecular weight (HM) hyaluronic acid (HA) fabricated by bioenzymatic synthesis was evaluated. The presence of a proteoglycan aggregate with processes of the core protein in the absence of sulfated glycosaminoglycans was established. The effect of irradiation with a light flux on the ordering of the HA matrix was observed. Intermolecular consolidation of high molecular HA was established. The elastic modulus of the matrix with LM and HM HA has also been determined.

Astrocyte extracellular matrix modulates neuronal dendritic development.

Major developmental events occurring in the hippocampus during the third trimester of human gestation and neonatally in altricial rodents include rapid and synchronized dendritic arborization and astrocyte proliferation and maturation. We tested the hypothesis that signals sent by developing astrocytes to developing neurons modulate dendritic development in vivo. We altered neuronal development by neonatal (third trimester-equivalent) ethanol exposure in mice; this treatment increased dendritic arborization in hippocampal pyramidal neurons. We next assessed concurrent changes in the mouse astrocyte translatome by translating ribosomal affinity purification (TRAP)-seq. We followed up on ethanol-inhibition of astrocyte Chpf2 and Chsy1 gene translation because these genes encode for biosynthetic enzymes of chondroitin sulfate glycosaminoglycan (CS-GAG) chains (extracellular matrix components that inhibit neuronal development and plasticity) and have not been explored before for their roles in dendritic arborization. We report that Chpf2 and Chsy1 are enriched in astrocytes and their translation is inhibited by ethanol, which also reduces the levels of CS-GAGs measured by Liquid Chromatography/Mass Spectrometry. Finally, astrocyte-conditioned medium derived from Chfp2-silenced astrocytes increased neurite branching of hippocampal neurons in vitro. These results demonstrate that CS-GAG biosynthetic enzymes in astrocytes regulates dendritic arborization in developing neurons.

Biosynthesis of animal-free recombinant chondroitin sulfate E using a functional chondroitin sulfotransferase in E. coli

Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli. Moreover, there is no experimentally derived three-dimensional structure available for this specific sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks.Key points• Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished.• First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.

Therapeutic potential of sulfated glycosaminoglycan from seafood Asian green mussel (Perna viridis): Insights from an in vivo study

The Asian green mussel, Perna viridis, is widely consumed as seafood throughout the coastal regions of the Indo-Pacific area owing to its significant pharmaceutical and biomedical benefits. A pharmacologically active (1 → 3)/(1 → 4) linked sulfated glycosaminoglycan (PVP-2) with β-(1 → 3)-GlcNSp, and α-(1 → 4)-GlcAp units was isolated from P. viridis. PVP-2 (at 1–10 μg/mL) demonstrated a suppression of nitric oxide (NO) secretion in lipopolysaccharide-induced RAW 264.7 cells. Notably, PVP-2 at 5 μg/mL effectively restored NO levels (0.32 μg/mL) to homeostasis by downregulating excessive production (1.22 μg/mL). Furthermore, PVP-2 (at 22 mg/kg body weight) showed effective reduction of carrageenan-induced paw edema (82% inhibition at 5 h) in time dependent manner. This reduction was found to be comparable with standard treatment (87% inhibition of paw edema at 5 h). Treatment with PVP-2 (at 110 mg/kg body weight) resulted substantial inhibition of formalin-induced paw edema up to 10 days. At the end of the 10th day, the reduction in paw thickness was significantly greater (a four-fold increase) in the PVP-2 treated group compared to the formalin-induced group. Furthermore, PVP-2 displayed potent anti-inflammatory effect by attenuating cyclooxygenases (COX-1, 2) and 5-lipoxygenase (5-LOX) (IC50 < 2 mg/mL) enzymes with a higher selectivity index (IC50 COX-1/IC50 COX-2 ∼ 1.7) towards inducible pro-inflammatory COX-2. Therefore, inhibition of the NO production along with the reduction of paw edema, highlights the therapeutic potential of sulfated glycosaminoglycan from seafood Asian green mussel, as a naturally derived functional food to mitigate inflammation related disorders.

Thrombin-derived C-terminal peptides bind and form aggregates with sulfated glycosaminoglycans

Glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) play crucial roles in inflammation and wound healing, serving as regulators of growth factors and pro-inflammatory mediators. In this study, we investigated the influence of heparin/HS on thrombin proteolysis and its interaction with the generated 11 kDa thrombin-derived C-terminal peptides (TCPs). Employing various biochemical and biophysical methods, we demonstrated that 11 kDa TCPs aggregate in the presence of GAGs, including heparin, heparan sulfate, and chondroitin sulfate-B. Circular dichroism analysis demonstrated that 11 kDa TCPs, in the presence of GAGs, adopt a β-sheet structure, a finding supported by thioflavin T1 (ThT) fluorescence measurements and visualization of 11 kDa TCP-heparin complexes using transmission electron microscopy (TEM). Furthermore, our investigations revealed a stronger binding affinity between 11 kDa TCPs and GAGs with higher sulfate group contents. Congruently, in silico simulations showed that interactions between 11 kDa TCPs and heparin/HS are predominantly electrostatic in nature. Collectively, our study suggests that 11 kDa TCPs have the capacity to aggregate in the presence of GAGs, shedding light on their potential roles in inflammation and wound healing.

Attenuation of arsenic-induced gut injury in mice by sulfated glycosaminoglycan from swim bladder via regulation of gut microbiota, metabolites, and intestinal barrier integrity

Attenuation of arsenic-induced gut injury in mice by sulfated glycosaminoglycan from swim bladder via regulation of gut microbiota, metabolites, and intestinal barrier integrity

Extracellular Matrix Sulfation in the Tumor Microenvironment Stimulates Cancer Stemness and Invasiveness.

Tumor extracellular matrices (ECM) exhibit aberrant changes in composition and mechanics compared to normal tissues. Proteoglycans (PG) are vital regulators of cellular signaling in the ECM with the ability to modulate receptor tyrosine kinase (RTK) activation via their sulfated glycosaminoglycan (sGAG) side chains. However, their role on tumor cell behavior is controversial. Here, it is demonstrated that PGs are heavily expressed in lung adenocarcinoma (LUAD) patients in correlation with invasive phenotype and poor prognosis. A bioengineered human lung tumor model that recapitulates the increase of sGAGs in tumors in an organotypic matrix with independent control of stiffness, viscoelasticity, ligand density, and porosity, is developed. This model reveals that increased sulfation stimulates extensive proliferation, epithelial-mesenchymal transition (EMT), and stemness in cancer cells. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K) signaling axis is identified as a mediator of sulfation-induced molecular changes in cells upon activation of a distinct set of RTKs within tumor-mimetic hydrogels. The study shows that the transcriptomic landscape of tumor cells in response to increased sulfation resembles native PG-rich patient tumors by employing integrative omics and network modeling approaches.

BMP-Binding Polysulfonate Brushes to Control Growth Factor Presentation and Regulate Matrix Remodelling.

Bone morphogenetic proteins (BMPs) are important targets to incorporate in biomaterial scaffolds to orchestrate tissue repair. Glycosaminoglycans (GAGs) such as heparin allow the capture of BMPs and their retention at the surface of biomaterials at safe concentrations. Although heparin has strong affinities for BMP2 and BMP4, two important types of growth factors regulating bone and tissue repair, it remains difficult to embed stably at the surface of a broad range of biomaterials and degrades rapidly in vitro and in vivo. In this report, biomimetic poly(sulfopropyl methacrylate) (PSPMA) brushes are proposed as sulfated GAG mimetic interfaces for the stable capture of BMPs. The growth of PSPMA brushes via a surface-initiated activator regenerated by electron transfer polymerization is investigated via ellipsometry, prior to characterization of swelling and surface chemistry via X-ray photoelectron spectroscopy and Fourier transform infrared. The capacity of PSPMA brushes to bind BMP2 and BMP4 is then characterized via surface plasmon resonance. BMP2 is found to anchor particularly stably and at high density at the surface of PSPMA brushes, and a strong impact of the brush architecture on binding capacity is observed. These results are further confirmed using a quartz crystal microbalance with dissipation monitoring, providing some insights into the mode of adsorption of BMPs at the surface of PSPMA brushes. Primary adsorption of BMP2, with relatively little infiltration, is observed on thick dense brushes, implying that this growth factor should be accessible for further binding of corresponding cell membrane receptors. Finally, to demonstrate the impact of PSPMA brushes for BMP2 capture, dermal fibroblasts were then cultured at the surface of functionalized PSPMA brushes. The presence of BMP2 and the architecture of the brush are found to have a significant impact on matrix deposition at the corresponding interfaces. Therefore, PSPMA brushes emerge as attractive coatings for scaffold engineering and stable capture of BMP2 for regenerative medicine applications.

Multiple Administration of Dexamethasone Possesses a Deferred Long-Term Effect to Glycosylated Components of Mouse Brain.

Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.

Sulphated glycosaminoglycan isolated from the edible slipper oyster Magallana bilineata (Röding, 1798) attenuates inflammatory cytokines on lipopolysaccharide-prompted macrophages

The slipper oyster Magallana bilineata (Ostreidae) is considered as culinary delicacy among marine bivalves, and a sulphated glycosaminoglycan, 4,6-O-SO3-β-(1→3)-GalNAcp (unit A) and β-(1→4)-GlcAp (unit B) as principle structural motif containing laterally branched 4-O-SO3-β-glucopyranose (unit C) (MBP-3) was isolated from this species. Nuclear magnetic resonance (NMR), Fourier transform infra-red (FTIR), and mass spectroscopy techniques were used to characterise MBP-3. MBP-3 exhibited anti-inflammatory activities against inflammatory 5-lipoxygenase (IC50 0.11 mg mL−1) and cyclooxygenase-2 (IC50 0.12 mg mL−1) enzymes. MBP-3 (at 100 μg mL−1) showed effective downregulation against pro-inflammatory cytokines generation, namely interleukins-6, 1β, (IL-6, 1β) (1–1.7 pg mL−1) and tumour necrosis factor-α (TNF-α) (4 pg mL−1) along with substantial downregulation of ROS production in lipopolysaccharide (LPS)-inflamed cells. MBP-3 blocked the mRNA of NF-κB, cyclooxygenase-2 (COX-2), and other cytokines, in lipopolysaccharide-induced macrophages. The potential to constrain inflammatory cytokine production revealed its application to develop functional food to attenuate inflammation-associated disorders.

The chondrocyte "mechanome": Activation of the mechanosensitive ion channels TRPV4 and PIEZO1 drives unique transcriptional signatures.

The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition invitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis.

Monoclonal Antibody chP3R99 Reduces Subendothelial Retention of Atherogenic Lipoproteins in Insulin-Resistant Rats: Acute Treatment Versus Long-Term Protection as an Idiotypic Vaccine for Atherosclerosis.

Atherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to low-density lipoprotein, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported antiatherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. Solid-phase assays demonstrated that chP3R99 effectively blocked >50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix invitro. The preperfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by ~60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose dependent (25-250 μg/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a 5-week vaccination study in insulin resistant rats with (200 μg subcutaneously, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of antichondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. Both acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced low-density lipoprotein and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting proatherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis.

Physiologic Doses of TGF-β Improve the Composition of Engineered Articular Cartilage.

Conventionally, for cartilage tissue engineering applications, TGF-β is administered at doses that are several orders of magnitude higher than those present during native cartilage development. While these doses accelerate extracellular matrix (ECM) biosynthesis, they may also contribute to features detrimental to hyaline cartilage function, including tissue swelling, type-I collagen (COL-I) deposition, cellular hypertrophy, and cellular hyperplasia. In contrast, during native cartilage development, chondrocytes are exposed to moderate TGF-β levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-β excesses. Here, we examine the hypothesis that physiologic doses of TGF-β can yield neocartilage with a more hyaline-cartilage-like composition and structure relative to conventionally-administered supraphysiologic doses. This hypothesis was examined on a model system of reduced-size constructs (Ø2×2mm or Ø3×2mm) comprised of bovine chondrocytes encapsulated in agarose, which exhibit mitigated TGF-β spatial gradients allowing for an evaluation of the intrinsic effect of TGF-β doses on tissue development. Reduced-size (Ø2×2mm or Ø3×2mm) and conventional-size constructs (Ø4-Ø6mm×2mm) were subjected to a range of physiologic (0.1, 0.3, 1ng/mL) and supraphysiologic (3, 10ng/mL) TGF-β doses. At day 56, the physiologic 0.3ng/mL dose yielded reduced-size constructs with native-cartilage-matched Young's modulus (EY) (630±58kPa) and sulfated GAG (sGAG) content (5.9±0.6%) while significantly increasing the sGAG-to-collagen ratio, leading to significantly reduced tissue swelling relative to constructs exposed to the supraphysiologic 10ng/mL TGF-β dose. Further, reduced-size constructs exposed to the 0.3ng/mL dose exhibited a significant reduction in fibrocartilage-associated COL-I and a 77% reduction in the fraction of chondrocytes present in a clustered morphology, relative to the supraphysiologic 10ng/mL dose (p<0.001). EY was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-β transport limitations in these larger tissues (p<0.001). Overall, physiologic TGF-β appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating features detrimental to hyaline cartilage function. While reduced-size constructs are not suitable for the repair of clinical-size cartilage lesions, insights from this work can inform TGF-β dosing requirements for emerging scaffold-release or nutrient-channel delivery platforms capable of achieving uniform delivery of physiologic TGF-β doses to larger constructs required for clinical cartilage repair.

Alterations in the Structure, Composition, and Organization of Galactosaminoglycan-Containing Proteoglycans and Collagen Correspond to the Progressive Stages of Dupuytren's Disease.

Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.

The Application of Sulfated Glycosaminoglycans in Pathologies of Vitreomacular and Retinal Interfaces. Literature Review

Perfection of technologies along with extreme diagnostic potential cause high clinical and anatomical effect of surgical intervention for vitreoretinal pathology. However final functional result is not always comparable with the prognostic expectations. Having the knowledge about the changes of the eye tissue at retinal pathology and their interrelation with the stages of healing in response to the operative trauma, it is possible to carry out timely preventive activity to optimize the healing process. Up to date the recognized method of treatment of resistant macular edema and increasing of visual acuity in patients is intravitreal injection of corticosteroids. However, their application is limited both in acute and distant period due to a wide range of side effects. Therefore, the problem of inhibition and complex treatment of traumatic effects on the retina in the course of surgical treatment remains topical. A promising direction in solving this problem is the study of the influence of sulfated glycosaminoglycans (sGAG) on the inflammatory response and repair processes of vitreomacular and retinal interface structures. The purpose of this study was to summarize the experience of experimental studies of the effect of sGAG preparations on pathological processes of various tissues to create a substrate for further studies of their application in the field of vitreomacular and retinal interfaces.

Dengue virus infection induces complement factor H but protein remains cell-associated, with changes intracellularly and in cell surface binding

Aim: Severe dengue is correlated with a decrease in the circulating complement regulator, factor H (FH) and prior work has shown that dengue virus (DENV) infection induces FH mRNA but not FH protein release. Here, the mechanisms of this phenomenon were defined. Methods: HEK293 cells were infected with DENV-2 and changes in FH mRNA and protein were analyzed by real time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and fluorescent microscopy. Additionally, cells were stimulated with size fractionated supernatants from DENV-infected cells, supernatant containing DENV non-structural protein-1 (NS1) without virus particles, and infections performed with or without the toll-like receptor-4 (TLR4) antagonist, TAK-242. Mass spectrometry was used to define the protein content of the fractionated supernatant, and treatment of cells with sialidase or heparinase was used to define cell-associated FH protein. Results: DENV-infection induced full-length FH mRNA and cell-associated FH protein. Microscopy demonstrated membrane and intracellular-associated FH with a cytoskeletal and perinuclear localisation, in both DENV positive and uninfected neighboring bystander cells. Fractionation of cultured supernatant from DENV-infected cells demonstrated that secreted factors &gt; 50 (kilodaltons) kDa induced FH mRNA and this could be blocked with TAK-242 but was not simulated by the TLR4 agonist, DENV NS1. Mass spectrometry detected DENV envelope, membrane and NS1, complement component 5 (C5), and complement FB, and indicated a &gt; 20-fold increase in C4, inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), and alpha-2-macroglobulin in the &gt; 50 kDa fraction from DENV-infected compared with conditioned media from uninfected cells. Sialic acid levels were unchanged and cleavage did not affect release of FH from DENV-infected compared to uninfected cells. In contrast, sulphated glycosaminoglycans (GAGs) were reduced in the cultured supernatant and cell lysates following DENV-infection, and heparinase cleavage released significantly more FH from DENV-compared with uninfected cells. Conclusions: Following DENV-infection, secreted molecules induce FH that remains intracellular and with increased binding to cell surface heparan sulphate. The mediators of induction of FH mRNA act in trans and via TLR4 but this is not likely to be via DENV NS1. The retention of FH in the local environment of the infected cell could benefit the virus by negating local complement killing of cells, and/or benefit the host by inhibition of heparan sulphate-mediated DENV infection to restrict viral spread.

The Biomechanical, Biochemical, and Morphological Properties of 19 Human Cadaveric Lower Limb Tendons and Ligaments: An Open-Access Data Set

Background: Methodological heterogeneity hinders data comparisons across isolated studies of tendon and ligament properties, limiting clinical understanding and affecting the development and evaluation of replacement materials. Purpose: To create an open-access data set on the morphological, biomechanical, and biochemical properties of clinically important tendons and ligaments of the lower limb, using consistent methodologies, to enable direct tendon/ligament comparisons. Study Design: Descriptive laboratory study. Methods: Nineteen distinct lower limb tendons and ligaments were retrieved from 8 fresh-frozen human cadavers (5 male, 3 female; aged 49-65 years) including Achilles, tibialis posterior, tibialis anterior, fibularis (peroneus) longus, fibularis (peroneus) brevis, flexor hallucis longus, extensor hallucis longus, plantaris, flexor digitorum longus, quadriceps, patellar, semitendinosus, and gracilis tendons; anterior cruciate, posterior cruciate, medial collateral, and lateral collateral ligaments; and 10 mm–wide grafts from the contralateral quadriceps and patellar tendons. Outcomes included morphology (tissue length, ultrasound-quantified cross-sectional area [CSAUS], and major and minor axes), biomechanics (failure load, ultimate tensile strength [UTS], failure strain, and elastic modulus), and biochemistry (sulfated glycosaminoglycan [sGAG] and hydroxyproline contents). Tissue differences were analyzed using mixed-model regression. Results: There was a range of similarities and differences between tendons and ligaments across outcomes. A key finding relating to potential graft tissue suitability was the comparable failure loads, UTS, CSAUS, sGAG, and hydroxyproline present between hamstring tendons (a standard graft source) and 5 tendons not typically used for grafting: fibularis (peroneus) longus and brevis, flexor and extensor hallucis longus, and flexor digitorum longus tendons. Conclusion: This study of lower limb tendons and ligaments has enabled direct comparison of morphological, biomechanical, and biochemical human tissue properties—key factors in the selection of suitable graft tissues. This analysis has identified 6 potential new donor tissues with properties comparable to currently used grafts. Clinical Relevance: This extensive data set reduces the need to utilize data from incompatible sources, which may aid surgical decisions (eg, evidence to expand the range of tendons considered suitable for use as grafts) and may provide congruent design inputs for new biomaterials and computational models. The complete data set has been provided to facilitate further investigations, with the capacity to expand the resource to include additional outcomes and tissues.

Evaluation of the effects of decellularized umbilical cord Wharton's Jelly ECM on polyhydroxy butyrate electrospun scaffolds: A new strategy for cartilage tissue engineering

The field of regenerative medicine has recently witnessed the emergence of the allogeneic decellularized extracellular matrix as a scaffold with natural biological signals in tissue engineering. In the present study, to prevent immune rejection and disease transmission, Wharton's Jelly extracellular matrix (WJM) derived from the human umbilical cord was first decellularized (DWJM). Eventually, the fibrous scaffolds based on poly 3-hydroxybutyrate (PHB) and five different concentrations (0, 1, 3, 5, and 10 wt%) of DWJM were fabricated through electrospinning. The results demonstrate reducing the diameter of the fibers, improved mechanical properties, hydrophilicity, and degradation rate in the scaffold containing 3 wt % DWJM. Excellent biocompatibility and increased chondrocyte cell adherence rate on the substrate of PHB-DWJM 3 % scaffolds compared with pure PHB scaffolds can be attributed to the high amounts of Collagen, sulfated glycosaminoglycans (GAGs), and hyaluronic acid (HA) in the DWJM. In conclusion, the physicochemical properties and biological activities of PHB-DWJM scaffolds indicate that blending DWJM with PHB electrospun scaffold can provide a suitable environment for articular cartilage tissue regeneration.

Potential prebiotic activity and improvement effect on intestinal injury of sulfated glycosaminoglycan from swim bladder

Recently, the exploration of bioactive substances for improving intestinal function has attracted wide attention. In this study, a sulfated glycosaminoglycan from the swim bladder (SBSG) was used to explore the prebiotic activities and its improvement effect on arsenic-induced intestinal injury. In vitro digestion experiments showed that SBSG was not degraded in the upper digestive tract and it could promote the growth of several lactic acid bacteria, which showed that it possessed potential prebiotic activities. In vivo study showed that SBSG could reduce cytokine (IL-2, IL-1β and TNF-α) to alleviate the level of inflammation, and decrease intestinal permeability by reducing endotoxin levels. SBSG greatly alleviated the macroscopic and microscopic pathological damage of the jejunum and colon induced by arsenic. Besides, SBSG could directly intervene in intestinal injury by reducing oxidative stress and acute inflammation. Our results indicated that SBSG could be developed as a new prebiotic resource against intestinal injury and regulate microecological balance.